Use of active ingredient DST from yam of dioscorea septemloba in preparation of medicine for treating psoriasis

Abstract

The application belongs to the technical field of biological medicine and natural medicine, and discloses a use of an active ingredient DST from Dioscorea septemloba Thunb. in preparation of a medicine for treating psoriasis. Research shows that the DST can regulate LYN related signals, reduce the phosphorylation level of STAT3 protein at Y705 site, down-regulate the expression of LRG1 in keratinocytes and the LRG1 level in extracellular vesicles of the keratinocytes, and then weaken the activation of macrophage ERK / CREB1 signals and M1 type pro-inflammatory polarization. The DST can also inhibit abnormal proliferation and migration of keratinocytes, reduce epidermal thickening, inflammatory cell infiltration and various pro-inflammatory factor levels of psoriasis-like skin lesions, and thus improve psoriasis-like inflammatory reactions. The application provides an experimental basis and a new technical scheme for the application of the DST in the medicine for treating psoriasis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and natural medicine technology, specifically relating to the application and mechanism of action of active ingredients derived from Dioscoreae Septemloba in anti-psoriasis drugs, particularly the use of the active ingredients to improve psoriasis-related inflammatory responses by regulating the LYN / STAT3 / LRG1 signaling axis, inhibiting abnormal proliferation and migration of keratinocytes, and regulating intercellular communication between keratinocytes and macrophages and macrophage M1 polarization. Background Technology

[0002] Psoriasis is a chronic, relapsing, immune-mediated inflammatory skin disease. Its main pathological features include abnormal proliferation and differentiation of keratinocytes, epidermal thickening, and extensive infiltration of immune cells in the lesions. The development of psoriasis involves complex interactions between keratinocytes, macrophages, and various inflammatory cytokines, and may be accompanied by abnormal expression of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17A (IL-17A), and interleukin-22 (IL-22). Psoriasis has a prolonged course and is prone to recurrence, affecting not only the patient's skin barrier function and appearance but also having a lasting impact on their quality of life.

[0003] Currently, psoriasis treatment mainly includes topical medications, systemic medications, and biologics targeting specific inflammatory factors or immune pathways. In recent years, biologics targeting specific cytokines and their related inflammatory cascades have achieved good therapeutic effects in improving psoriatic lesions. However, existing treatments still have certain limitations. For example, some biologics are expensive, and long-term use may be associated with immunosuppression risks. Furthermore, the development of psoriasis is not driven by a single inflammatory factor, and single-target interventions may still have insufficient efficacy, relapse, or require long-term maintenance therapy in some patients. Therefore, developing drugs with good safety profiles that can comprehensively regulate the abnormal proliferation and immune inflammatory microenvironment associated with psoriasis remains of great significance.

[0004] Abnormal proliferation and migration of keratinocytes are one of the important pathological bases of psoriasis. Meanwhile, increasing research indicates that macrophage polarization imbalance also participates in the formation and maintenance of the inflammatory microenvironment in psoriasis. Macrophages can undergo functional polarization in different directions according to the local microenvironment, among which pro-inflammatory M1 macrophages can produce various inflammatory mediators and further promote local inflammatory responses. In psoriasis patient lesions and related animal models, an increased proportion of pro-inflammatory macrophages and abnormal M1 / M2 polarization balance have been observed. Therefore, how to regulate macrophage-related inflammatory responses while inhibiting abnormal keratinocyte activation is an important direction in psoriasis drug research.

[0005] Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in the regulation of cell proliferation, differentiation, and inflammatory responses. Previous studies have shown that abnormal activation of STAT3 signaling is closely related to excessive proliferation of keratinocytes and inflammatory responses associated with psoriasis. However, in the pathological environment of psoriasis, the upstream regulatory processes of abnormal STAT3 activation and its interactions with keratinocytes and immune cells remain complex, and further research is needed to develop safe and effective interventional drugs targeting these regulatory networks.

[0006] Furthermore, extracellular vesicles, especially exosomes, serve as important carriers of intercellular communication, carrying various bioactive substances such as proteins and nucleic acids, and participating in signal exchange between local skin cells. Intercellular communication between keratinocytes and macrophages, established through secreted factors and extracellular vesicles, may be involved in the sustained maintenance of the inflammatory microenvironment in psoriasis. Therefore, identifying drugs that can simultaneously intervene in abnormal proliferation and pro-inflammatory immune responses, from the perspective of keratinocyte-immune cell interactions, holds potential value for psoriasis treatment.

[0007] Traditional Chinese medicine and its natural active ingredients are characterized by multi-component, multi-target, and multi-pathway regulation, providing a certain research foundation for the intervention of chronic inflammatory diseases. Dioscoreae Septemloba, a plant of the Dioscoreaceae family, has active ingredients mainly including steroidal saponins and their analogues. Previous studies have shown that active ingredients from Dioscorea species possess pharmacological activities such as anti-inflammatory, analgesic, and regulation of multiple cell signaling pathways, and can regulate pathways such as HIF-1α, EZH2 / STAT3, and COX-2 / PPAR-γ.

[0008] However, current technologies still lack systematic research on the specific effects of active ingredients derived from Dioscorea hypoglauca in the treatment of psoriasis, particularly regarding their ability to simultaneously regulate the abnormal proliferation and migration of psoriasis-related keratinocytes and the skin's immune-inflammatory microenvironment. Sufficient mechanistic studies and experimental verification are still lacking. Further elucidating the regulatory role of active ingredients derived from Dioscorea hypoglauca in key pathological processes of psoriasis, and developing new anti-psoriasis drugs accordingly, remains a pressing technical problem to be solved in this field. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide the use of DST, an active ingredient derived from Dioscorea hypoglauca, in the preparation of drugs for treating psoriasis, so as to improve the abnormal proliferation, migration and inflammatory response of psoriasis-related keratinocytes, and regulate the inflammatory-related signal transduction between keratinocytes and macrophages, thereby achieving the regulation of psoriatic skin lesions and the immune inflammatory microenvironment.

[0010] This invention further regulates the LYN / STAT3 / LRG1 signaling axis by using the active ingredient DST derived from Dioscorea hypoglauca, thereby reducing the phosphorylation level of tyrosine residue 705 (Y705) of the STAT3 protein, downregulating the expression of LRG1 in keratinocytes, and further regulating extracellular vesicle-related signals from keratinocytes, as well as ERK / CREB1 signals and M1-type pro-inflammatory polarization in macrophages, in order to inhibit psoriasis-related abnormal epidermal proliferation and inflammatory response.

[0011] To achieve the above objectives, the present invention provides the following technical solution: In one possible implementation, the use of DST, an active ingredient derived from Dioscorea hypoglauca, in the preparation of a medicament for treating psoriasis.

[0012] In one possible implementation, the drug exerts its anti-psoriatic effect by modulating the LYN / STAT3 / LRG1 signaling axis.

[0013] In one possible implementation, the DST reduces the phosphorylation level of tyrosine residue 705 of the STAT3 protein by inhibiting LYN-mediated signal transduction and downregulates the transcription and / or protein expression of LRG1 in keratinocytes.

[0014] In one possible implementation, the DST reduces the level of phosphorylated STAT3 without reducing the expression level of total STAT3 protein.

[0015] In one possible implementation, the drug inhibits the abnormal proliferation and / or migration of psoriasis-associated keratinocytes by reducing the phosphorylation level of tyrosine residue 705 of the STAT3 protein and downregulating the expression of LRG1 in keratinocytes.

[0016] In one possible implementation, the drug reduces the epidermal thickness and / or the number of Ki-67 positive cells in psoriatic lesions, and reduces the expression and / or secretion of at least one pro-inflammatory factor among TNF-α, IL-1β, IL-6, IL-17A and IL-22.

[0017] In one possible implementation, the drug reduces the LRG1 content in extracellular vesicles derived from keratinocytes by downregulating the expression of LRG1 in keratinocytes, the extracellular vesicles including exosomes.

[0018] In one possible implementation, the drug inhibits macrophage polarization toward the M1 pro-inflammatory phenotype by reducing the content of LRG1 in keratinocyte-derived exosomes and decreasing the phosphorylation levels of ERK and CREB1 in macrophages.

[0019] In one possible implementation, the drug reduces the expression of at least one M1 polarization-related marker among CD80, iNOS, and CD86 in macrophages, and reduces the expression of at least one pro-inflammatory factor among TNF-α and IL-6.

[0020] In one possible implementation, the drug attenuates the promoting effect of M1 macrophages on abnormal proliferation, migration, and / or inflammatory responses of keratinocytes by inhibiting macrophage polarization toward the M1 pro-inflammatory phenotype.

[0021] Based on the above technical solution, this invention provides the use of DST, an active ingredient derived from Dioscorea hypoglauca, in the preparation of drugs for treating psoriasis. Specifically, this invention utilizes DST to regulate psoriasis-related signaling processes. By inhibiting LYN-mediated signal transduction, it reduces the phosphorylation level of tyrosine residue 705 of the STAT3 protein, i.e., the STAT3 Y705 site, thereby inhibiting abnormal STAT3 activation and further downregulating the transcription and / or protein expression of leucine-rich α-2-glycoprotein 1 (LRG1) in keratinocytes.

[0022] Phosphorylation at STAT3 Y705 is involved in the transcriptional regulation of LRG1. By regulating the LYN / STAT3 / LRG1 signaling axis, DST can reduce the abnormal expression of LRG1 in keratinocytes under psoriasis-associated conditions and inhibit the abnormal proliferation and / or migration of keratinocytes. Correspondingly, DST can reduce the epidermal thickness and the number of Ki-67 positive cells in psoriasis-like lesions, and reduce the expression or secretion of pro-inflammatory factors such as TNF-α, IL-1β, IL-6, IL-17A, and IL-22, thereby alleviating psoriasis-associated epidermal hyperplasia and inflammatory response.

[0023] Furthermore, the regulation of psoriasis-related pathological processes by DST also involves inflammation-related intercellular communication between keratinocytes and macrophages. Under psoriasis-like stimuli, LRG1 expression and its extracellular output increase in keratinocytes. Extracellular vesicles derived from keratinocytes can carry LRG1, including exosomes. DST reduces the LRG1 content in these extracellular vesicles by downregulating LRG1 expression in keratinocytes.

[0024] LRG1 in keratinocyte-derived exosomes can participate in the regulation of macrophage-related signals. DST reduces the phosphorylation levels of ERK and CREB1 in macrophages by decreasing the content of LRG1 in keratinocyte-derived exosomes, thereby weakening ERK / CREB1 signaling activation and inhibiting macrophage polarization towards the M1 pro-inflammatory phenotype.

[0025] Furthermore, after macrophage M1 polarization is inhibited, the expression of M1 polarization-related markers such as CD80, iNOS, and CD86, as well as pro-inflammatory factors such as TNF-α and IL-6, is reduced accordingly, thereby alleviating macrophage-related pro-inflammatory responses.

[0026] Meanwhile, paracrine signaling produced by M1 macrophages can promote abnormal proliferation, migration, and inflammatory responses of keratinocytes. DST can further attenuate the promoting effect of M1 macrophages on abnormal proliferation, migration, and / or inflammatory responses of keratinocytes by inhibiting macrophage polarization towards the M1 pro-inflammatory phenotype, thereby regulating the mutually reinforcing inflammation-related intercellular communication between keratinocytes and macrophages.

[0027] Therefore, this invention uses the LYN / STAT3 / LRG1 signaling axis as an important link. On the one hand, it inhibits the abnormal proliferation and / or migration of keratinocytes by reducing phosphorylation at the STAT3Y705 site and LRG1 expression. On the other hand, it reduces the LRG1 content in extracellular vesicles derived from keratinocytes, weakens the activation of macrophage ERK / CREB1 signaling and M1 pro-inflammatory polarization, and further weakens the promoting effect of M1 macrophages on the abnormal activation of keratinocytes, thereby regulating psoriasis-related epidermal hyperplasia and immune inflammatory response.

[0028] The experimental results of this invention show that DST treatment can reduce LYN-related signal activation, STAT3 Y705 phosphorylation, and LRG1 expression in psoriatic lesions, decrease the number of Ki-67 positive cells and the level of related pro-inflammatory factors, and improve IMQ-induced psoriatic lesions, epidermal thickening, and inflammatory cell infiltration. Simultaneously, related cell experiments show that DST can reduce LRG1-related signaling in extracellular vesicles derived from keratinocytes, weaken ERK and CREB1 phosphorylation and M1-type pro-inflammatory polarization in macrophages, and further alleviate the promoting effect of macrophage-related pro-inflammatory signals on abnormal proliferation, migration, and inflammatory responses of keratinocytes. These experimental results support the technical solution of using DST for the preparation of drugs for treating psoriasis at the molecular, cellular, and animal levels.

[0029] Therefore, this invention provides a psoriasis drug application scheme based on DST, an active ingredient derived from Dioscorea hypoglauca. By regulating the LYN / STAT3 / LRG1 related signaling process and further influencing the LRG1-related extracellular vesicles from keratinocytes, the ERK / CREB1 signaling of macrophages, and the M1 polarization process, it achieves the technical effects of inhibiting abnormal proliferation and migration of keratinocytes, reducing pro-inflammatory macrophage responses, and improving psoriasis-related inflammatory responses. This provides a new application scheme for the drug treatment of psoriasis using an active ingredient derived from Dioscorea hypoglauca. Attached Figure Description

[0030] Figure 1 The experimental results of DST improving IMQ-induced psoriasis-like mouse skin lesions and inflammatory responses are shown in the figure, including clinical manifestations of skin lesions, weight changes, PASI score, H&E staining, epidermal thickness, serum inflammatory factor levels, and Ki-67 immunofluorescence detection results.

[0031] Figure 2 The figure shows the experimental results of DST regulating the LYN / STAT3 / LRG1 signaling pathway in vivo and in vitro. It includes the immunohistochemical detection results of LYN, p-STAT3 and LRG1 in skin tissue, the detection results of the concentration of DST on keratinocytes, and the detection results of the expression levels of LYN / p-STAT3 / LRG1, inflammatory factors, VEGF, CX43 and Vimentin in keratinocytes.

[0032] Figure 3 The figure shows the experimental results of DST inhibiting psoriatic inflammation and abnormal proliferation of keratinocytes by regulating LYN. It includes LYN / p-STAT3 / LRG1 signaling, inflammatory factors and expression levels of VEGF, CX43 and Vimentin in keratinocytes, as well as epidermal thickness, PASI score, histopathology, inflammatory factors and Ki-67 detection results in psoriasis-like mice.

[0033] Figure 4 The figure shows the experimental results of DST inhibiting the inflammatory response and abnormal migration of psoriatic keratinocytes by regulating STAT3 Y705 phosphorylation and LRG1 expression. It includes a schematic diagram of STAT3 Y705 mutation, LRG1 promoter dual-luciferase detection results, p-STAT3 / LRG1 and related inflammatory factor expression results, and the effect of LRG1 overexpression on keratinocyte migration.

[0034] Figure 5 The figure shows the experimental results of DST inhibiting macrophage M1 polarization by reducing LRG1 in extracellular vesicles derived from keratinocytes. It includes transmission electron microscopy images of extracellular vesicles, particle size distribution, detection of exosome markers, detection of LRG1 content, and detection results of macrophage M1 polarization-related proteins, mRNA, immunofluorescence and flow cytometry.

[0035] Figure 6 The figure shows the experimental results of LRG1 promoting macrophage M1 polarization by regulating the ERK / CREB1 signaling pathway. It includes the expression detection results of p-CREB1 / CREB1, p-ERK / ERK, CD80 and TNF-α in macrophages, as well as the effect of keratinocyte conditioned medium under different conditions on macrophage polarization.

[0036] Figure 7 The experimental results of promoting keratinocyte proliferation, migration and inflammatory response by M1 macrophages treated with LRG1 are shown in the figure. The results include the expression detection results of VEGF, CX43 and inflammation-related factors in keratinocytes, cell proliferation detection results, and migration and invasion experimental results.

[0037] Figure 8 The results of DST active ingredient screening and target prediction analysis are shown in the figure, including DST chromatograms of Dioscorea hypoglauca samples and controls, screening results of disease-related active ingredients, differential protein analysis results, DST target and differential protein intersection results, molecular docking results of DST and LYN, and differential expression and functional enrichment analysis results of psoriasis-related transcriptomes.

[0038] Figure 9 This image shows the results of bioinformatics analysis of key regulatory molecules in LYN-related psoriasis, including transcriptome differential gene and proteome differential protein intersection analysis, LYN-related key gene screening, functional enrichment analysis, LYN-LRG1 correlation analysis, and protein-protein interaction network analysis.

[0039] Figure 10The image shows the supplementary experimental results of DST acting on HaCaT keratinocytes, including DST concentration screening and the detection results of protein and mRNA expression of LYN / p-STAT3 / LRG1, inflammatory factors, VEGF, CX43 and Vimentin in HaCaT cells.

[0040] Figure 11 Supplementary experimental results for STAT3 Y705 phosphorylation, LRG1 expression, and macrophage polarization regulated by keratinocyte-derived conditioned medium are shown. These include the detection results of p-STAT3 / LRG1, inflammatory factors, VEGF, CX43, and Vimentin expression in HaCaT cells, as well as the flow cytometry, immunofluorescence, protein, and mRNA detection results of M1 polarization-related indicators such as CD80 and iNOS in macrophages.

[0041] Figure 12 The image shows the RNA sequencing and bioinformatics analysis results of LRG1 regulating macrophage transcriptional characteristics, including principal component analysis, differentially expressed gene analysis, gene intersection analysis, KEGG pathway enrichment analysis, and ERK / CREB1 related pathway screening results.

[0042] Figure 13 Supplementary experimental results for LRG1 regulation of macrophage ERK / CREB1 signaling and M1 polarization are shown, including the results of p-CREB1 / CREB1, p-ERK / ERK, CD80 and TNF-α expression detection, as well as flow cytometry and immunofluorescence detection results of macrophage M1 polarization after treatment with different keratinocyte conditioned media.

[0043] Figure 14 Supplementary experimental results are shown for the effects of different macrophage conditioned media on the proliferation, migration, invasion, and inflammatory response of HaCaT keratinocytes. These include the results of VEGF, CX43 and inflammation-related factor expression detection, CCK-8 cell proliferation detection, and cell migration and invasion experiments.

[0044] Figure 15 This image shows the detection results of relevant molecules and inflammatory markers in clinical samples from psoriasis patients, including the detection results of LYN, p-STAT3, LRG1 and Ki-67 in skin tissues of psoriasis patients and healthy controls, the immunofluorescence co-localization results of CD80 and CD68, and the expression levels of LRG1, TNF-α, IL-1β, IL-6, IL-17A, IL-22 and IL-23 in serum. Detailed Implementation

[0045] I. General Description of Detailed Implementation Methods The technical solution of the present invention will be further described in detail below with reference to specific examples and the accompanying drawings. It should be noted that the following examples are only used to illustrate the technical solution of the present invention, and are not intended to limit the protection scope of the present invention. Without departing from the technical concept of the present invention, equivalent substitutions, routine adjustments or improvements made by those skilled in the art based on the present specification shall all be deemed to fall within the scope covered by the technical solution of the present invention.

[0046] The present invention takes yamogenin, an active ingredient screened from components derived from Dioscorea septemloba Thunb., as the research object. In the original experimental studies and relevant experimental data, this active ingredient is uniformly labeled as "DST", and the relevant original research records also contain the experimental identifier "Dioscorea septemloba Thunb A". After corresponding confirmation, the Chinese name of the active ingredient according to the present invention is yamogenin, the common English name is Yamogenin, and the molecular formula is C 27 H 42 O3, CAS No. 512-06-1, IUPAC name is (25S)-spirost-5-en-3β-ol, which is an isomer of diosgenin. To maintain consistency between the present specification and the identifiers used in the original experimental data, pharmacodynamic evaluation results and mechanism of action studies, the above-mentioned yamogenin is hereinafter uniformly abbreviated as DST.

[0047] Unless otherwise explicitly stated, the "DST" referred to in the present specification specifically refers to the above-mentioned yamogenin, that is, the active compound screened and determined by the present invention and actually used in in vitro cell experiments, in vivo animal experiments and mechanism of action studies; the "DST" is not used to refer to the Dioscorea septemloba Thunb. plant itself, the whole Dioscorea septemloba Thunb. medicinal material, nor is it used to generally refer to the Dioscorea septemloba Thunb. extract or a mixture thereof without explicit component limitation.

[0048] The present invention conducts systematic research on the intervention effect of DST on psoriasis-related pathological phenotypes and its potential molecular mechanism of action. First, the component corresponding to DST in the Dioscorea septemloba Thunb. sample is detected and quantified by liquid chromatography analysis, and combined with herb-related databases and target prediction methods, the disease-related candidate active components derived from Dioscorea septemloba Thunb. and the possible action targets involved in DST are screened. On this basis, combined with proteomics, transcriptomics and protein-protein interaction network analysis, integrated analysis of psoriasis-related differentially expressed molecules, candidate targets and related signaling pathways is carried out to establish a candidate molecular network that DST may participate in regulating.

[0049] Following the above screening and integration analysis, candidate target proteins such as LYN, ALOX5, CA2, and CDK6 were identified. Based on the candidate target protein screening results and the direction of subsequent experimental verification, this invention focuses on LYN as a key research object and conducts further research on the relationship between LYN-related signaling and DST's improvement of abnormal activation and inflammatory responses in psoriasis-related cells.

[0050] Based on this, this invention verifies the regulatory relationship between DST and LYN-related signaling through molecular docking, cell experiments, and animal experiments. An in vitro research system was established using mKC and HaCaT keratinocytes, and the causal relationship between DST and LYN, STAT3, and LRG1 was analyzed by combining LYN overexpression, LRG1 overexpression and knockdown, and STAT3 Y705F phosphorylation defect mutation. Experimental results show that DST can inhibit LYN-related signal transduction, reduce the phosphorylation level of STAT3 protein at the Y705 site, but has no significant effect on the total STAT3 protein level, and further downregulates LRG1 expression in keratinocytes.

[0051] To further evaluate the regulatory effects of DST on psoriasis-related skin inflammation and abnormal keratinocyte proliferation, this invention employed an imiquimod (IMQ)-induced psoriasis-like mouse model for in vivo validation. The anti-psoriatic efficacy of DST was evaluated by observing changes in clinical manifestations of skin lesions, PASI scores, epidermal thickness, histopathological changes, Ki-67 expression, and various inflammatory factors. Results showed that DST could improve IMQ-induced erythema, scaling, and epidermal thickening, reduce PASI scores and inflammatory cell infiltration, and decrease the levels of inflammatory factors such as IL-17A, IL-22, IL-6, IL-1β, and TNF-α, while also alleviating abnormal keratinocyte proliferation.

[0052] In addition to its effects on keratinocytes themselves, this invention further investigates intercellular communication between keratinocytes and macrophages. Extracellular vesicles were isolated from keratinocyte conditioned medium using differential ultracentrifugation and identified using transmission electron microscopy, nanoparticle tracking analysis, and exosome marker protein detection. Furthermore, conditioned medium or exosomes derived from keratinocytes under different treatment conditions were applied to bone marrow-derived macrophages or THP-1-derived macrophages to evaluate LRG1 and its mediated macrophage polarization changes. Experimental results show that LRG1 from psoriatic keratinocytes can participate in transmitting pro-inflammatory signals to macrophages via extracellular vesicles, and DST can reduce LRG1 levels in keratinocyte-derived exosomes and weaken macrophage polarization towards the M1 pro-inflammatory phenotype.

[0053] Furthermore, this invention investigates the downstream mechanisms of LRG1-regulated macrophage polarization by performing RNA sequencing and pathway enrichment analysis on bone marrow-derived macrophages under different treatment conditions. The results show that LRG1 promotes phosphorylation of ERK and CREB1 in macrophages and promotes the formation of the M1 phenotype. After reducing LRG1 levels in keratinocytes, the activation of ERK / CREB1 signaling and M1 polarization were correspondingly weakened. On the other hand, conditioned medium for M1-type macrophages stimulated by LRG1 further promotes keratinocyte proliferation, migration, and the release of inflammatory mediators, suggesting that keratinocytes and macrophages can form mutually reinforcing inflammatory cell communication.

[0054] Furthermore, this invention validates the results obtained from the aforementioned cell and animal experiments using clinical skin tissue and serum samples from psoriasis patients. Changes in the expression of LYN, p-STAT3, LRG1, Ki-67, macrophage-related markers, and various inflammatory factors were detected using immunohistochemistry, immunofluorescence, and ELISA to evaluate the consistency of LYN / STAT3 / LRG1 signaling and related inflammatory phenotypes in clinical psoriasis. Clinical samples included skin tissue and serum samples from psoriasis patients and healthy subjects.

[0055] Based on the above research, the specific embodiments of the present invention are described in the following order: 1. Detection and active ingredient screening of DST: DST was detected and its potential targets were screened using liquid chromatography, herbal databases, target prediction and proteomics methods. 2. Therapeutic effect of DST on psoriasis-like mice: The effects of DST on skin lesions, PASI score, histopathology, keratinocyte proliferation and inflammatory factors were evaluated using an IMQ-induced psoriasis-like mouse model. 3. Screening and confirmation of DST target sites and the LYN / STAT3 / LRG1 signaling axis: Through multi-omics analysis, molecular docking and in vitro and in vivo experiments, LYN / STAT3 / LRG1 was confirmed as an important signaling axis for DST to regulate psoriasis; 4. Verification of the anti-psoriasis effect of DST targeting LYN: Through experiments such as LYN overexpression and LYN inhibitor control, the regulatory effect of DST on LYN-related signals and psoriasis phenotype was verified; 5. Verification of the causal mechanism of STAT3 Y705 phosphorylation and LRG1: The relationship between STAT3 Y705 phosphorylation and LRG1 expression and abnormal phenotype of keratinocytes was verified by STAT3 Y705F mutation, dual-luciferase reporter assay, and LRG1 overexpression and knockdown assay. 6. Validation of the regulation of macrophage M1 polarization by keratinocyte-derived exosome LRG1: The effect of keratinocyte-derived LRG1 on the pro-inflammatory phenotype of macrophages was validated by exosome isolation and identification, conditioned culture, and macrophage polarization detection. 7. Verification of LRG1 regulation of macrophage M1 polarization via ERK / CREB1 signaling: The regulatory role of LRG1 on ERK / CREB1 signaling and macrophage M1 polarization was verified by RNA sequencing, pathway analysis and protein expression detection. 8. Feedback effect of M1 macrophages on keratinocyte proliferation, migration and inflammatory response: Keratinocytes were treated with macrophage conditioned medium, and changes in keratinocyte proliferation, migration, invasion and inflammatory mediators were detected; 9. Validation of LYN / STAT3 / LRG1 and related inflammatory phenotypes in clinical psoriasis samples: Clinical validation of LYN / STAT3 / LRG1 signals and related inflammatory markers was performed using skin and serum samples from psoriasis patients and healthy controls.

[0056] Through the above embodiments, the effects and molecular mechanisms of DST in treating psoriasis are verified from multiple levels, including active ingredients, efficacy evaluation, target sites, signaling pathways, intercellular communication, and clinical samples, so as to form complete experimental support for the technical solution of the present invention.

[0057] II. General Experimental Materials and Methods Unless otherwise stated, the experimental methods described below are applicable to all subsequent embodiments. The experimental conditions, reagents, instruments, and detection parameters used shall be set according to the following methods.

[0058] 1. Detection and quantification of DST in Dioscorea hypoglauca samples 1.1 Sample Preparation Accurately weigh 10.0 g of *Dioscorea hypoglauca* sample and place it in 50 mL of 2 mol / L hydrochloric acid solution. Hydrolyze under reflux for 3 h. After hydrolysis, allow the sample to cool naturally to room temperature and filter, collecting the insoluble residue. Wash the residue repeatedly with distilled water until the washing solution is neutral.

[0059] The washed residue was dried at 80°C and then transferred to a Soxhlet extractor. 30 mL of petroleum ether with a boiling range of 60–90°C was added, and extraction was continued for 4 h. After extraction, the petroleum ether was evaporated, and the resulting residue was redissolved in methanol and quantitatively transferred to a 50.0 mL volumetric flask, then diluted to the mark with methanol. The resulting solution was filtered through a 0.45 μm microporous membrane and used as the test solution.

[0060] 1.2 Liquid Chromatography Conditions Liquid chromatography analysis was performed using an Inertsil ODS-3 reversed-phase column (4.6 mm × 250 mm). The mobile phase consisted of methanol, acetonitrile, and water in a volume ratio of 80:10:10. Detection was performed using a UV detector at a wavelength of 210 nm. The flow rate was 1.0 mL / min, the injection volume was 20 μL, and the column temperature was ambient temperature.

[0061] Take 20 μL of the test solution and the reference solution respectively for liquid chromatography detection. By comparing the retention time and peak area of ​​the sample peak and the reference peak, the DST is qualitatively and quantitatively analyzed.

[0062] The liquid chromatography results showed that the retention time of the reference standard was 20.455 min, the peak area was 2,424,552, and the theoretical plate number was 16,139; the retention time of the corresponding peak in the test sample was 20.302 min, the peak area was 1,062,119, and the theoretical plate number was 16,389. Based on the detection results, the DST content in the sample was calculated to be 1.93 mg / g.

[0063] The liquid chromatography detection results are shown below. Figure 8 A and Table 1.

[0064] 2. Cell Culture 2.1 Keratinocyte and THP-1 cell culture The experiment used mKC mouse keratinocytes, HaCaT human keratinocytes, and THP-1 mononuclear cells.

[0065] All cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 50 μM 2-mercaptoethanol. Cells were cultured routinely at 37°C in a humidified environment with 5% CO2.

[0066] In subsequent experiments, mKC or HaCaT cells were subjected to DST, LPS, gene overexpression, gene knockdown or other corresponding treatments according to specific conditions. After treatment, cells, cell lysates or conditioned medium were collected for subsequent detection.

[0067] 2.2 THP-1 cell differentiation induction Adjust THP-1 monocytes to 5 × 10 5 Cells / mL were inoculated in complete RPMI-1640 medium containing 80 nM phorbol 12-myristate 13-acetate, i.e., PMA.

[0068] THP-1 monocytes were cultured at 37℃ and 5% CO2 for 72 h to induce differentiation into adherent macrophages. The resulting macrophages were used for subsequent stimulation with keratinocyte conditioned medium, macrophage polarization, and detection of related protein and gene expression.

[0069] 3. Isolation and culture of bone marrow-derived macrophages Bone marrow-derived macrophages, or BMDMs, were prepared using mouse bone marrow.

[0070] The femur and tibia of mice were harvested, the bones were cleaned, and the ends were trimmed. The bone marrow was flushed into a 15 mL centrifuge tube using a syringe, and 1–2 mL of PBS was added. The flushing was repeated until the bone marrow turned mostly white.

[0071] The obtained bone marrow cells were resuspended in 2 mL of room temperature erythrocyte lysis buffer for erythrocyte lysis. Subsequently, the resulting cell suspension was transferred to RPMI-1640 medium for pre-culture to reduce the influence of contaminated fibroblasts.

[0072] The treated bone marrow cells were further cultured for 5 days in RPMI-1640 medium containing 10% FBS and 10 ng / mL macrophage colony-stimulating factor, namely M-CSF, to induce the formation of bone marrow-derived macrophages.

[0073] In related experiments, RPMI-1640 medium containing 1% FBS was also used, and BMDMs were treated with 5 ng / mL TGF-β1 for 72 h for the corresponding protein induction experiments.

[0074] 4. Isolation of exosomes derived from keratinocytes 4.1 Collection of conditioned media Keratinocyte conditioned medium was collected, with each sample containing 50 mL, for subsequent separation of extracellular vesicles and exosomes.

[0075] 4.2 Differential centrifugation removes cells and large particles. First, centrifuge the conditioned medium at 300×g for 10 min at 4°C to remove intact cells.

[0076] Subsequently, the obtained supernatant was centrifuged at 10000×g for 20 min at 4°C to remove cell debris and larger extracellular vesicles.

[0077] After centrifugation, carefully collect the supernatant and transfer it to a sterile polycarbonate ultracentrifuge tube.

[0078] 4.3 Exosome sedimentation and washing The supernatant was centrifuged at 100,000 × g for 70 min at 4 °C. The supernatant was discarded and the precipitate rich in exosomes was collected.

[0079] The resulting precipitate was gently resuspended in sterile 1×PBS buffer and filtered through a 0.22 μm syringe filter to remove any aggregated particles.

[0080] Subsequently, the sample was washed by two consecutive 100,000×g ultracentrifugations, each lasting 1 hour, both at 4°C.

[0081] The resulting exosome precipitate was resuspended in an appropriate amount of 1×PBS for subsequent transmission electron microscopy observation, particle size and concentration detection, exosome marker protein detection, and LRG1-related functional experiments.

[0082] 5. Identification of exosomes 5.1 Nanoparticle Tracking Analysis Nanoparticle tracking analysis was performed using the NanoSight NS300 system to determine the particle size distribution and concentration of exosome particles.

[0083] Before testing, the obtained exosome samples were resuspended in sterile PBS and diluted 1:1000 to achieve a working concentration of approximately 6 × 10⁻⁶. 7 Particles / mL. All measurements were repeated three times.

[0084] In addition, the hydrodynamic diameter and zeta potential of exosome particles were detected using the ELsz-2000 analyzer via dynamic light scattering.

[0085] 5.2 Observation using transmission electron microscopy Transmission electron microscopy was used to observe the morphology of exosomes in order to evaluate whether the obtained particles have typical exosome morphology and size characteristics.

[0086] Experimental results showed that the isolated extracellular vesicles were mainly spherical granules with a diameter of about 80-100 nm and had a typical membrane structure.

[0087] 5.3 Detection of exosome marker proteins Western blot was used to detect exosome marker proteins such as CD63 and TSG101 to further confirm the exosome nature of the isolated particles.

[0088] Exosome identification results correspond Figure 5 A~C.

[0089] 6. CCK-8 cell viability and proliferation assay The Cell Counting Kit-8 (CCK-8 reagent) was used to evaluate the effect of DST on cell viability and the proliferation capacity of keratinocytes under different treatment conditions.

[0090] Cells were exposed to different concentrations of DST ranging from 0 to 100 μM and cultured for 24 h. After treatment, 10 μL of CCK-8 reagent was added to each well, and incubation was continued for another 2 h.

[0091] The absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader. Cell viability was evaluated based on the changes in absorbance, and a suitable DST treatment concentration was selected accordingly.

[0092] The corresponding concentration screening results were used for subsequent mechanism experiments; the relevant results are shown in [link to relevant results]. Figure 2 G and Figure 10 A.

[0093] 7. Quantitative real-time fluorescence PCR detection Total RNA was extracted from cells using TRIzol reagent. After RNA extraction was completed according to the reagent procedure, the total RNA was reverse transcribed into cDNA using the TaKaRa reverse transcription kit.

[0094] Real-time quantitative PCR was performed using a 7900HT rapid real-time PCR system with SYBR Premix EX Taq as the amplification system to detect the mRNA expression level of the target gene in cells of different treatment groups.

[0095] Gene detection includes, but is not limited to: Il-1β; Tnf-α; Il-6; Il-17A; Il-22; Cd80; Cd86; Inos; Vegf; Cx43 and their corresponding human homologs. GAPDH was used as an internal reference gene. The specific forward and reverse primer sequences for each target gene were amplified according to the sequences shown in Table 2.

[0096] Table 2: Primer sequences used for real-time quantitative PCR analysis

[0097] 8. Western blot protein expression detection 8.1 Total Protein Extraction Cells were lysed using RIPA lysis buffer. After complete lysis and vortexing, the cells were centrifuged at 4°C and 15000×g for 25 min, and the supernatant was collected as the total protein sample.

[0098] 8.2 Protein isolation and transfer Total protein was separated by SDS-PAGE electrophoresis at 100 V for 1.5 h.

[0099] After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane under the following conditions: 50 V for 80 min.

[0100] 8.3 Blocking and Antibody Incubation After the transfer was completed, the membrane was blocked with 5% skim milk at room temperature for 1 hour.

[0101] The membrane was then incubated overnight at 4°C with the corresponding primary antibody.

[0102] The primary antibodies involved in the experiment include: LYN; STAT3; p-STAT3; LRG1; TNF-α; IL-1β; IL-17A; IL-22; VEGF; CX43; Vimentin; CD80; iNOS; CD63; TSG101; GAPDH.

[0103] Primary antibodies against LYN, STAT3, p-STAT3, LRG1, and most target proteins were diluted at a ratio of 1:500, while primary antibodies against GAPDH were diluted at a ratio of 1:1000.

[0104] After incubation with the appropriate secondary antibody, the target protein band was detected, and GAPDH or the corresponding total protein was used as an internal control or normalization basis.

[0105] 9. ELISA testing Commercially available ELISA kits were used to detect cytokine and LRG1 levels in different experimental samples according to the kit instructions.

[0106] The detection indicators include: TNF-α; IL-1β; IL-6; IL-17A; IL-22; LRG1.

[0107] Depending on the experimental purpose, the samples tested include: cell culture supernatant; conditioned medium; mouse serum; and human serum.

[0108] The corresponding protein concentrations for each sample were calculated based on the standard curve of the kit and used to compare the changes between different experimental treatment groups.

[0109] 10. Gene overexpression, gene knockdown, and cell transfection The siRNA targeting LRG1 and the LRG1 overexpression vector were designed and provided by GeneChem.

[0110] Cell transfection was performed using Lipofectamine 3000 transfection reagent. Depending on the experimental purpose, the corresponding siRNA, LRG1 overexpression vector, or other plasmids required for the experiment were introduced into keratinocytes.

[0111] In the LYN mechanism verification experiment, a LYN overexpression cell model was constructed to observe the changes in STAT3 phosphorylation, LRG1 expression, and DST intervention under enhanced LYN expression conditions.

[0112] In the LRG1 function validation experiment, the effects of increased or decreased LRG1 levels on abnormal activation, migration and related inflammatory phenotypes of keratinocytes were evaluated by overexpression of LRG1 and knockdown of LRG1 mediated by siRNA.

[0113] 11. STAT3 Y705 site mutation and dual-luciferase reporter assay To verify the relationship between STAT3 Y705 phosphorylation and LRG1 transcription, a STAT3 Y705F phosphorylation defect mutant was constructed.

[0114] Wild-type or Y705F mutant STAT3 was introduced into the corresponding cells, and the transcriptional activity of the LRG1 promoter was further detected.

[0115] Luciferase activity was detected using the Dual-Luciferase Reporter Assay System. Cells from different treatment groups were collected and lysed, and firefly luciferase and Renilla luciferase signals were measured according to the reagent instructions.

[0116] Using Renilla luciferase as an internal control, the firefly luciferase signal was normalized, and the change in transcriptional activity of the LRG1 promoter was finally expressed as the relative luciferase activity compared with the negative control group.

[0117] The regulatory role of STAT3 Y705 phosphorylation on LRG1 transcription was evaluated by comparing the LRG1 promoter activity under different conditions, including wild-type STAT3, STAT3 Y705F, and DST treatment. Related experimental results are shown below. Figure 4 A~B.

[0118] 12. Transwell migration and invasion experiments Cell migration and invasion were detected using 24-well plates equipped with 6.5 mm diameter and 8.0 μm pore size Transwell chambers.

[0119] In the invasion experiment, 50 μL of Matrigel was pre-laid on the surface of each Transwell chamber membrane.

[0120] The cells to be tested were added to the upper chamber, and the lower chamber was filled with complete culture medium containing 10% FBS as chemotactic conditions.

[0121] After culturing for 24 h, the cells were fixed and stained with 0.1% crystal violet for 30 min.

[0122] Gently remove cells that have not migrated or invaded from the upper surface of the membrane, while retaining cells that have migrated or invaded to the lower surface of the membrane.

[0123] Six fields of view were randomly selected under a microscope to manually count and statistically analyze migrating or invading cells.

[0124] 13. Scratch Healing Experiment Keratinocytes were arranged in a pattern of 2 × 10 cells per well. 5 Cells were seeded at a density of 100 cells per well in 6-well plates and cultured until the cells formed a complete monolayer and reached full confluence.

[0125] Standardized scratches were prepared on the surface of a cell monolayer using a sterile 200 μL pipette tip.

[0126] After scratch formation, gently wash with 1×PBS to remove detached cells and cell debris.

[0127] Imaging of the same field of view was performed at 100x magnification using a Nikon Eclipse Ti inverted microscope at 0 h and 24 h after scratch formation.

[0128] Cell migration and wound closure ability were evaluated based on changes in the scratched area at different time points.

[0129] 14. Flow cytometry detection of macrophage polarization Macrophages exposed to different keratinocyte conditioned media were collected and analyzed by flow cytometry.

[0130] Take approximately 1×10 from each sample. 6 Cells were collected by centrifugation and resuspended in 100 μL of 1× binding buffer.

[0131] Add the corresponding fluorescent dye-conjugated antibody and stain at 37°C in the dark for 30 min.

[0132] The experiment mainly used CD80 antibody to detect M1 macrophage-related phenotypes.

[0133] After staining, data were acquired using a Becton-Dickinson FACSCalibur flow cytometer and analyzed using FlowJo v10.4.2 software.

[0134] The degree of M1 polarization of macrophages under different treatment conditions was evaluated based on the proportion of CD80 positive cells and changes in related fluorescence signals.

[0135] 15. Immunohistochemical detection Paraffin-embedded tissue sections were prepared into sections with a thickness of 5 μm, fixed on positively charged glass slides, and dried at 37 °C.

[0136] After dewaxing with xylene, the sections were rehydrated with decreasing concentrations of ethanol and then subjected to antigen retrieval.

[0137] Subsequently, the cells were incubated with primary antibodies targeting the following proteins: LYN; p-STAT3; and LRG1.

[0138] Biotin-labeled secondary antibody was added after incubation with primary antibody.

[0139] After staining, the tissue was counterstained with hematoxylin, dehydrated with graded ethanol and xylene, mounted, and the expression changes of the corresponding proteins in the skin tissue were observed and analyzed under a microscope.

[0140] 16. Immunofluorescence detection After appropriate pretreatment and antigen retrieval, tissue sections were stained with immunofluorescence using fluorescently labeled antibodies.

[0141] The main biomarkers involved in the experiment included: CD80; iNOS; CD68; F4 / 80; Ki-67.

[0142] The antibody incubation process is carried out under light-protected conditions.

[0143] Fluorescence images were observed and acquired using a Nikon Eclipse 90i microscope.

[0144] Depending on the different experimental objectives: Ki-67 was used to evaluate keratinocyte proliferation; CD80 and iNOS were used to evaluate the M1 macrophage phenotype; F4 / 80 was used to label mouse macrophages; CD68 is used to label macrophages in clinical tissues.

[0145] The corresponding biological changes are evaluated by the intensity of positive signals, the proportion of positive cells, or co-localization.

[0146] 17. RNA sequencing Total RNA was extracted from cells, and RNA concentration was detected using a Qubit 2.0 fluorometer. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer, and samples with an RNA integrity index (RIN) ≥ 7.5 were selected for subsequent library construction.

[0147] First-strand cDNA was synthesized using the SMARTer V4 Ultra Low Input kit and then fragmented, ligated with adapters, and tagged with Nextera XT.

[0148] Library size screening was performed using Ampure XP magnetic beads. Different libraries were normalized, mixed in equimolar proportions, and sequenced on an Illumina HiSeq 3000 platform.

[0149] The sequencing mode was 75 bp paired-end sequencing, with an average sequencing depth of approximately 60 million reads per sample.

[0150] The obtained transcriptome data were used for principal component analysis, differentially expressed gene analysis, and subsequent functional pathway analysis.

[0151] 18. Proteomics Analysis Cells were lysed using a lysis buffer containing 8 M urea, 50 mM Tris-HCl, protease inhibitors, and phosphatase inhibitors.

[0152] After being sonicated on ice, the samples were centrifuged at 4℃ and 14000×g for 15 min, the supernatant was collected, and the protein concentration was determined by the BCA method.

[0153] 100 μg of protein was taken from each sample, reduced with 10 mM dithiothreitol at 37 °C for 1 h, and then alkylated with 50 mM iodoacetamide at room temperature in the dark for 45 min.

[0154] Diluted 4 times with 50 mM ammonium bicarbonate, then add trypsin and hydrolyze overnight at 37°C at an enzyme-to-substrate mass ratio of 1:50.

[0155] The obtained peptides were desalted by C18 StageTips, vacuum dried, and then resuspended in 0.1% formic acid.

[0156] The detection was performed using an Easy-nLC 1200 system coupled with a Q Exactive HF-X mass spectrometer.

[0157] The raw data were processed using MaxQuant v2.1.4.0 and searched using the UniProt human proteome and reverse bait databases.

[0158] The false discovery rates at both peptide and protein levels were controlled to be below 1%.

[0159] Label-free quantitative data were analyzed in Perseus v1.6.15.0 after log2 transformation. The differential protein screening criteria were a p-value less than 0.05 after Benjamini-Hochberg correction and a |log2 fold change| > 0.58.

[0160] 19. Bioinformatics and Protein-Protein Interaction Network Analysis The mRNA expression profiles in psoriatic lesion tissues and non-lesion tissues were analyzed using the GSE30999 dataset from the NCBI GEO database.

[0161] Principal component analysis was used to evaluate the overall distribution and clustering of different samples and to screen for differentially expressed genes.

[0162] Functional and pathway enrichment analyses were performed using the Gene Ontology and KEGG databases, and the results were visualized using R software.

[0163] Gene set enrichment analysis was performed using the fgsea software package.

[0164] The level of immune cell infiltration was assessed using the single-sample gene set enrichment analysis method in the GSVA software package.

[0165] In addition, protein-protein interaction network analysis was performed using the STRING database to screen key regulatory molecules related to LYN and their possible signaling connections.

[0166] 20. Molecular docking analysis between DST and target protein Based on the screening results of potential targets of DST, molecular docking analysis was performed on DST and candidate proteins.

[0167] We used the UniProt database to obtain relevant protein information and the ProteinPLUS database to predict the potential drug binding pocket of the target protein.

[0168] PyMOL 2.5.4 was used for molecular docking analysis and structural model visualization.

[0169] By comparing the predicted binding of different candidate target proteins to DST, this method can be used to help screen potential key targets.

[0170] The relevant results were used in the design of subsequent experiments on the LYN mechanism.

[0171] 21. Statistical Analysis All continuous variables are expressed as mean ± standard deviation, or mean ± SD.

[0172] To assess the differences between multiple experimental groups, one-way ANOVA was used for statistical comparison, and Tukey's honest significance test was used for post-hoc pairwise comparisons between groups.

[0173] All statistical analyses were performed using GraphPad Prism 8.0 software.

[0174] A two-tailed test was used, and a p-value < 0.05 was considered statistically significant between groups.

[0175] In the attached figures, different significance levels are indicated by “*”, “**”, or “***”, depending on the specific statistical results.

[0176] Example 1: Detection of DST, screening of active ingredients and identification of candidate targets: In this embodiment, DST in samples derived from Dioscorea hypoglauca was detected by liquid chromatography. Combined with herbal databases, target prediction, proteomics, and molecular docking analysis, active ingredients related to psoriasis and potential targets of DST were screened to identify key targets for subsequent mechanism research.

[0177] 1.1 Liquid Chromatography Detection of DST Following the sample preparation method described in the aforementioned "General Experimental Materials and Methods", 10.0 g of Dioscorea hypoglauca sample was accurately weighed and added to 50 mL of 2 mol / L hydrochloric acid solution. The sample was hydrolyzed under reflux for 3 h. After hydrolysis, the sample was cooled to room temperature and filtered. The insoluble residue was repeatedly washed with distilled water until neutral, and then dried at 80 °C.

[0178] The dried sample was placed in a Soxhlet extractor, and 30 mL of petroleum ether with a boiling range of 60–90 °C was added for continuous extraction for 4 h. After extraction, the petroleum ether was evaporated, and the resulting residue was redissolved in methanol and quantitatively transferred to a 50.0 mL volumetric flask. The solution was then diluted to the mark with methanol and filtered through a 0.45 μm microporous membrane. The resulting filtrate was used as the test solution.

[0179] Liquid chromatography was performed using an Inertsil ODS-3 reversed-phase column (4.6 mm × 250 mm). The mobile phase consisted of methanol, acetonitrile, and water in a volume ratio of 80:10:10. The detection wavelength was set to 210 nm, the flow rate to 1.0 mL / min, the injection volume to 20 μL, and the column temperature to ambient temperature.

[0180] Take 20 μL each of the DST reference solution and the test solution for detection, and record the retention time, peak area, and theoretical plate number of the corresponding chromatographic peaks. The detection results are shown in Table 1.

[0181] Table 1. Liquid Chromatography Results of DST Reference Standard and Test Sample

[0182] The results showed that the retention time of the DST reference standard was 20.455 min, and the test sample showed a characteristic chromatographic peak at 20.302 min, similar to the retention time of the reference standard. Based on the above chromatographic conditions and the comparison of the corresponding chromatographic peaks of the reference standard and the test sample, the DST-corresponding components in the test sample obtained after the aforementioned acid hydrolysis and extraction treatment were qualitatively and quantitatively analyzed. The content was determined to be 1.93 mg / g, based on the DST reference standard.

[0183] The liquid chromatograms of DST reference standard and Smilax glabra test sample are shown below. Figure 8 A. The above results indicate that, after the aforementioned sample hydrolysis, extraction, and chromatographic analysis, components corresponding to the DST reference standard can be detected and quantified, providing an experimental basis for subsequent studies on the efficacy and mechanism of action of DST.

[0184] 1.2 Screening of active ingredients related to diseases of Dioscorea opposita To screen for candidate components related to inflammatory skin diseases from the active ingredients contained in Dioscorea hypoglauca, this embodiment analyzes the active ingredients of Dioscorea hypoglauca and their association with atopic dermatitis and psoriasis based on herbal medicine databases.

[0185] First, a search was conducted in the database for natural active ingredients related to atopic dermatitis, yielding 731 relevant ingredients; a search was conducted for natural active ingredients related to psoriasis, yielding 723 relevant ingredients.

[0186] Further, an intersection analysis was conducted between the above two types of disease-related active ingredients and 120 active ingredients identified in Dioscorea hypoglauca to screen for candidate ingredients that are simultaneously associated with Dioscorea hypoglauca, atopic dermatitis, and psoriasis.

[0187] The results of the intersection analysis are shown below. Figure 8 B. The results showed that among the 120 active ingredients of Dioscorea hypoglauca, a total of 8 active ingredients were associated with both atopic dermatitis and psoriasis.

[0188] Based on the above screening results, among the eight candidate active ingredients, DST was identified as the main candidate effective active ingredient according to the comprehensive score. Therefore, subsequent in vivo and in vitro efficacy and mechanism of action studies will focus on DST.

[0189] Therefore, by analyzing the intersection of disease-related active ingredients and the component profiles of Dioscorea hypoglauca, the candidate range can be further narrowed down from a large number of natural active ingredients, and DST can be identified as the main candidate active ingredient for subsequent anti-psoriasis mechanism research.

[0190] 1.3 Screening of potential targets for DST 1.3.1 Prediction of Potential Targets for DST To determine the potential protein targets of DST, this embodiment uses databases such as Herb and Swiss TargetPrediction to predict the targets of DST.

[0191] Based on the chemical information used to characterize DST in the original study, the chemical information corresponding to DST was input into the Herb database and target prediction platforms such as Swiss Target Prediction to obtain a set of potential proteins that act on DST.

[0192] 1.3.2 Screening of differentially expressed proteins in keratinocytes of a psoriasis model The protein expression differences between HaCaT keratinocytes (a psoriasis model) and normal control HaCaT keratinocytes were compared using proteomics methods.

[0193] The aforementioned proteomics methods were used to extract, reduce, alkylate, digest with trypsin, desalt peptides, and perform liquid chromatography-mass spectrometry (LC-MS) detection of total cellular protein. Raw mass spectrometry data were analyzed using MaxQuant software and quantified without labeling.

[0194] Differentially expressed proteins were screened based on an adjusted P-value of less than 0.05 and a |log2 fold change| greater than 0.58.

[0195] Volcano diagram of differentially expressed proteins between HaCaT cells in a psoriasis model and normal control cells is shown below. Figure 8 C.

[0196] 1.3.3 Intersection analysis of DST predicted targets and differentially expressed proteins Intersection analysis was performed between potential targets of DST predicted by the database and differentially expressed proteins obtained from proteomics of HaCaT cells, a psoriasis model.

[0197] The results of the intersection analysis are shown below. Figure 8 D. The results showed that four candidate proteins were obtained that simultaneously met the criteria of "potential target of DST" and "differential protein associated with psoriasis", namely: LYN, ALOX5, CA2 and CDK6.

[0198] The above results suggest that LYN, ALOX5, CA2, and CDK6 may be candidate targets for DST to participate in the regulation of psoriasis-related pathological processes.

[0199] 1.3.4 Molecular docking analysis of DST with candidate target proteins To further evaluate the potential binding ability between DST and candidate target proteins, this embodiment performs molecular docking analysis on DST and candidate proteins.

[0200] The UniProt database was used to obtain structural information of candidate proteins, and the ProteinPLUS database was used to predict potential drug-binding pockets. Molecular docking analysis and structural visualization were then performed using PyMOL software.

[0201] The predicted binding affinity of different candidate proteins to DST was compared. The results showed that among the four candidate proteins LYN, ALOX5, CA2 and CDK6, LYN showed a stronger predicted binding affinity to DST.

[0202] The molecular docking results of DST and LYN are shown in Figure 8 E.

[0203] Based on target prediction, differential proteomics analysis of keratinocytes in a psoriasis model, and molecular docking results, this embodiment ultimately identifies LYN as the key candidate target for DST action to be validated in the future.

[0204] 1.4 Analysis of Results in This Example In this embodiment, the *Dioscorea hypoglauca* sample was first subjected to acid hydrolysis, extraction, and liquid chromatography analysis. Characteristic chromatographic peaks corresponding to the DST reference standard were detected in the obtained test sample, and the content of the corresponding component was calculated to be 1.93 mg / g according to the DST reference standard. Subsequently, through screening of disease-related active ingredients, eight candidate components related to both atopic dermatitis and psoriasis were obtained from 120 identified active ingredients of *Dioscorea hypoglauca*, and DST was further selected as the main research object.

[0205] In terms of target screening, intersection analysis was performed between predicted targets from the DST database and differentially expressed proteomic proteins from the HaCaT cell psoriasis model, resulting in four candidate proteins: LYN, ALOX5, CA2, and CDK6. Further molecular docking results showed that LYN exhibited a strong predicted binding affinity to DST.

[0206] Therefore, this embodiment systematically screened DST and its potential targets from multiple levels, including "active ingredient identification, disease-related component screening, potential target prediction, disease-differential protein screening, intersection analysis, and molecular docking," providing an experimental and analytical basis for subsequent mechanism verification of LYN and LYN-related signaling pathways.

[0207] It should be noted that the database target prediction and molecular docking results in this embodiment are used to screen candidate targets and cannot prove that DST has a direct inhibitory effect on LYN. The actual regulatory effect of DST on LYN-related signals and downstream STAT3 / LRG1 pathways will be further verified through cell and animal experiments in subsequent embodiments.

[0208] Example 2: DST improves IMQ-induced psoriatic dermatitis This embodiment uses an imiquimod (IMQ)-induced psoriasis-like mouse model to evaluate the in vivo anti-psoriatic effect of DST. The ameliorative effect of DST on psoriasis-like dermatitis was comprehensively evaluated by observing the appearance of mouse skin lesions, weight changes, Psoriasis Area and Severity Index (PASI), skin histopathological changes, epidermal thickness, inflammatory factor levels, and Ki-67 expression.

[0209] 2.1 Establishment of a psoriasis-like mouse model and experimental grouping Male C57BL / 6 mice aged 7–8 weeks were selected for the experiment. Before the experiment, the fur on the back of the mice was shaved to expose a skin area of ​​approximately 2 cm × 3 cm on the back.

[0210] The experimental mice were randomly divided into 5 groups of 10 mice each, as follows: (1) Solvent control group: White petroleum jelly was applied topically to the back skin after shaving once a day for 7 consecutive days; (2) IMQ model group: 5% IMQ cream was applied topically to the back skin once a day, with each mouse receiving 62.5 mg each time, for 7 consecutive days to induce psoriatic dermatitis; (3) IMQ+DST low-dose group (DST-L group): DST 20 mg / kg was administered on the basis of IMQ induction; (4) High-dose IMQ+DST group (DST-H group): DST 40 mg / kg was administered on the basis of IMQ induction; (5) IMQ+Bafetinib group: Bafetinib 10 mg / kg was administered on the basis of IMQ induction as a LYN-related pharmacological control.

[0211] Both DST and Bafetinib were administered orally via gavage 30 minutes before each topical application of IMQ.

[0212] All animal experiments were conducted in accordance with the relevant regulations for animal experiments conducted by the institution and were approved by the Animal Ethics Committee of Shenyang Medical College. The experiment number is SYYXY2024041201.

[0213] 2.2 Macroscopic observation and PASI score of psoriasis-like lesions From the beginning of the model's establishment, researchers unaware of the group assignments of each experimental group observed the changes in skin lesions on the backs of mice daily and evaluated the severity of the lesions.

[0214] The main observation indicators include: the degree of erythema; the degree of scaling; and the degree of thickening of the skin and epidermis.

[0215] The above indicators are scored on a scale of 0 to 4, where: A score of 0 indicates no obvious abnormalities; A score of 4 indicates an extremely severe lesion.

[0216] Simultaneously, mouse weight changes were recorded, and PASI evaluation of psoriatic lesions was performed by combining various skin lesion indicators.

[0217] The appearance of representative skin lesions in each group of mice is shown in the figure. Figure 1 A, See weight changes. Figure 1 B, changes in PASI and related skin lesion indicators are shown in [the table]. Figure 1 C~F.

[0218] The results showed that mice treated with IMQ developed obvious psoriasis-like skin changes starting from day 2 of the experiment, mainly manifested as increased erythema, thickened skin, and the formation of silvery-white scales. With continued IMQ treatment, these skin lesions gradually worsened.

[0219] Compared with the IMQ model group, DST treatment significantly alleviated the above-mentioned psoriasis-like skin manifestations and showed a certain dose-dependent effect.

[0220] Among them, the skin color and texture of mice in the DST-H group were closer to normal, and the erythema, scaling and skin thickening were significantly reduced; the DST-L group also showed some improvement, but the degree of improvement was between that of the IMQ model group and the DST-H group.

[0221] The PASI-related scores were consistent with the results observed by the naked eye. The PASI scores of mice in the IMQ model group increased significantly with the progress of the experiment, while the increase in PASI scores was inhibited after DST treatment, with the improvement effect being more obvious in the DST-H group.

[0222] The results of weight change showed that IMQ treatment was accompanied by a decrease in mouse weight, while DST-H treatment had a certain ameliorative effect on IMQ-induced weight loss, indicating that the mice in the DST-H group had better overall tolerance under the dosage conditions used in this example.

[0223] The above results indicate that DST can improve IMQ-induced psoriasis-like skin lesions in mice, and the improvement effect of high-dose DST is more significant than that of low-dose DST.

[0224] 2.3 Skin histopathology and evaluation of epidermal thickness After completing the appropriate treatment, skin tissue from the back of the mouse was taken for histopathological examination.

[0225] Skin tissue was fixed in neutral buffered formalin, embedded in paraffin, and then prepared into 5 μm thick tissue sections. Hematoxylin-eosin (H&E) staining was used to observe the pathological changes in skin tissue of different experimental groups.

[0226] The following pathological features should be observed in detail: degree of epidermal hyperplasia; abnormal keratinization and parakeratosis; changes in the granular layer structure; and inflammatory cell infiltration in the dermis and lesion area.

[0227] Representative H&E staining results for each group are shown below. Figure 1 G, the results of quantitative analysis of epidermal thickness are shown in [the table]. Figure 1 H.

[0228] The results showed that the skin tissue structure of mice in the solvent control group was basically normal, the epidermis was thin, the tissue layers were clear, and no obvious abnormal inflammatory cell infiltration was observed.

[0229] In contrast, the IMQ model group showed typical psoriasis-like histopathological changes, mainly manifested as obvious epidermal hyperplasia, parakeratosis, and relatively dense inflammatory cell infiltration in the dermal region, with a significant increase in epidermal thickness.

[0230] After administration of DST, the aforementioned histopathological changes were improved to varying degrees. In the DST-H group, epidermal hyperplasia was significantly reduced, epidermal thickness decreased to near the level of the normal control group, the granular layer structure was restored to some extent, and inflammatory foci and inflammatory cell infiltration were significantly reduced. The DST-L group also showed improvement, but the overall degree of improvement was lower than that of the DST-H group.

[0231] Quantitative results of epidermal thickness further confirmed that DST can alleviate IMQ-induced abnormal epidermal thickening, and the effect of high-dose DST is more obvious.

[0232] Therefore, the histopathological results were consistent with the aforementioned gross skin lesion and PASI evaluation results, indicating that DST can not only improve the appearance of psoriasis-like skin lesions in mice, but also alleviate their typical histopathological damage.

[0233] 2.4 Effects of DST on the levels of inflammatory factors in psoriasis-like mice To evaluate the effect of DST on IMQ-induced inflammatory response, the protein levels of related inflammatory factors in different experimental groups were detected by ELISA.

[0234] The detection indicators include: interleukin-17A (IL-17A); interleukin-22 (IL-22); and interleukin-6 (IL-6). Interleukin-1β (IL-1β); Tumor necrosis factor-α (TNF-α).

[0235] The detection results of IL-17A, IL-22, IL-6, IL-1β and TNF-α are shown in the following figures. Figure 1 I~M.

[0236] The results showed that, compared with the solvent control group, the levels of the above-mentioned inflammatory factors in the IMQ model group were significantly increased, indicating that IMQ treatment induced a significant inflammatory response.

[0237] After DST treatment, the elevation of the above-mentioned inflammatory factors was suppressed to varying degrees. Among them, the levels of IL-17A, IL-22, IL-6, IL-1β and TNF-α in the DST-H group were significantly lower than those in the IMQ model group, and all the above comparison results reached the level of statistical significance.

[0238] The above results indicate that DST can inhibit the increase of multiple pro-inflammatory mediators induced by IMQ, thereby alleviating the inflammatory response associated with psoriasis-like dermatitis.

[0239] 2.5 Effect of DST on Ki-67 expression in skin lesions Abnormal proliferation of keratinocytes is one of the important histopathological features of psoriasis. To further evaluate the effect of DST on abnormal cell proliferation in psoriatic lesions, this example used immunofluorescence to detect Ki-67 expression in mouse skin tissue.

[0240] Skin tissue sections were prepared according to the aforementioned immunofluorescence assay and stained with Ki-67 antibody. Ki-67-positive cells in the skin tissues of different experimental groups were observed using a fluorescence microscope, and the Ki-67 positive signals were statistically analyzed.

[0241] Representative immunofluorescence images and quantitative results are shown in Figure 1 N~O.

[0242] The results showed that the number of Ki-67 positive cells in the skin tissue of the solvent control group was relatively small; after IMQ treatment, the number of Ki-67 positive cells in the skin tissue increased significantly, suggesting that IMQ can induce abnormal proliferation of keratinocytes.

[0243] DST treatment can reduce IMQ-induced Ki-67 positive signal. In particular, the number of Ki-67 positive cells in the DST-H group was significantly reduced, and its Ki-67 level was close to that of the control group.

[0244] The above results further demonstrate that DST can inhibit the abnormal proliferation of keratinocytes in psoriatic lesions.

[0245] 2.6 Results Analysis of this Example In this embodiment, the anti-psoriasis effect of DST was evaluated from multiple aspects, including skin lesion appearance, PASI score, histopathological changes, epidermal thickness, inflammatory factors, and cell proliferation, using an IMQ-induced psoriasis-like mouse model.

[0246] Experimental results showed that continuous administration of DST could improve IMQ-induced psoriasis-like dermatitis and exhibited a certain dose-dependent relationship.

[0247] Specifically, compared to the IMQ model group, after DST processing: (1) The degree of skin erythema, scaling and thickening is reduced; (2) PASI-related scores decreased; (3) Abnormal epidermal thickening, parakeratosis, and reduced inflammatory cell infiltration; (4) The levels of pro-inflammatory factors such as IL-17A, IL-22, IL-6, IL-1β and TNF-α are decreased; (5) The number of Ki-67 positive cells in the skin lesion tissue decreased.

[0248] Among them, the improvement effect of the high-dose DST, i.e., the 40 mg / kg DST treatment group was generally better than that of the 20 mg / kg DST treatment group.

[0249] The above experimental results demonstrate at the in vivo level that DST can improve IMQ-induced psoriasis-like lesions, inhibit abnormal proliferation of keratinocytes, and reduce related inflammatory responses, providing experimental evidence for the use of DST in the preparation of drugs for the treatment of psoriasis.

[0250] Meanwhile, this embodiment mainly demonstrates that DST has a clear anti-psoriasis effect, but its direct molecular target cannot be determined solely based on the above phenotypic experiments. Therefore, in subsequent embodiments, multi-omics analysis, LYN / STAT3 / LRG1 signal detection, LYN overexpression, STAT3 Y705 site mutation, and LRG1 functional intervention will be used to further verify the specific molecular mechanism by which DST exerts the above-mentioned anti-psoriasis effect.

[0251] Example 3: DST control of LYN / STAT3 / LRG1 signal axes Based on the aforementioned clear anti-psoriasis efficacy of DST, this embodiment further combines clinical psoriasis transcriptome data, proteomics data, functional enrichment analysis, protein-protein interaction network analysis, and in vivo and in vitro molecular biology experiments to screen and verify the key signaling pathways through which DST exerts its anti-psoriasis effect, in order to clarify the association between LYN, STAT3, and LRG1 in the process of DST action.

[0252] 3.1 Psoriasis-related transcriptome and multi-omics analysis 3.1.1 Transcriptome Data Analysis of Clinical Skin Lesions in Psoriasis To analyze the abnormally expressed genes and related signaling pathways in psoriatic lesions, this embodiment selected the GSE30999 dataset from the NCBI GEO database to analyze the mRNA expression profiles of psoriatic lesions and control skin tissues.

[0253] First, principal component analysis was performed on the overall gene expression characteristics of different samples to evaluate the clustering relationship and overall expression differences between the psoriasis group and the control group.

[0254] Further differential expression analysis was performed. The results showed that, compared with control skin, 871 upregulated genes and 536 downregulated genes were detected in psoriatic lesion tissue. The volcano plot and heatmap analysis results of the differentially expressed genes are shown below. Figure 8 F~G.

[0255] Subsequently, GO and KEGG pathway enrichment analyses were performed on the differentially expressed genes. The results showed that psoriasis-related differentially expressed genes were significantly enriched in multiple inflammation and immune-related pathways, including: (1) IL-17 signaling pathway; (2) Cytokine-cytokine receptor interaction pathways; (3) JAK-STAT signaling pathway.

[0256] The corresponding enrichment analysis results are shown in Figure 8 H.

[0257] Further gene set enrichment analysis was used to evaluate the overall set of differentially expressed genes related to psoriasis, and single-sample gene set enrichment analysis was combined to analyze the characteristics of related immune cell infiltration.

[0258] The above results indicate that psoriatic lesions exhibit significant inflammation-related transcriptional remodeling, with STAT-related signaling pathways in an abnormally activated state, providing a basis for subsequent analysis of the regulatory relationship between LYN and STAT3.

[0259] 3.1.2 Integrated analysis of transcriptome and proteome data To screen key molecules related to psoriasis from different omics levels, this embodiment further integrates and analyzes the differentially expressed gene of GSE30999 transcriptome with the differentially expressed proteins obtained from the proteomics of keratinocytes in the aforementioned psoriasis model.

[0260] Molecules showing consistent trends at both the transcriptional and protein levels were subjected to intersection screening.

[0261] The analysis results are shown below. Figure 9 A~B.

[0262] The results showed that a total of 43 core molecules showed an upward trend and 4 core molecules showed a downward trend.

[0263] The above results indicate that the screened molecules not only changed at the transcriptional level in clinical psoriasis samples, but also at the protein level in keratinocytes of psoriasis models, thus they are worthy of further functional analysis.

[0264] 3.2 Screening of key LYN-related genes and the regulatory relationship between LYN / STAT3 / LRG1 3.2.1 Screening of LYN-related candidate genes In Example 1, through DST target prediction, screening of differentially expressed proteins in keratinocytes of a psoriasis model, and molecular docking analysis, LYN was identified as a key candidate target for DST.

[0265] To further analyze the regulatory network of LYN in psoriasis, this embodiment focuses on LYN and further screens LYN-related genes in the psoriasis group and the control group.

[0266] Intersection analysis ultimately identified 12 candidate genes that may be involved in the regulation of LYN-dependent psoriasis, namely: MPZL2, RAB38, CEBPD, LRG1, SAMD8, SERPINB1, LRP8, DHRS9, KRT6B, APOL2, SLC30A9 and CDH3.

[0267] The relevant screening results can be found Figure 9 C.

[0268] 3.2.2 Functional enrichment analysis of LYN-related candidate genes Further GO functional enrichment analysis was performed on the above 12 LYN-related candidate genes.

[0269] The analysis results are shown below. Figure 9 F~G.

[0270] The results showed that the candidate genes were significantly associated with biological processes related to keratinocyte proliferation, and were significantly enriched in functional items related to the positive regulation of keratinocyte proliferation.

[0271] Among the candidate molecules mentioned above, LRG1 is a node with important associations, so LRG1 will be included in subsequent research on LYN-related signaling pathways.

[0272] 3.2.3 Correlation analysis between LYN and LRG1 expression Further analysis was conducted to determine the correlation between LYN and LRG1 expression in clinical psoriasis samples. Results are shown below. Figure 9 H~I.

[0273] The analysis showed that there was a significant positive correlation between LYN expression level and LRG1 expression level in psoriasis patient samples; in contrast, no similar correlation was observed in healthy control samples.

[0274] The above results suggest that there may be a regulatory relationship between LYN and LRG1 that is related to the pathological state of psoriasis.

[0275] It should be noted that correlation analysis alone can only suggest an expression association between LYN and LRG1 in psoriasis, and cannot prove that LYN directly regulates LRG1. Therefore, this embodiment further combines protein-protein interaction network analysis and subsequent functional experiments to verify the possible signaling relationship between the two.

[0276] 3.2.4 Filtering STAT3 as a candidate intermediate node between LYN and LRG1 A protein-protein interaction network was constructed using the STRING database to analyze potential signaling connections between LYN and LRG1. Results are shown below. Figure 9 J.

[0277] Protein-protein interaction network analysis results show that STAT3 can serve as an important candidate intermediate node between LYN and LRG1.

[0278] Based on the aforementioned results showing significant enrichment of JAK-STAT-related pathways in the clinical transcriptome of psoriasis, this embodiment proposes and further verifies the following candidate signal regulatory relationship: LYN→STAT3→LRG1.

[0279] The above multi-omics and bioinformatics analyses were used to establish candidate mechanism models, and the specific signal relationships were further verified through the following in vivo and in vitro experiments.

[0280] 3.3 The effect of DST on LYN / p-STAT3 / LRG1 signals 3.3.1 Effects of DST on the expression of LYN, p-STAT3 and LRG1 in skin tissue of psoriasis-like mice To verify whether LYN / STAT3 / LRG1-related signals are altered in psoriatic lesions and to evaluate the effect of DST on these signals, this embodiment uses immunohistochemistry to detect the following in the skin tissues of mice in different treatment groups: (1) LYN; (2) p-STAT3, mainly detects the phosphorylation form of STAT3 at the Y705 site; (3) LRG1.

[0281] The relevant immunohistochemical staining and quantitative results are shown in [link to relevant data]. Figure 2 A~F.

[0282] The results showed that, compared with the normal control group, the detection signals of LYN, p-STAT3(Y705) and LRG1 in IMQ-induced psoriasis-like skin tissue were significantly increased.

[0283] After DST treatment, the above-mentioned abnormal increases were suppressed, and the improvement showed a trend of decreasing with increasing DST treatment dose, with the improvement being more obvious in the high-dose DST group.

[0284] The above results indicate that phosphorylation at LYN and STAT3 Y705 sites, as well as LRG1 expression, are all associated with IMQ-induced psoriasis-like pathological states, and DST can regulate these signal changes.

[0285] 3.3.2 Screening of DST concentrations To determine the appropriate concentration of DST for subsequent cell mechanism experiments, the CCK-8 assay was used to detect the effect of different concentrations of DST on keratinocyte viability.

[0286] Cells were exposed to 0–100 μM DST for 24 h, followed by the addition of CCK-8 reagent. Cell viability was evaluated by absorbance at 450 nm. Results are shown below. Figure 2 G and Figure 10 A.

[0287] Based on the cell viability test results, a DST concentration range that does not produce significant cytotoxic effects was selected for subsequent cell mechanism experiments to minimize the impact of non-specific cytotoxicity on protein expression and inflammatory phenotype detection results.

[0288] 3.3.3 Effects of DST on LYN / STAT3 / LRG1-related proteins in keratinocytes Western blot was used to detect the protein levels of LYN, STAT3, p-STAT3, and LRG1 in keratinocytes under different treatment conditions. The relevant results are shown below. Figure 2 H~K and Figure 10 B~E.

[0289] The results showed that under psoriasis-like stimulation conditions, the expression levels of p-STAT3 and LRG1 in keratinocytes were increased.

[0290] DST treatment can reduce STAT3 phosphorylation levels and LRG1 expression levels.

[0291] It is noteworthy that DST treatment primarily affects the phosphorylation state of STAT3, but does not show a corresponding reduction in the total STAT3 protein level.

[0292] That is, after DST treatment, p-STAT3 decreased, while the total STAT3 protein remained relatively stable overall.

[0293] These results indicate that DST primarily affects STAT3-related signals by inhibiting their activation state, rather than by reducing the total expression level of STAT3 protein.

[0294] Based on the aforementioned screening results of LYN candidate targets and the network analysis results of LYN / STAT3 / LRG1, the above data further support that DST may reduce phosphorylation of STAT3 Y705 site and further downregulate LRG1 by regulating LYN-related signals.

[0295] It should be noted that this part of the experiment mainly demonstrates the correspondence between DST treatment and changes in LYN / p-STAT3 / LRG1 signaling; the functional role of LYN in this signaling chain will be further verified in subsequent LYN overexpression experiments.

[0296] 3.4 Effects of DST on inflammatory response and indicators of abnormal keratinocyte activation To further analyze whether DST regulation of LYN / STAT3 / LRG1-related signals is accompanied by changes in psoriasis-related inflammation and abnormal keratinocyte phenotypes, this embodiment further detected related inflammatory factors and keratinocyte activation-related indicators.

[0297] 3.4.1 Effects of DST on the expression of inflammation-related factors Inflammation-related factors in keratinocytes and their culture supernatant under different treatment conditions were detected using Western blot, ELISA, and qRT-PCR.

[0298] The main testing indicators include: (1) TNF-α; (2) IL-1β; (3) IL-17A; (4) IL-22.

[0299] Results of relevant protein detection, culture supernatant detection, and mRNA detection are shown below. Figure 2 M~U and Figure 10 F~N.

[0300] The results showed that, under psoriasis-like stimulation conditions, the protein levels and / or mRNA expression of the above-mentioned pro-inflammatory factors were significantly increased.

[0301] After DST treatment, the abnormal increases in TNF-α, IL-1β, IL-17A and IL-22 were significantly suppressed.

[0302] The above results indicate that DST modulates LYN / STAT3 / LRG1-related signals while simultaneously reducing psoriasis-related pro-inflammatory responses.

[0303] 3.4.2 Effects of DST on indicators related to abnormal activation and migration of keratinocytes Further methods, such as Western blot, were used to detect proteins associated with abnormal activation and migration of keratinocytes, including: (1) Vascular endothelial growth factor (VEGF); (2) Connector protein 43, i.e., CX43; (3) Vimentin.

[0304] Test results are shown Figure 2 V~Y and Figure 10 O~R.

[0305] The results showed that the expression of the above-mentioned related proteins changed significantly under psoriasis-like stimulation conditions, and DST treatment could reduce the expression levels of VEGF, CX43 and Vimentin.

[0306] The above results are consistent with the effect of DST in inhibiting the abnormal activation and migration of psoriasis-associated keratinocytes.

[0307] 3.5 Analysis of Results in This Example This embodiment screened and validated key signaling axes related to psoriasis that may be regulated by DST through clinical transcriptome analysis of psoriasis, integration of proteomics data, screening of LYN-related genes, functional enrichment analysis of GO and KEGG, expression correlation analysis, protein-protein interaction network analysis, and in vivo and in vitro molecular experiments.

[0308] First, GSE30999 data analysis showed that there were significant differences in transcriptional expression between psoriatic lesions and control skin, with a total of 871 upregulated genes and 536 downregulated genes detected. Moreover, the differentially expressed genes were significantly involved in inflammatory signaling pathways such as IL-17, cytokine-cytokine receptor interaction, and JAK-STAT.

[0309] Further integration of transcriptomic and proteomic data yielded 43 co-upregulated molecules and 4 co-downregulated molecules. Further screening around the DST candidate target LYN identified 12 candidate genes potentially involved in the regulation of LYN-related psoriasis. Among them, LRG1 showed a strong association with keratinocyte proliferation-related functions.

[0310] Clinical sample correlation analysis showed that LYN and LRG1 expression were positively correlated in the psoriasis state; protein interaction network further suggested that STAT3 may serve as an important candidate intermediate node connecting LYN and LRG1.

[0311] This forms the candidate signal chain: LYN→STAT3→LRG1.

[0312] In in vivo experiments, LYN, p-STAT3 (Y705) and LRG1 were significantly elevated in IMQ-induced psoriasis-like skin, and DST treatment could attenuate these abnormal changes.

[0313] In keratinocyte experiments, DST reduced the phosphorylation level of STAT3 at the Y705 site and LRG1 expression without significantly reducing the total STAT3 protein level, indicating that its role mainly involves the regulation of STAT3 activation state.

[0314] At the same time, DST can also reduce the expression of inflammation-related factors such as TNF-α, IL-1β, IL-17A and IL-22, and reduce indicators related to abnormal activation and migration of keratinocytes such as VEGF, CX43 and Vimentin.

[0315] Therefore, this embodiment supports the following regulatory relationship from both multi-omics analysis and in vitro / in vivo experiments: DST → Regulates LYN-related signals → Reduces STAT3 Y705 phosphorylation → Downregulates LRG1 expression → Inhibits abnormal activation of keratinocytes and related inflammatory responses.

[0316] The above results provide experimental evidence for the anti-psoriasis effect of DST by regulating the LYN / STAT3 / LRG1 signal axis in this invention.

[0317] However, the connections between LYN, STAT3, and LRG1 obtained in this embodiment still include evidence such as multi-omics associations, protein-protein interaction network predictions, and changes in expression after treatment. To further demonstrate the causal relationship in this signaling chain, subsequent embodiments will further verify the signal transduction relationship LYN→STAT3(Y705)→LRG1 through LYN overexpression, STAT3 Y705F phosphorylation defect mutation, and LRG1 overexpression and knockdown experiments.

[0318] Example 4: DST inhibits psoriasis-related inflammation and abnormal proliferation by regulating LYN. In Example 3, multi-omics analysis, protein-protein interaction network analysis, and in vitro and in vivo experiments preliminarily identified LYN / STAT3 / LRG1 as an important candidate signaling axis for the anti-psoriasis effect of DST. To further verify the functional role of LYN in the DST process, this example constructed a LYN-overexpressing keratinocyte model and, combined with LPS stimulation and intervention with different concentrations of DST, observed changes in STAT3 phosphorylation, LRG1 expression, inflammatory factors, and indicators related to abnormal keratinocyte activation under enhanced LYN expression conditions. Simultaneously, in an IMQ-induced psoriasis-like mouse model, the in vivo effects of DST and LYN inhibitors were compared using the LYN inhibitor Bafetinib as a pharmacological control.

[0319] 4.1 Establishment of LYN overexpression cell model and DST intervention 4.1.1 Experimental Grouping and Processing An in vitro experimental model was established using keratinocytes. The expression level of LYN in the cells was increased by transfecting them with a LYN overexpression vector, and LPS was used to stimulate the cells to enhance the psoriasis-related inflammation and signal activation.

[0320] According to the experimental objectives, corresponding control groups, LPS stimulation groups, LYN overexpression combined with LPS stimulation groups, and DST treatment groups with different concentrations on the basis of LYN overexpression and LPS stimulation were set up.

[0321] After each group was processed, total cellular protein was collected for subsequent Western blot analysis.

[0322] In this embodiment, different concentrations of DST were compared. The DST concentrations of each treatment group were set in a gradient range within the concentration range that did not produce obvious cytotoxic effects, based on the aforementioned CCK-8 cell viability detection results.

[0323] 4.1.2 Detection of LYN and STAT3 phosphorylation and LRG1 expression The expression levels of the following proteins in cells from different experimental groups were detected using Western blot: (1) LYN; (2) Phosphorylation of STAT3, i.e., p-STAT3; (3) Total STAT3 protein; (4) LRG1.

[0324] The relevant experimental results are shown in Figure 3 A~D and Figure 10 S~V.

[0325] The results showed that in LYN-overexpressing cells, the expression of p-STAT3 and LRG1 was significantly increased after LPS stimulation, indicating that enhanced LYN expression was accompanied by STAT3 signaling activation and increased LRG1 expression.

[0326] After adding DST, the expression of both p-STAT3 and LRG1 was inhibited, and the expression gradually decreased with increasing DST concentration.

[0327] Meanwhile, DST treatment did not cause a corresponding decrease in the total STAT3 protein level and p-STAT3 level, indicating that DST mainly affects the phosphorylation activation state of STAT3, rather than simply reducing the total STAT3 protein expression.

[0328] The above results indicate that when LYN expression is artificially enhanced, STAT3 phosphorylation and LRG1 expression are correspondingly enhanced, and DST can still reverse these changes, further supporting the regulatory role of DST on LYN-related signaling pathways from a functional experimental perspective.

[0329] Based on the DST candidate target screening results in the previous embodiments, the results of this embodiment further support the following regulatory relationship: Increased LYN expression → Increased STAT3 phosphorylation → Increased LRG1 expression; DST treatment can suppress these changes.

[0330] It should be noted that this experiment mainly uses the signal response relationship under LYN overexpression conditions to prove that LYN participates in the process of DST regulating STAT3 / LRG1. Whether there is a direct physical binding between DST and LYN and its specific binding mode should still be further evidenced by corresponding direct binding or enzyme activity experiments.

[0331] 4.2 Effects of DST on inflammatory factors in the context of LYN overexpression To further analyze whether DST regulation of LYN-related signals can reduce keratinocyte inflammatory response, this embodiment detected changes in the expression of related inflammatory factors under different treatment conditions.

[0332] The testing indicators include: (1) TNF-α; (2) IL-1β; (3) IL-17A; (4) IL-22.

[0333] The evaluation was conducted using a combination of protein expression detection and transcriptional level detection.

[0334] See relevant results Figure 3 E~I and Figure 10 W.

[0335] The results showed that under LPS stimulation and LYN overexpression, the expression of the above-mentioned inflammatory factors was significantly increased, indicating that enhanced LYN-related signaling can be accompanied by aggravation of keratinocyte inflammation.

[0336] Compared with the LPS combined with LYN overexpression group, the protein expression and / or transcription levels of TNF-α, IL-1β, IL-17A and IL-22 were significantly reduced after DST treatment, and the overall inflammatory response was suppressed as the concentration of DST increased.

[0337] The above results indicate that DST can not only inhibit LYN-related STAT3 phosphorylation and LRG1 expression, but also simultaneously reduce the abnormal increase of inflammatory factors in the context of LYN overexpression.

[0338] Therefore, it can be observed from both the signal and functional phenotype levels: DST → Inhibits LYN-related signaling → Reduces STAT3 phosphorylation and LRG1 expression → Alleviates keratinocyte-related inflammatory response.

[0339] 4.3 Effects of DST on indicators related to abnormal keratinocyte activation One of the important pathological features of psoriasis is the abnormal activation of keratinocytes, accompanied by phenotypic changes related to cell proliferation and migration.

[0340] To further evaluate the effects of LYN-related signals and DST treatment on the abnormal activation state of keratinocytes, this embodiment used Western blot to detect the following related proteins: (1) Vascular endothelial growth factor (VEGF); (2) Connector protein 43, i.e., CX43; (3) Vimentin.

[0341] See relevant results Figure 3 J~M and Figure 10 X.

[0342] The results showed that under LPS stimulation and LYN overexpression conditions, the indicators related to abnormal activation of keratinocytes changed significantly; after DST intervention, the expression levels of VEGF, CX43 and Vimentin were reduced.

[0343] The decreased expression levels of VEGF, CX43, and Vimentin are consistent with the aforementioned results showing that DST weakens the phenotypes associated with abnormal keratinocyte activation and migration.

[0344] Based on the LYN / p-STAT3 / LRG1 detection results, it can be seen that the regulation of LYN-related signals by DST is not only reflected at the molecular signal level, but also accompanied by the improvement of functional indicators related to abnormal activation of keratinocytes.

[0345] 4.4 Comparison of the effects of DST and LYN inhibitor bafetinib in psoriasis-like mice To further verify the relationship between the anti-psoriasis effects of LYN and DST, this embodiment compared the effects of DST treatment with Bafetinib as a LYN inhibitor control in an IMQ-induced psoriasis-like mouse model.

[0346] 4.4.1 Animal handling The animal model and basic treatment methods were carried out in accordance with the methods described in Example 2 above.

[0347] Select 7-8 week old male C57BL / 6 mice and shave the hair in an area of ​​about 2 cm × 3 cm on the back.

[0348] Psoriasis-like dermatitis was induced by topical treatment with 5% IMQ cream at a dose of 62.5 mg per mouse per day for 7 consecutive days.

[0349] The low dose of DST is 20 mg / kg, and the high dose of DST is 40 mg / kg.

[0350] The treatment dose of Bafetinib was 10 mg / kg.

[0351] Both DST and Bafetinib were administered orally by gavage 30 minutes before each IMQ local treatment.

[0352] 4.4.2 Evaluation of skin thickness After the experiment, skin lesions were taken from the back of the mice, and the epidermal thickness of the mice in different experimental groups was measured.

[0353] Quantitative results of epidermal thickness are shown in […]. Figure 3 N.

[0354] The results showed that IMQ treatment significantly increased the thickness of the mouse epidermis; DST treatment significantly inhibited the IMQ-induced epidermal thickening.

[0355] Among them, the high-dose DST group showed a more significant improvement.

[0356] 4.4.3 Weight and PASI assessment Animal weight was recorded daily during the experiment, and PASI-related evaluations were performed based on skin erythema, scaling, and thickening.

[0357] Animal weight changes are shown in Figure 3 For PASI related scores, see [link / reference]. Figure 3 P~S.

[0358] The results showed that the IMQ model group developed obvious psoriasis-like skin lesions as the experiment progressed, while the degree of erythema, scaling and skin thickening was reduced after DST treatment, and the PASI related scores decreased accordingly.

[0359] Bafetinib treatment can also improve the IMQ-induced psoriasis-like phenotype.

[0360] The therapeutic effect of high-dose DST in the above animal models was comparable to that in the Bafetinib positive control.

[0361] 4.4.4 Skin tissue pathological examination Skin tissue was collected from the backs of mice in each group, fixed, embedded in paraffin, sectioned, and stained with H&E.

[0362] Histopathological results are shown in Figure 3 T.

[0363] The IMQ model group exhibited typical psoriasis-like histological changes, including significant epidermal hyperplasia, abnormal tissue structure, and inflammatory cell infiltration.

[0364] After DST treatment, the pathological damage to the skin tissue was significantly reduced, and the degree of epidermal thickening and inflammatory cell infiltration decreased.

[0365] Bafetinib treatment can also improve the above-mentioned pathological changes.

[0366] The above results further demonstrate that DST and LYN inhibitors exhibit a consistent ameliorative effect in IMQ-induced psoriasis-like dermatitis.

[0367] 4.4.5 Detection of inflammatory factors ELISA was used to detect psoriasis-related inflammatory factors in different animal experimental groups.

[0368] The testing indicators include: (1) IL-17A; (2) IL-22; (3) IL-6; (4) IL-1β; (5) TNF-α. See related results. Figure 3 U.

[0369] The results showed that the expression levels of the above-mentioned inflammatory factors were significantly increased after IMQ treatment, while their expression levels were inhibited to varying degrees after DST treatment.

[0370] Bafetinib treatment can also reduce IMQ-induced inflammatory responses.

[0371] This result is consistent with the aforementioned improvement in skin lesions and histopathology, indicating that both DST and LYN inhibitors can alleviate the psoriatic inflammatory state induced by IMQ.

[0372] 4.4.6 Ki-67 Immunofluorescence Detection To evaluate the effects of different treatments on the abnormal proliferation of keratinocytes in psoriatic lesions, Ki-67 immunofluorescence staining was used to detect the cell proliferation status in skin tissues of each group.

[0373] The relevant immunofluorescence and statistical results are shown in [link to relevant data]. Figure 3 V~W.

[0374] The results showed that the Ki-67 positive signal was significantly enhanced in the skin lesions of the IMQ model group, indicating that the keratinocytes were in an abnormal proliferative state.

[0375] After DST treatment, the Ki-67 positive signal decreased significantly, indicating that the abnormal proliferation of keratinocytes was inhibited.

[0376] Bafetinib treatment also showed a similar inhibitory effect.

[0377] 4.5 Analysis of Results in This Example This embodiment uses a LYN overexpression cell model and an animal control experiment with the LYN inhibitor Bafetinib to verify the relevant function of LYN in the anti-psoriasis effect of DST from two different perspectives.

[0378] In in vitro LYN overexpression experiments, enhanced LYN expression combined with LPS stimulation resulted in: (1) Increased p-STAT3 expression; (2) Increased LRG1 expression; (3) Increased levels of inflammatory factors such as TNF-α, IL-1β, IL-17A, and IL-22; (4) Corresponding changes occurred in keratinocyte abnormal activation-related indicators such as VEGF, CX43 and Vimentin.

[0379] DST treatment was able to reverse the above changes, with p-STAT3 and LRG1 decreasing in a dose-dependent manner with DST treatment, while total STAT3 protein did not decrease to a corresponding degree.

[0380] Therefore, this embodiment further supports LYN being located upstream of the DST regulating STAT3 / LRG1 signal.

[0381] In in vivo experiments, the LYN inhibitor bafetinib improved IMQ-induced psoriatic dermatitis, while DST treatment produced a concordant therapeutic effect. DST can: (1) Reduce epidermal thickness; (2) Improve PASI-related scores; (3) Reduce pathological damage to skin tissue; (4) Reduce the level of inflammatory factors; (5) Reduce Ki-67 positive signal.

[0382] Among them, the therapeutic effect of high-dose DST was generally comparable to that of the Bafetinib positive control.

[0383] Based on the combined results of in vitro and in vivo experiments, the following experimental relationship can be obtained: Increased LYN expression → Increased STAT3 phosphorylation → Increased LRG1 expression → Exacerbation of inflammation and abnormal activation of keratinocytes; DST treatment → inhibits LYN-related signaling → reduces STAT3 Y705 phosphorylation → reduces LRG1 expression → alleviates inflammatory response and abnormal keratinocyte activation.

[0384] Meanwhile, DST and the LYN inhibitor Bafetinib produced a consistent improvement in psoriasis-like animal models, further supporting the involvement of LYN-related signaling in the anti-psoriasis effect of DST.

[0385] Therefore, this embodiment provides further functional experimental evidence for the anti-psoriasis effect of DST by regulating the LYN / STAT3 / LRG1 signal axis described in this invention, and forms mutual verification with Example 3.

[0386] Based on this, in order to further determine whether the specific phosphorylation sites of STAT3 are involved in the transcriptional regulation of LRG1, and whether LRG1 is a functional downstream molecule that leads to abnormal phenotypes of keratinocytes, the next embodiment further uses STAT3Y705F phosphorylation defect mutation, dual-luciferase reporter assay, and LRG1 overexpression and knockdown experiments to verify the causal relationship of STAT3(Y705)→LRG1.

[0387] Example 5: Phosphorylation of STAT3 Y705 and Causal Verification of LRG1 in the Mechanism of Action of DST In Examples 3 and 4, multi-omics analysis, LYN overexpression experiments, and DST intervention experiments demonstrated that enhanced LYN expression was accompanied by increased STAT3 phosphorylation and LRG1 expression, while DST treatment reduced STAT3 phosphorylation levels and LRG1 expression. To further verify the causal relationship between STAT3 specific site phosphorylation and LRG1 transcription, and to clarify the function of LRG1 in abnormal activation and migration of keratinocytes, this example further constructed a STAT3 Y705F phosphorylation-deficient mutant. Combined with dual-luciferase reporter assays, Western blot detection, and LRG1 overexpression and knockdown experiments, the relationship between "STAT3 Y705 phosphorylation—LRG1 expression—abnormal keratinocyte phenotype" was verified.

[0388] 5.1 Construction of STAT3 Y705F phosphorylation defective mutant The tyrosine residue at position 705 of the STAT3 protein, also known as the Y705 site, is an important site for STAT3 phosphorylation activation.

[0389] To verify whether phosphorylation at this site is involved in the regulation of DST-related signals and LRG1 expression, this embodiment constructs a STAT3 Y705F phosphorylation-deficient mutant, that is, mutating the 705th tyrosine residue of the STAT3 protein to phenylalanine, so that the corresponding tyrosine phosphorylation cannot occur at this site.

[0390] A schematic diagram of the STAT3 Y705 site and the Y705F mutation is shown below. Figure 4 A.

[0391] By introducing the STAT3 Y705F mutant into keratinocytes and comparing it with wild-type STAT3, the effects of STAT3 Y705 phosphorylation on downstream LRG1 expression and related cell phenotypes can be evaluated without directly altering other signaling nodes.

[0392] In this embodiment, STAT3 phosphorylation, LRG1 expression, inflammation-related factors, and keratinocyte activation and migration-related indicators were subsequently detected to determine the function of the Y705 site in the above signal regulation.

[0393] 5.2 Effect of STAT3 Y705 phosphorylation on LRG1 transcription 5.2.1 Dual-luciferase reporter assay To analyze the relationship between STAT3 Y705 phosphorylation and LRG1 transcriptional activity, this embodiment uses a dual-luciferase reporter system to detect LRG1 promoter transcriptional activity.

[0394] Following the aforementioned general experimental method, cells from different treatment groups were collected and lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System.

[0395] Firefly luciferase and Renilla luciferase signals were detected separately, and Renilla luciferase was used as an internal control to normalize the firefly luciferase signal.

[0396] The transcriptional output of the LRG1 promoter was ultimately represented by relative luciferase activity compared to the negative control group.

[0397] The changes in LRG1 promoter activity were compared between wild-type STAT3 and STAT3 Y705F mutant conditions. The relevant results are shown in [link to relevant results]. Figure 4 B.

[0398] 5.2.2 Experimental Results The results showed that, compared with wild-type STAT3, the relative transcriptional activity of the LRG1 promoter was significantly reduced after the STAT3 Y705F mutation.

[0399] These results indicate that phosphorylation at the STAT3 Y705 site is closely related to LRG1 transcriptional activity; when phosphorylation at the Y705 site fails to occur normally, the transcriptional output of LRG1 is significantly inhibited.

[0400] Therefore, this embodiment further supports the view from the perspective of promoter transcriptional activity that phosphorylation at the STAT3 Y705 site is involved in the transcriptional regulation of LRG1.

[0401] Based on the results in the foregoing examples that DST can reduce phosphorylation at the STAT3 Y705 site and simultaneously reduce LRG1 expression, it can be further inferred that the regulation of LRG1 by DST is at least related to the phosphorylation state of the STAT3 Y705 site.

[0402] 5.3 Effects of DST and STAT3 Y705F mutations on inflammatory signaling To further evaluate the role of STAT3 Y705 phosphorylation in DST-induced suppression of the inflammatory response, this study compared changes in p-STAT3, LRG1, and related inflammatory factors under LPS stimulation, DST treatment, and STAT3 Y705F mutation conditions.

[0403] 5.3.1 Detection of p-STAT3 and LRG1 Western blot was used to detect the following in keratinocytes under different treatment conditions: (1) p-STAT3; (2) LRG1.

[0404] See relevant results Figure 4 C~E and Figure 11 A~C.

[0405] The results showed that LPS stimulation significantly increased the expression levels of p-STAT3 and LRG1.

[0406] When DST was added to LPS stimulation, the increase of p-STAT3 and LRG1 was suppressed and showed a decreasing trend with DST treatment.

[0407] Under the STAT3 Y705F mutation condition, the level of p-STAT3 was significantly reduced, and the expression of LRG1 also decreased accordingly.

[0408] The above results are consistent with the results of the dual-luciferase reporter assay, further indicating that phosphorylation at the Y705 site plays an important role in STAT3 activation and LRG1 expression.

[0409] 5.3.2 Detection of inflammatory factors Further investigation was conducted to detect inflammation-related factors in keratinocytes under different treatment conditions.

[0410] This section mainly observes: (1) TNF-α; (2) IL-1β; (3) IL-6 and other related inflammatory factors.

[0411] In addition, the inflammatory response under different treatment conditions was evaluated by combining the Western blot and related test results shown in the attached figure.

[0412] See relevant results Figure 4 F~K and Figure 11 D~H.

[0413] The results showed that LPS stimulation significantly increased the production of inflammatory factors.

[0414] DST treatment can significantly reduce the increase of LPS-induced inflammatory factors.

[0415] Meanwhile, the STAT3 Y705F mutant group showed a similar trend to the DST treatment group, namely, the levels of inflammation-related factors were significantly lower than those in the LPS stimulation group.

[0416] These results indicate that blocking phosphorylation at the STAT3 Y705 site can attenuate LPS-induced inflammatory responses, while DST treatment can produce a directional effect.

[0417] Therefore, the results in this section further support that the inhibition of the inflammatory response by DST is at least partly related to the inhibition of phosphorylation at the STAT3 Y705 site.

[0418] 5.4 Effects of STAT3 Y705F mutation on keratinocyte activation and migration To further analyze whether phosphorylation at the STAT3 Y705 site is involved in abnormal activation and migration of keratinocytes, this embodiment detects relevant functional indicators under different treatment conditions.

[0419] 5.4.1 Detection of proteins related to abnormal activation of keratinocytes Western blot was used to detect: (1) VEGF; (2) CX43; (3) Vimentin.

[0420] The relevant test results are shown below. Figure 4 L~O and Figure 11 I~L.

[0421] The results showed that, under LPS stimulation, the above-mentioned indicators related to abnormal activation and migration of keratinocytes were significantly elevated.

[0422] After DST treatment, levels of VEGF, CX43, and Vimentin decreased.

[0423] Under the STAT3 Y705F mutation condition, the above-mentioned related indicators also showed a downward trend, and their direction of change was consistent with that of the DST treatment group.

[0424] 5.4.2 Evaluation of keratinocyte migration ability Combined with corresponding cell migration experiments, the migration ability of keratinocytes under different treatment conditions was compared.

[0425] The results showed that LPS stimulation enhanced the migration ability of keratinocytes, while DST treatment weakened this effect.

[0426] After the STAT3 Y705F mutation, the migration ability of keratinocytes was also significantly reduced, which was consistent with the changes observed under DST treatment.

[0427] The above results indicate that phosphorylation at the STAT3 Y705 site is not only related to LRG1 expression and the production of inflammatory factors, but also involved in the abnormal activation of keratinocytes and the formation of migration-related phenotypes.

[0428] Therefore, the following relationship can be further established: Increased STAT3 Y705 phosphorylation → Increased LRG1 expression and related inflammatory response → Increased abnormal activation and migration of keratinocytes; DST treatment or STAT3 Y705F mutation → decreased STAT3 Y705 phosphorylation → decreased LRG1 and inflammation-related markers → weakened abnormal activation and migration of keratinocytes.

[0429] 5.5 Effects of LRG1 overexpression and knockdown on keratinocyte function After confirming in the aforementioned experiments that phosphorylation at the STAT3 Y705 site is related to LRG1 transcription and expression, this embodiment further verifies whether LRG1 is a functional downstream molecule of abnormal activation and migration of keratinocytes by directly regulating the expression level of LRG1.

[0430] 5.5.1 LRG1 Overexpression Experiment Keratinocytes were transfected with an LRG1 overexpression vector to increase the intracellular LRG1 expression level.

[0431] Under appropriate stimulation conditions, the activation and migration abilities of keratinocytes in the LRG1 overexpression group and the control group were compared.

[0432] The results showed that, in addition to basal stimulation, ectopic overexpression of LRG1 further enhanced the abnormal activation and migration of keratinocytes. (See related results below.) Figure 4 P~U and Figure 11 M~P.

[0433] The above results indicate that elevated LRG1 levels can promote the formation of a more pronounced pathological activation phenotype in keratinocytes.

[0434] 5.5.2 LRG1 Knockdown Experiment Keratinocytes were transfected with siRNA targeting LRG1 to reduce LRG1 expression.

[0435] The abnormal activation and migration abilities of cells in the LRG1 knockdown group and the corresponding control group were compared.

[0436] The results showed that siRNA-mediated LRG1 knockdown significantly reduced keratinocyte activation and migration phenotypes.

[0437] This result shows the opposite trend to the LRG1 overexpression experiment, namely, increased LRG1 promotes abnormal activation and migration, while decreased LRG1 inhibits abnormal activation and migration.

[0438] Therefore, LRG1 is not merely a companion molecule that changes synchronously with STAT3 phosphorylation, but a functional downstream factor involved in the abnormal activation and migration of keratinocytes.

[0439] 5.6 Analysis of Results in This Example In this embodiment, the causal relationship between STAT3 Y705 phosphorylation and LRG1 and keratinocyte pathological phenotypes was further verified by STAT3 Y705F phosphorylation defect mutation, LRG1 promoter dual-luciferase reporter assay, inflammatory factor detection, keratinocyte function detection, and LRG1 overexpression and knockdown experiments.

[0440] First, dual-luciferase reporter assays showed that the STAT3 Y705F mutation significantly reduced the transcriptional activity of the LRG1 promoter, indicating that phosphorylation at the STAT3 Y705 site is involved in the transcriptional regulation of LRG1.

[0441] Secondly, under LPS stimulation, p-STAT3 and LRG1 were significantly increased, while DST treatment could reduce p-STAT3 and LRG1; STAT3 Y705F mutation could also reduce p-STAT3 and LRG1, and its inflammatory factors and cell migration-related phenotypes showed similar trends to those in the DST-treated group.

[0442] The above results indicate that: Inhibiting phosphorylation at the STAT3 Y705 site can itself produce signaling and functional effects consistent with the direction of DST treatment.

[0443] Furthermore, LRG1 overexpression can enhance abnormal activation and migration of keratinocytes, while LRG1 knockdown can attenuate the above phenotype, thus proving that LRG1 is an important functional molecule involved in the pathological changes of keratinocytes.

[0444] Based on the results of Examples 3 and 4, this example further establishes the following continuous signal modulation relationship: LYN → Phosphorylation at STAT3 Y705 → Increased LRG1 transcription and expression → Abnormal activation, migration, and enhanced inflammatory response of keratinocytes.

[0445] DST processing can suppress the above signal processes, as shown in the following ways: DST → Inhibits LYN-related signaling → Reduces STAT3 Y705 phosphorylation → Downregulates LRG1 expression → Alleviates abnormal activation, migration, and inflammatory responses of keratinocytes.

[0446] Therefore, this embodiment further validates the causal relationship of the “LYN→STAT3(Y705)→LRG1” signaling chain from two levels: phosphorylation site mutation and downstream molecular functional intervention. This provides more direct experimental support for the anti-psoriasis effect of DST by regulating this signaling axis in this invention.

[0447] Based on this, in order to further clarify whether LRG1 acts only on keratinocytes themselves or can affect the psoriasis immune microenvironment through intercellular communication, subsequent examples further verified LRG1 in extracellular vesicles derived from keratinocytes and its effect on macrophage M1 polarization.

[0448] Example 6: DST inhibits macrophage M1 polarization by reducing keratinocyte-derived exosome LRG1. In Example 5, phosphorylation at the STAT3 Y705 site was demonstrated to participate in LRG1 transcriptional regulation through STAT3 Y705F phosphorylation defect mutation, LRG1 promoter dual-luciferase reporter assay, and LRG1 overexpression and knockdown experiments, and it was also demonstrated that LRG1 can promote abnormal activation and migration of keratinocytes. To further clarify whether keratinocyte-derived LRG1 can affect the psoriasis-related immune microenvironment through intercellular communication, extracellular vesicles were isolated from keratinocyte conditioned medium and their morphology, particle size, and marker proteins were identified. Furthermore, the effects of keratinocyte-derived LRG1 on macrophage M1 polarization under different treatment conditions were evaluated using mKC-BMDMs and HaCaT-THP-1 cell systems, and in vivo verification was performed using IMQ-induced psoriasis-like mouse skin lesions.

[0449] 6.1 Isolation and identification of exosomes derived from keratinocytes 6.1.1 Collection of conditioned medium for keratinocytes mKC mouse keratinocytes were used as extracellular vesicle source cells.

[0450] mKC cells were cultured according to the aforementioned cell culture method and treated accordingly based on the experimental design. After treatment, conditioned medium was collected in batches of 50 mL for subsequent isolation of extracellular vesicles.

[0451] To reduce interference from intact cells, cell debris, and larger particles in subsequent experiments, the conditioned medium was subjected to differential centrifugation.

[0452] First, the conditioned medium was centrifuged at 300×g for 10 min at 4°C to remove intact cells; then the supernatant was collected and centrifuged at 10000×g for 20 min at 4°C to remove cell debris and larger extracellular vesicles.

[0453] The resulting supernatant was further transferred to a sterile polycarbonate ultracentrifuge tube and centrifuged at 100,000 × g for 70 min at 4 °C to collect the precipitate.

[0454] 6.1.2 Washing of exosome precipitates The precipitate obtained by centrifugation at 100,000×g was gently resuspended in sterile 1×PBS and filtered through a 0.22 μm filter membrane to reduce the impact of larger aggregated particles.

[0455] The washing process was then carried out by two consecutive 100,000×g ultracentrifugations, each lasting 1 hour, both at 4°C.

[0456] The resulting precipitate was resuspended in an appropriate amount of 1×PBS for subsequent transmission electron microscopy observation, particle size detection, exosome marker protein detection, and functional experiments.

[0457] 6.1.3 Observation using transmission electron microscopy The morphology of the extracellular vesicles was observed using transmission electron microscopy. Representative images are shown below. Figure 5 A.

[0458] The results showed that the isolated vesicles were mainly round or nearly round granules with typical membrane vesicle structures. The particle diameter was mainly about 80-100 nm, and their morphology and size were consistent with the characteristics of exosomes.

[0459] 6.1.4 Nanoparticle Tracking Analysis The isolated extracellular vesicles were further analyzed using the NanoSight NS300 system for nanoparticle tracking, i.e., NTA detection.

[0460] Before testing, the sample was diluted with sterile PBS at a ratio of 1:1000 to achieve a working concentration of approximately 6 × 10⁻⁶. 7 Particle size distribution was measured per mL, and repeated measurements were performed. See [link to particle size distribution results]. Figure 5 B.

[0461] NTA results showed that the particle size distribution of the obtained particles was consistent with the aforementioned transmission electron microscopy observations, mainly falling within the typical extracellular vesicle / exosome particle size range.

[0462] 6.1.5 Detection of exosome marker proteins Western blot was used to detect the typical exosome marker proteins CD63 and TSG101 in the isolated vesicles.

[0463] Test results are shown Figure 5 C.

[0464] The results showed that CD63 and TSG101 signals could be detected in the obtained vesicles, further supporting the fact that the separated particles had exosome characteristics.

[0465] Based on the combined results of transmission electron microscopy, NTA, and exosome marker protein detection, this embodiment obtained extracellular vesicles with typical exosome characteristics from mKC conditioned medium through differential ultracentrifugation, which can be used for subsequent research on LRG1-mediated keratinocyte-macrophage communication.

[0466] 6.2 Detection of LRG1 secretion in keratinocytes under different treatment conditions To evaluate the effects of psoriasis-like stimulation and DST treatment on LRG1 secretion in keratinocytes, this example further examined the LRG1 level in the culture supernatant of mKC cells under different treatment conditions.

[0467] mKC cells were treated according to the experimental design, and the culture supernatant was collected. The LRG1 protein level was detected using ELISA. The relevant results are shown below. Figure 5 D.

[0468] The results showed that the level of LRG1 in the culture supernatant of mKC cells was significantly increased after LPS stimulation, indicating that inflammatory stimulation can promote the release of LRG1 from keratinocytes into the extracellular environment.

[0469] Compared with the LPS stimulation group, the DST combined treatment reduced the LRG1 level in the culture supernatant, suggesting that DST can inhibit the abnormal secretion of LRG1 by keratinocytes under inflammatory stimulation.

[0470] Further detection was performed in human HaCaT keratinocytes, and the results of LRG1 detection in the culture supernatant are shown below. Figure 11 Q's trend is consistent with that of the mKC system.

[0471] Based on the results of DST reducing LRG1 transcription and protein expression in keratinocytes in Examples 3-5, this example further demonstrates that DST regulates LRG1 not only in terms of intracellular expression levels but also in terms of reduced extracellular LRG1 output.

[0472] 6.3 Effect of conditioned medium for keratinocytes on M1 polarization of BMDMs 6.3.1 Preparation and co-culture treatment of BMDMs Bone marrow cells were isolated from the femur and tibia of mice using the aforementioned method. After erythrocyte lysis, the cells were cultured for 5 days in RPMI-1640 medium containing 10% FBS and 10 ng / mL M-CSF to obtain bone marrow-derived macrophages (BMDMs).

[0473] Conditioned culture media of mKC cells under different treatment conditions were collected and applied to BMDMs to simulate the paracrine regulation of macrophages by keratinocytes.

[0474] The study focuses on comparing the effects of keratinocyte conditioned medium under psoriasis-like stimulation conditions and keratinocyte conditioned medium treated with DST on the polarization state of BMDMs.

[0475] 6.3.2 Detection of M1-type polarization-related proteins Western blot was used to detect M1 polarization-related proteins in BMDMs: (1) CD80; (2) iNOS. See relevant results. Figure 5 E~G.

[0476] The results showed that the conditioned medium from LPS-activated mKC cells significantly increased the expression of CD80 and iNOS in BMDMs, indicating that soluble factors and / or extracellular vesicles produced by psoriatic keratinocytes can promote macrophage shift toward the M1 pro-inflammatory phenotype.

[0477] However, when mKC cells were treated with DST on the basis of LPS stimulation, the effect of the conditioned medium on increasing CD80 and iNOS in BMDMs was significantly weakened.

[0478] The results suggest that DST treatment of keratinocytes can alter the pro-inflammatory signals they transmit to macrophages.

[0479] 6.3.3 Detection of M1 polarization-related gene expression The expression levels of M1 polarization and inflammation-related genes in BMDMs were further detected by qRT-PCR.

[0480] The detection indicators include: (1) Cd80; (2) Inos; (3) Cd86; (4) Tnf-α; (5) Il-6.

[0481] Test results are shown Figure 5 H~L.

[0482] The results showed that LPS-activated conditioned medium derived from keratinocytes significantly increased the expression of the aforementioned M1 polarization and pro-inflammatory genes in BMDMs.

[0483] In comparison, the conditioned medium for keratinocytes treated with DST showed a significantly reduced induction of the expression of the aforementioned genes.

[0484] The protein and mRNA detection results showed a consistent trend, indicating that DST can inhibit macrophage M1 polarization and related inflammatory responses by affecting paracrine signals produced by keratinocytes.

[0485] 6.3.4 CD80 Immunofluorescence Detection Immunofluorescence was used to further detect CD80 expression in BMDMs treated with different culture media.

[0486] Representative images are shown Figure 5 M.

[0487] The results showed that after treatment with conditioned medium for psoriatic keratinocytes, the CD80 positive fluorescence signal in BMDMs was significantly enhanced; after stimulation with conditioned medium obtained by treating keratinocytes with DST, the CD80 positive signal was weakened.

[0488] The results were consistent with those of Western blot and qRT-PCR.

[0489] 6.3.5 Flow cytometry detection Flow cytometry was used to further analyze CD80 expression in BMDMs.

[0490] Collect BMDMs after different treatments, taking approximately 1 × 10⁻⁶. 6 Cells were stained with fluorescently labeled CD80 antibody, detected by FACSCalibur flow cytometry, and analyzed using FlowJo software. See [link to relevant results]. Figure 5 N~O.

[0491] The results showed that LPS-activated keratinocyte conditioned medium treatment increased the proportion of CD80-positive macrophages, while DST-treated keratinocyte conditioned medium reduced the proportion of CD80-positive macrophages.

[0492] The combined results of Western blot, qRT-PCR, immunofluorescence, and flow cytometry indicate that paracrine signals generated by psoriatic keratinocytes can promote the polarization of BMDMs towards the M1 pro-inflammatory phenotype, while DST treatment can significantly weaken this effect.

[0493] 6.4 Validation in human HaCaT-THP-1 cell system To evaluate whether the aforementioned keratinocyte-macrophage communication phenomenon can also be observed in human cell systems, this embodiment uses HaCaT human keratinocytes and PMA-induced THP-1-derived macrophages for verification.

[0494] 6.4.1 THP-1 cell induction Adjust THP-1 cells to 5 × 10 5 Cells / mL were cultured in complete RPMI-1640 medium containing 80 nM PMA for 72 h to differentiate into adherent macrophages.

[0495] 6.4.2 Conditioned Culture Medium Treatment Conditioned culture media of HaCaT cells under different treatment conditions were collected and applied to PMA-induced THP-1-derived macrophages.

[0496] Subsequently, indicators related to M1 type polarization of macrophages were detected.

[0497] 6.4.3 Flow cytometry and immunofluorescence detection CD80 expression in macrophages was detected by flow cytometry. Results are shown below. Figure 11 R~S.

[0498] The changes in the M1 phenotype of macrophages under different treatment conditions were observed using CD80 immunofluorescence. Representative images are shown below. Figure 11 T.

[0499] The results showed that the conditioned medium for psoriasis-like HaCaT cells could promote the increase of CD80 expression in THP-1-derived macrophages, while the ability of DST-treated HaCaT conditioned medium to induce CD80 increase was significantly weakened.

[0500] 6.4.4 Detection of M1 polarized proteins and mRNA The expression of CD80 and iNOS proteins in macrophages was further detected by Western blot, and the results are shown in [Figure number missing]. Figure 11 U~V.

[0501] Detection was performed using qRT-PCR: (1) CD80; (2) iNOS; (3) CD86; (4) TNF-α; (5) IL-6. Related results can be found in [link to relevant results]. Figure 11 W.

[0502] Overall, the test results showed that after treatment with psoriasis-like HaCaT cell conditioned medium, THP-1-derived macrophages exhibited obvious M1-type pro-inflammatory polarization characteristics; while DST treatment could reduce macrophage M1-type related indicators by altering the paracrine output of HaCaT cells.

[0503] The above results corroborate the mKC-BMDMs system, indicating that the role of DST in regulating keratinocyte-macrophage communication is not limited to mouse cell systems.

[0504] 6.5 Validation of IMQ-induced macrophage polarization in psoriasis-like mouse skin lesions To further evaluate the effect of DST on pro-inflammatory polarization of macrophages in psoriatic lesions at the in vivo level, this embodiment used immunofluorescence to detect F4 / 80 and iNOS in the skin lesions of mice in different treatment groups.

[0505] Among them, F4 / 80 was used to label mouse macrophages; iNOS was used as a marker for M1 type pro-inflammatory macrophages.

[0506] Skin tissue sections were stained with F4 / 80 and iNOS immunofluorescence, respectively, and the co-localization of the two was observed.

[0507] See relevant results Figure 5 P~Q.

[0508] The results showed that the colocalization signal of F4 / 80 and iNOS was significantly increased in the psoriatic lesion tissue induced by IMQ, suggesting that there were a large number of macrophages with M1 pro-inflammatory characteristics in the lesion area.

[0509] After DST treatment, the colocalization signals of F4 / 80 and iNOS in the lesion area decreased significantly, indicating that DST can reduce the infiltration of pro-inflammatory macrophages and / or M1 polarization in psoriatic lesions.

[0510] The improvement observed in the high-dose DST group was similar to that in the LYN inhibitor Bafetinib group.

[0511] These in vivo results are consistent with the aforementioned in vitro results for mKC-BMDMs and HaCaT-THP-1, further supporting the ability of DST to regulate macrophage polarization in the immune microenvironment of psoriatic skin.

[0512] 6.6 Analysis of Results in This Example In this embodiment, the role of DST in regulating intercellular communication between keratinocytes and macrophages was verified through exosome isolation and identification, co-culture of mouse keratinocytes and bone marrow-derived macrophages, human HaCaT-THP-1 cell system, and detection of skin lesions from IMQ psoriasis-like mice.

[0513] First, extracellular vesicles were isolated from mKC conditioned medium by differential ultracentrifugation. TEM results showed that the obtained vesicles were mainly spherical granules with a diameter of approximately 80–100 nm; NTA assay further confirmed their particle size distribution; Western blot analysis showed that the obtained vesicles expressed typical exosome markers such as CD63 and TSG101, indicating that the isolated particles had typical exosome characteristics.

[0514] Secondly, ELISA results showed that LPS stimulation increased LRG1 levels in keratinocyte culture supernatant, while DST treatment reduced extracellular output of LRG1.

[0515] Further stimulation of BMDMs with conditioned medium containing keratinocytes under different treatment conditions revealed: Conditioned culture medium for psoriasis-like keratinocytes → Increased levels of CD80, iNOS, CD86, TNF-α, and IL-6 → Macrophages shift towards the M1 pro-inflammatory phenotype; The conditioned medium for keratinocytes after DST treatment: → Decreased expression of M1-related proteins and genes → Decreased proportion of CD80-positive macrophages → Suppressed macrophage pro-inflammatory polarization.

[0516] Consistent directional results were also observed in the human HaCaT-THP-1 system, further verifying the above phenomenon.

[0517] Furthermore, in IMQ-induced psoriasis-like mouse lesions, DST was able to reduce F4 / 80 and iNOS colocalization signals, further demonstrating that DST can alleviate the pro-inflammatory macrophage-related phenotype in psoriatic lesions in vivo.

[0518] Based on the examples established in Examples 3-5: DST→LYN-related signaling is suppressed→STAT3 Y705 phosphorylation decreases→LRG1 expression decreases In this embodiment, the upstream mechanism is further extended to intercellular communication between keratinocytes and macrophages: DST→ LYN / STAT3(Y705) / LRG1 signaling is suppressed→ Keratinocyte LRG1 expression and extracellular output are reduced→ Keratinocyte-derived exosome-related pro-inflammatory signals are weakened→ Macrophage M1 polarization is suppressed.

[0519] Therefore, this embodiment shows that DST can not only directly improve abnormal activation of keratinocytes, but also regulate macrophage pro-inflammatory polarization by reducing LRG1-related exosome signals from keratinocytes, thereby regulating the immune-inflammatory microenvironment of psoriatic lesions.

[0520] This embodiment has not yet determined the specific downstream signaling pathway by which LRG1 promotes M1 polarization after entering macrophages. To further clarify this process, the next embodiment will analyze the role of LRG1-related ERK / CREB1 signaling in macrophage M1 polarization through BMDM RNA sequencing, pathway enrichment analysis, and protein expression verification.

[0521] Example 7: LRG1 regulates macrophage M1 polarization via ERK / CREB1 signaling In Example 6, through the isolation and identification of extracellular vesicles derived from keratinocytes, LRG1 detection, and keratinocyte-macrophage conditioned culture experiments, it was found that LRG1-related signals from psoriatic keratinocytes could promote macrophage shift towards the M1 pro-inflammatory phenotype. DST treatment could attenuate this effect by reducing LRG1 expression and extracellular output in keratinocytes. To further clarify the downstream signaling processes involved in LRG1's influence on macrophage M1 polarization, this example first used RNA sequencing and pathway enrichment analysis to screen candidate signaling pathways altered after LRG1 treatment. Subsequently, changes in ERK / CREB1 signaling and macrophage M1 polarization phenotype were verified through direct LRG1 stimulation, keratinocyte conditioned medium stimulation, and LRG1 knockdown experiments.

[0522] 7.1 Macrophage RNA Sequencing and Screening of Candidate Downstream Signaling Pathways 7.1.1 Experimental Grouping and RNA Sequencing Bone marrow-derived macrophages (BMDMs) were isolated and induced from mouse bone marrow using the aforementioned method. Resting macrophage groups, LPS-treated groups, LRG1-treated groups, and LPS / LRG1 combined treatment groups were established according to different stimulation conditions. The resting macrophage group received no LPS or LRG1 stimulation and was used to characterize the basal transcriptional state of BMDMs; the LPS-treated group was used to establish a pro-inflammatory stimulation background; the LRG1-treated group was used to evaluate the effect of LRG1 itself on the macrophage transcriptional expression profile; and the LPS / LRG1 combined treatment group was used to evaluate the transcriptional changes produced by further action of LRG1 on macrophages under a pro-inflammatory stimulation background.

[0523] After each group's treatment, BMDMs were collected and total RNA was extracted. RNA quality was assessed using the aforementioned RNA sequencing methods, and RNA samples meeting library construction requirements were selected for cDNA synthesis, library construction, and high-throughput sequencing. The resulting sequencing data, after quality control and standardization, were used for principal component analysis, differentially expressed gene analysis, and subsequent pathway enrichment analysis. The experimental group setup and analysis procedures are detailed below. Figure 12 A.

[0524] 7.1.2 Principal Component Analysis Principal component analysis was performed on the transcriptomic data of each group of BMDMs to evaluate the differences in the overall transcriptional expression characteristics of macrophages under different treatment conditions. The results are shown in […]. Figure 12 B.

[0525] The results showed that the distribution of samples from different treatment groups differed in the principal component space. The LRG1 treatment group and the LPS and LRG1 combined treatment group formed different clusters with the resting macrophage group, indicating that LRG1 stimulation can alter the overall transcriptional expression profile of BMDMs. Further administration of LRG1 under the background of LPS pro-inflammatory stimulation also resulted in further changes in the transcriptional expression characteristics of macrophages, suggesting that LRG1 can regulate the transcriptional program of macrophages under both basal and pro-inflammatory conditions.

[0526] 7.1.3 Screening of differentially expressed genes and core genes Further differential expression analysis was performed on the RNA sequencing results of different treatment groups, and the distribution of differentially expressed genes was displayed using a volcano plot. The relevant results are shown in [link to relevant data]. Figure 12 B~D.

[0527] Compared with the resting macrophage group, both LPS treatment and LRG1 treatment induced a series of gene expression changes; further administration of LRG1 on top of LPS stimulation also revealed significant differentially expressed gene changes. To screen for common molecules potentially associated with LRG1-mediated macrophage pro-inflammatory phenotypes from these differentially expressed genes, an intersection analysis was performed on differentially expressed genes obtained from different groups. The results are shown below. Figure 12 E~F.

[0528] Intersection analysis identified 21 core genes with a common upregulation trend. These core genes showed a consistent upward trend under LRG1-related treatment, suggesting their potential involvement in LRG1-induced macrophage transcriptional responses. The downregulated transcripts identified in the intersection analysis mainly involved non-coding RNAs and / or pseudogene-related sequences; therefore, subsequent analysis will focus on these commonly upregulated core genes and their associated signaling pathways.

[0529] 7.1.4 KEGG pathway enrichment and ERK / CREB1 candidate signal screening KEGG pathway enrichment analysis was performed on differentially expressed genes obtained from different experimental groups, and the intersection of enriched pathways between different groups was further analyzed. The relevant results are shown in [link to relevant data]. Figure 12 G~J.

[0530] Analysis revealed 82 commonly enriched pathways, indicating that LRG1-related stimulation-induced macrophage transcriptional changes involve multiple signaling regulatory networks. Further analysis, combining the characteristics of commonly upregulated core genes, macrophage pro-inflammatory polarization functions, and detectable signal nodes for subsequent experiments, screened candidate pathways, identifying ERK / CREB1-related signals as the primary targets for future validation. In the pathway analysis, ERK and CREB1-related molecules were found to be located within the signaling network of the KEGG circadian rhythm synchronization pathway Hsa04713. Corresponding pathway diagrams are provided. Figure 12 K.

[0531] It should be noted that RNA sequencing and KEGG enrichment analysis are used to screen candidate signals that LRG1 may influence at the transcriptome level, and cannot alone prove that ERK / CREB1 signaling mediates LRG1-induced M1 polarization. Therefore, this embodiment further experimentally verifies the above candidate signals by detecting the phosphorylation status of ERK and CREB1 and macrophage M1 polarization-related indicators.

[0532] 7.2 Effects of LRG1 on ERK / CREB1 signaling and M1 polarization in macrophages 7.2.1 Detection of ERK and CREB1 phosphorylation Different treatment conditions were applied to BMDMs, and p-ERK, total ERK protein, p-CREB1, and total CREB1 protein were detected by Western blot. Total ERK and total CREB1 protein were used as normalized references for their respective phosphorylated proteins. The activation status of ERK and CREB1 signaling was evaluated by comparing the relative levels of p-ERK / ERK and p-CREB1 / CREB1. The relevant results are shown in […]. Figure 6 A~C and Figure 13 A~C.

[0533] The results showed that, compared with the control condition without LRG1 treatment, the phosphorylation levels of ERK and CREB1 in BMDMs were increased after LRG1 treatment; further administration of LRG1 under LPS pro-inflammatory stimulation further enhanced the above phosphorylation signals. These results indicate that LRG1 treatment is accompanied by enhanced phosphorylation and activation of macrophage ERK and CREB1.

[0534] 7.2.2 Detection of M1-type polarization-related proteins Based on the detection of ERK / CREB1 signaling, Western blot was used to detect the expression levels of CD80 and TNF-α in BMDMs. CD80 serves as a phenotypic marker associated with M1 macrophages, while TNF-α reflects the pro-inflammatory state of macrophages. The relevant results are shown in [Figure number missing]. Figure 6 D~F and Figure 13 D~F.

[0535] The results showed that LRG1 treatment increased the expression of CD80 and TNF-α in BMDMs; further administration of LRG1 under LPS stimulation further enhanced the above-mentioned M1 polarization and pro-inflammatory indicators. The changes in CD80 and TNF-α were consistent with the changes in p-ERK and p-CREB1, indicating that LRG1-induced macrophage pro-inflammatory phenotype formation is accompanied by ERK / CREB1 signaling activation.

[0536] 7.2.3 CD80 Immunofluorescence and Flow Cytometry Detection To further evaluate the M1 polarization status of macrophages at the cellular level, CD80 in BMDMs under different treatment conditions was detected by immunofluorescence and flow cytometry.

[0537] Immunofluorescence results are shown in Figure 6 G and Figure 13 I. LRG1 treatment enhanced CD80-positive fluorescence signal in macrophages, and the CD80-positive signal was further enhanced under combined LPS and LRG1 treatment.

[0538] Flow cytometry results are shown in Figure 6 H~I and Figure 13 G~H. Compared with the corresponding control group, the proportion of CD80 positive macrophages increased after LRG1 treatment; after further administration of LRG1 in the context of LPS stimulation, the proportion of CD80 positive cells increased further.

[0539] The results from Western blot, immunofluorescence, and flow cytometry corroborated each other, indicating that LRG1 treatment can promote the shift of BMDMs towards the M1 pro-inflammatory phenotype, and this phenotypic change occurs synchronously with the increase in ERK and CREB1 phosphorylation levels.

[0540] 7.3 Effects of keratinocyte-derived paracrine signals on macrophage ERK / CREB1 signaling and M1 polarization To further determine whether the results obtained from direct LRG1 stimulation can reflect intercellular communication between psoriatic keratinocytes and macrophages, this embodiment further collected conditioned medium from mKC keratinocytes under different treatment conditions and used the obtained conditioned medium to treat BMDMs.

[0541] mKC cells were cultured according to the aforementioned keratinocyte culture and treatment methods. After completing the corresponding psoriasis-like stimulation or intervention, the culture supernatant was collected, and keratinocyte conditioned medium was obtained after removing cells and cell debris. Conditioned medium from different sources was applied to BMDMs, and macrophages were collected after treatment for ERK / CREB1 signaling and M1 polarization index detection.

[0542] Western blot was used to detect p-ERK, ERK, p-CREB1, and CREB1 in BMDMs, and the activation levels of the corresponding signals were evaluated by p-ERK / ERK and p-CREB1 / CREB1, respectively. The relevant results are shown in […]. Figure 6 J~L and Figure 13 J~L. The results showed that the conditioned medium derived from psoriatic keratinocytes could increase the phosphorylation levels of ERK and CREB1 in BMDMs, and the direction of change was consistent with the results obtained by direct treatment with LRG1.

[0543] Further analysis of CD80 and TNF-α expression in BMDMs yielded the following results: Figure 6 J~O and Figure 13 J~O. After treatment with conditioned medium for psoriatic keratinocytes (BMDMs), the expression of CD80 and TNF-α increased. Simultaneously, flow cytometry analysis showed an increased proportion of CD80-positive macrophages; related results are shown in [link to relevant results]. Figure 6 P~Q and Figure 13 P-Q; Immunofluorescence assay also showed enhanced CD80 positive signal, relevant results are shown in [link to results]. Figure 6 R and Figure 13 R.

[0544] This indicates that extracellular paracrine signals generated by psoriatic keratinocytes can simultaneously induce ERK / CREB1 signaling activation in macrophages and enhance the M1 pro-inflammatory phenotype. However, based on the conditioned medium experiment itself, it cannot be determined whether the above effects are entirely mediated by LRG1. Therefore, further verification was conducted using a keratinocyte LRG1 knockdown experiment.

[0545] 7.4 Effects of LRG1 knockdown in keratinocytes on ERK / CREB1 signaling and M1 polarization in macrophages Keratinocytes were transfected with siRNA targeting LRG1 to reduce LRG1 expression in keratinocytes, and a corresponding control group without LRG1 knockdown was set up. After completing the same psoriasis-like stimulation conditions, the conditioned medium of keratinocytes from the LRG1 knockdown group and the corresponding control group was collected, and after removing cells and cell debris, it was used to treat BMDMs.

[0546] After treatment, Western blot was used to detect p-ERK, ERK, p-CREB1, and CREB1 in BMDMs, and the p-ERK / ERK and p-CREB1 / CREB1 levels were compared. The results showed that compared with the conditioned medium for psoriatic keratinocytes without LRG1 knockdown, the conditioned medium obtained after LRG1 knockdown significantly reduced the phosphorylation of ERK and CREB1 in BMDMs. That is, as the LRG1 level from keratinocytes decreased, the levels of p-ERK and p-CREB1 in macrophages decreased accordingly.

[0547] Further examination was conducted on CD80, TNF-α, and related M1 polarization phenotypes in BMDMs. The results showed that knockdown of LRG1 in keratinocytes reduced the effect of conditioned medium in inducing an increase in CD80 and TNF-α in BMDMs; flow cytometry showed a decrease in the proportion of CD80-positive macrophages, and immunofluorescence also showed a decrease in CD80-positive signal.

[0548] The above results indicate that the effect of psoriatic keratinocyte conditioned medium on macrophage ERK / CREB1 signaling activation and M1-type pro-inflammatory polarization is at least partially dependent on keratinocyte-derived LRG1. Decreased LRG1 levels were accompanied by a simultaneous reduction in ERK and CREB1 phosphorylation and M1-type polarization-related phenotypes, further supporting a functional link between keratinocyte-derived LRG1 and macrophage ERK / CREB1 signaling and pro-inflammatory polarization.

[0549] 7.5 Analysis of Results in This Example This embodiment verified the relationship between LRG1 and macrophage ERK / CREB1 signaling and M1 polarization from four aspects: transcriptome screening, exogenous LRG1 stimulation, keratinocyte conditioned medium treatment, and LRG1 knockdown backtesting.

[0550] RNA sequencing results showed that LRG1-related treatment could alter the overall transcriptional expression profile of BMDMs, and 21 commonly upregulated core genes were identified. Further KEGG pathway enrichment and intersection analysis revealed 82 commonly enriched pathways, and based on this, ERK / CREB1-related signals were identified as key candidate validation targets.

[0551] In the direct LRG1 stimulation experiment, LRG1 treatment increased the phosphorylation levels of ERK and CREB1 in BMDMs, accompanied by increased expression of CD80 and TNF-α and an increased proportion of CD80-positive macrophages, indicating that the LRG1-induced M1 pro-inflammatory phenotype of macrophages has a consistent relationship with ERK / CREB1 signaling activation.

[0552] Further treatment of BMDMs with psoriatic keratinocyte-derived conditioned medium resulted in enhanced ERK and CREB1 phosphorylation and elevated M1 polarization-related indices. However, knockdown of LRG1 in keratinocytes significantly reduced the ERK / CREB1 signaling activation and M1 pro-inflammatory polarization induced by the corresponding conditioned medium. This indicates that the regulatory effect of psoriatic keratinocytes on macrophage ERK / CREB1 signaling and M1 polarization is at least partially dependent on keratinocyte-derived LRG1.

[0553] Based on the isolation and identification of extracellular vesicles derived from keratinocytes in Example 6 and the results of changes in LRG1 content, this example further supports the following interaction relationship: increased LRG1 expression and extracellular output in psoriatic keratinocytes, accompanied by enhanced phosphorylation of macrophage ERK and CREB1 and M1-type pro-inflammatory polarization; after reducing LRG1 derived from keratinocytes, the above signals and phenotypes are correspondingly weakened.

[0554] Further combining the upstream interactions verified in Examples 3-6, the following continuous regulatory chain can be formed: DST inhibits LYN-related signals, reduces phosphorylation at the STAT3 Y705 site, downregulates LRG1 expression in keratinocytes and its related extracellular vesicle signals, thereby weakening macrophage ERK / CREB1 signal activation and M1-type pro-inflammatory polarization.

[0555] Therefore, this embodiment provides experimental support for the regulatory relationship of "LRG1-ERK / CREB1-macrophage M1 polarization" at the downstream signaling mechanism level, and connects with the aforementioned experimental results of DST regulating LYN / STAT3 / LRG1 signaling and keratinocyte-macrophage communication, providing further experimental evidence for explaining the mechanism by which DST improves the psoriasis-related immune inflammatory microenvironment.

[0556] Example 8: LRG1-associated M1 macrophages promote keratinocyte proliferation, migration, and inflammatory response. The results of Examples 6 and 7 indicate that LRG1-related extracellular output from psoriasis-like keratinocytes can promote ERK / CREB1 signaling activation in macrophages, accompanied by a shift in macrophages towards the M1 pro-inflammatory phenotype. To further evaluate whether macrophages in different pro-inflammatory states can inversely influence keratinocytes via paracrine signals, thereby forming an inflammatory feedback loop between keratinocytes and macrophages, this example used conditioned medium containing bone marrow-derived macrophages (BMDMs) under different treatment conditions to treat mKC mouse keratinocytes and HaCaT human keratinocytes, respectively. The effect of macrophage pro-inflammatory states on abnormal keratinocyte phenotypes was evaluated by detecting keratinocyte activation-related indicators, expression and secretion of inflammatory factors, cell proliferation capacity, and migration and invasion abilities.

[0557] 8.1 Preparation of macrophage conditioned medium and treatment of keratinocytes 8.1.1 Macrophage treatment and preparation of conditioned medium Bone marrow-derived macrophages (BMDMs) were isolated and induced using the aforementioned method. Depending on the experimental objectives, the BMDMs were cultured under different treatment conditions to obtain macrophages with varying inflammatory states.

[0558] The experiment included a resting macrophage group, a pro-inflammatory stimulation group, an LRG1 treatment group, and a pro-inflammatory stimulation combined with LRG1 treatment group. The resting macrophage group received no pro-inflammatory stimulation or LRG1 treatment and was used to characterize the basal state of macrophages. The pro-inflammatory stimulation group was used to induce a more pronounced M1 pro-inflammatory phenotype in macrophages. The LRG1 treatment group was used to evaluate the effect of LRG1 on the paracrine phenotype of macrophages. The pro-inflammatory stimulation combined with LRG1 treatment group was used to evaluate whether the addition of LRG1 in a pro-inflammatory context further enhanced the paracrine function of macrophages.

[0559] After each group of BMDMs completed the corresponding treatment, they were cultured and the culture supernatant was collected. Residual cells and cell debris were removed by centrifugation, and the resulting supernatant was used as the conditioned medium for macrophages in the corresponding treatment group.

[0560] Subsequently, macrophage conditioned media from the above-mentioned different sources were used to culture mKC mouse keratinocytes and HaCaT human keratinocytes, respectively, to evaluate the effects of soluble paracrine signals produced by macrophages under different inflammatory states on abnormal activation, inflammatory response, proliferation, migration and invasion of keratinocytes.

[0561] 8.1.2 Keratinocyte Treatment After mKC or HaCaT keratinocytes were cultured to a suitable state, they were replaced with conditioned medium obtained from the different BMDMs treatment groups mentioned above for further culture.

[0562] After processing, keratinocytes and their culture supernatant were collected. Cell samples were used for Western blot, qRT-PCR, and related cell function experiments, while culture supernatant was used for ELISA detection. Cell proliferation was evaluated using the CCK-8 assay, and cell migration and invasion were evaluated using the Transwell assay.

[0563] Using the above-mentioned culture system, the effects of macrophage-derived paracrine signals on keratinocytes were evaluated without direct contact with macrophages, thereby simulating the intercellular regulation of keratinocytes by macrophages in the psoriasis-associated inflammatory microenvironment.

[0564] 8.2 Effects of macrophage conditioned medium on indicators related to abnormal keratinocyte activation To evaluate whether paracrine signals generated by macrophages under different inflammatory states can alter the abnormal activation state of keratinocytes, this embodiment uses Western blot to detect the expression of vascular endothelial growth factor (VEGF) and connexin 43 (CX43) in keratinocytes treated with different culture media.

[0565] The results of mKC cell-related detection are shown below. Figure 7 A-C, HaCaT cell validation results are shown in […]. Figure 14 A~C.

[0566] The results showed that, compared with resting macrophage-derived conditioned medium treatment, the expression levels of VEGF and CX43 in keratinocytes were increased after treatment with pro-inflammatory macrophage-derived conditioned medium, indicating that paracrine signals generated by pro-inflammatory macrophages can enhance the abnormal activation-related phenotype of keratinocytes.

[0567] Furthermore, after LRG1-related treatment enhanced the pro-inflammatory state of macrophages, the corresponding macrophage conditioned medium further enhanced the promoting effect of VEGF and CX43 expression in keratinocytes.

[0568] The above results indicate that the pro-inflammatory state of macrophages can affect keratinocytes through paracrine mechanisms, and cause keratinocytes to exhibit more pronounced abnormal activation characteristics; the enhanced pro-inflammatory phenotype of macrophages induced by LRG1-related treatment is consistent with the aforementioned enhanced paracrine effect.

[0569] 8.3 Effects of macrophage conditioned medium on keratinocyte inflammatory response 8.3.1 Detection of Inflammation-Related Gene Expression To evaluate the effect of macrophage-derived paracrine signals on the inflammatory transcriptional state of keratinocytes, qRT-PCR was used to detect the mRNA expression levels of related genes in keratinocytes after treatment with different macrophage conditioned media.

[0570] The detection indicators included Vegf, Cx43, Tnf-α, Il-1β, Il-17, and Il-22. Results of mKC cell-related detections are shown below. Figure 7 The validation results for HaCaT cells from D to F are shown in [the table]. Figure 14 D~F.

[0571] The results showed that, compared with resting macrophage-derived conditioned medium, pro-inflammatory macrophage conditioned medium could increase the expression levels of various inflammation and abnormal activation-related genes in keratinocytes.

[0572] When macrophages developed a more pronounced pro-inflammatory state under LRG1-related treatment, the conditioned medium further enhanced the promoting effect of the expression of partial inflammation and activation-related genes in keratinocytes, indicating that the enhanced pro-inflammatory phenotype of macrophages can further amplify the inflammatory transcriptional response of keratinocytes.

[0573] 8.3.2 Detection of inflammatory factor secretion Further, ELISA was used to detect the levels of inflammatory factors in the keratinocyte culture supernatant after treatment with different macrophage conditioned media, mainly TNF-α, IL-1β, IL-17A and IL-22.

[0574] The results of mKC cell-related detection are shown below. Figure 7 G-J, HaCaT cell-related detection results are shown in […]. Figure 14 G~J.

[0575] The results showed that after treatment with pro-inflammatory macrophage conditioned medium, the levels of inflammatory mediators such as TNF-α, IL-1β, IL-17A and IL-22 in the keratinocyte culture supernatant increased.

[0576] Following further enhancement of the pro-inflammatory state of macrophages by LRG1-related treatment, the conditioned medium further enhanced the promoting effect of keratinocyte inflammatory mediator production and release.

[0577] The above mRNA and protein level detection results corroborate each other, indicating that pro-inflammatory macrophages can enhance the inflammatory response of keratinocytes through their paracrine products and promote keratinocytes to further produce and release pro-inflammatory mediators, thereby providing conditions for the continuous amplification of local inflammatory signals.

[0578] 8.4 Effect of macrophage conditioned medium on keratinocyte proliferation capacity Abnormal proliferation of keratinocytes is an important cellular phenotype of psoriasis-associated epidermal hyperplasia. To evaluate the effect of macrophage pro-inflammatory status on keratinocyte proliferation, this study used the CCK-8 assay to detect the proliferation of mKC and HaCaT cells after treatment with different macrophage conditioned media.

[0579] Following the aforementioned CCK-8 assay method, keratinocytes were cultured in different macrophage conditioned media. After treatment, CCK-8 reagent was added for further incubation, and the absorbance of each well was measured at 450 nm to evaluate the proliferation status of keratinocytes under different treatment conditions.

[0580] The relevant results for mKC cells are shown below. Figure 7 The K,HaCaT cell validation results are shown in […]. Figure 14 K.

[0581] The results showed that, compared with resting macrophage-derived conditioned medium, pro-inflammatory macrophage conditioned medium significantly enhanced keratinocyte proliferation-related signals.

[0582] When macrophages exhibited a further enhanced pro-inflammatory state under LRG1-related treatment, the conditioned medium further enhanced the effect of promoting keratinocyte proliferation.

[0583] The above results indicate that macrophages in the M1 pro-inflammatory state can promote the abnormal proliferation of psoriasis-associated keratinocytes through paracrine effects, and the enhanced pro-inflammatory phenotype of LRG1-associated macrophages is consistent with the enhanced pro-proliferative effect described above.

[0584] 8.5 Effects of macrophage conditioned medium on keratinocyte migration and invasion ability To further evaluate the effects of macrophages in different states on the migration and invasion phenotypes of keratinocytes, the Transwell assay was used to detect the migration and invasion abilities of keratinocytes treated with different macrophage conditioned media.

[0585] Following the aforementioned Transwell assay, keratinocytes treated with different culture media were added to the upper chamber of a Transwell chamber. For invasion experiments, a corresponding matrix gel layer was pre-placed on the chamber membrane surface. After culture, cells that did not pass through the membrane pores were removed. Cells that migrated or invaded to the other side of the membrane were fixed and stained with crystal violet, and cell counting was performed by randomly selecting fields of view under a microscope.

[0586] The results of mKC cell migration and invasion experiments are shown in the figure. Figure 7 L~O, HaCaT cell validation results are shown in […]. Figure 14 L~O.

[0587] The results showed that, compared with the conditioned medium derived from resting macrophages, the conditioned medium for pro-inflammatory macrophages significantly improved the migration and invasion capabilities of keratinocytes.

[0588] The pro-inflammatory macrophages induced under LRG1-related treatment conditions exhibited a further enhanced effect of conditioned medium on the migration and invasion of keratinocytes.

[0589] This result is consistent with the aforementioned results of increased VEGF and CX43 expression and CCK-8 proliferation detection, indicating that macrophages in a strong pro-inflammatory state can promote the formation of more obvious abnormal phenotypes of keratinocytes through multiple levels such as abnormal activation, proliferation, migration and invasion.

[0590] 8.6 Analysis of Results in This Example In this embodiment, conditioned medium derived from BMDMs under different treatment conditions was used to stimulate mKC mouse keratinocytes and HaCaT human keratinocytes, respectively, to verify the role of macrophages in reverse regulation of keratinocytes from multiple levels, including abnormal activation, inflammatory response, cell proliferation, migration and invasion.

[0591] The results showed that, compared with the conditioned medium derived from resting macrophages, the M1 pro-inflammatory macrophage conditioned medium could increase abnormal activation-related indicators such as VEGF and CX43 in keratinocytes, promote the expression and / or release of inflammatory mediators such as TNF-α, IL-1β, IL-17A and IL-22, enhance the proliferation capacity of keratinocytes, and strengthen the migration and invasion capabilities of keratinocytes.

[0592] Furthermore, after LRG1-related treatment enhanced the pro-inflammatory phenotype of macrophages, the aforementioned macrophage conditioned medium further amplified its promoting effects on abnormal activation, inflammatory response, proliferation, migration, and invasion of keratinocytes. These results indicate that the pro-inflammatory state of LRG1-related macrophages is consistent with its enhanced paracrine promotion effect on keratinocytes.

[0593] Combining the experimental results of Examples 6 and 7, a continuous intercellular regulatory relationship can be established, from keratinocytes to macrophages and further back to keratinocytes: increased LRG1 expression and related extracellular output in psoriatic keratinocytes promote the activation of macrophage ERK / CREB1-related signals and the formation of the M1 pro-inflammatory phenotype; macrophages in the M1 pro-inflammatory state further promote abnormal proliferation, migration, and expression and release of inflammatory mediators of keratinocytes through paracrine signals.

[0594] Therefore, psoriasis-associated keratinocytes and macrophages can form mutually reinforcing inflammatory cell communication. Specifically, keratinocyte-derived LRG1-related signals promote macrophage pro-inflammatory polarization, while paracrine signals produced by pro-inflammatory macrophages in turn enhance abnormal keratinocyte activation and inflammatory response, resulting in a continuously amplified feedback process between the abnormal state of keratinocytes and the local immune inflammatory microenvironment.

[0595] Further combining the upstream regulatory mechanism of DST established in Examples 3-7, a relatively complete action relationship can be formed: DST regulates LYN-related signals, reduces phosphorylation at STAT3 Y705 site and LRG1 expression in keratinocytes, reduces LRG1-related extracellular vesicle signals from keratinocytes, thereby weakening macrophage ERK / CREB1 signal activation and M1-type pro-inflammatory polarization, and further weakening the feedback promotion effect of pro-inflammatory macrophages on abnormal proliferation, migration and inflammatory response of keratinocytes.

[0596] Therefore, this embodiment further verifies the existence of an inflammatory feedback loop between keratinocytes and macrophages from the perspective of "macrophages inversely regulating keratinocytes". It also connects with the aforementioned experimental results of DST regulating LYN / STAT3 / LRG1 signaling and macrophage M1 polarization, providing further experimental evidence for explaining the mechanism by which DST improves psoriasis-related epidermal abnormal proliferation and immune inflammatory microenvironment.

[0597] Example 9: Validation of LYN / STAT3 / LRG1 and related inflammatory phenotypes in clinical samples from psoriasis patients In the aforementioned embodiments, experiments were conducted using an IMQ-induced psoriasis-like mouse model, a keratinocyte model, LYN overexpression, STAT3 Y705F phosphorylation defect mutation, LRG1 overexpression / knockdown, exosome isolation, and macrophage co-culture to establish experimental evidence that DST regulates the LYN / STAT3 / LRG1 signaling axis and further affects abnormal keratinocyte activation and macrophage M1 polarization. To further evaluate whether the aforementioned signal changes also exist in clinical psoriasis patients, this embodiment collected skin tissue and serum samples from psoriasis patients and healthy subjects. LYN, p-STAT3, LRG1, Ki-67, macrophage-related markers, and various inflammatory factors were detected to verify the consistency between the aforementioned animal and cell experimental results and the clinical disease state.

[0598] 9.1 Source of Clinical Samples Clinical samples were collected in this study between January 2024 and December 2025.

[0599] The psoriasis group included 30 psoriasis patients, from whom skin biopsies and serum samples were collected. The included patients met the inclusion criteria in terms of psoriasis area, severity index, and affected area, and had used emollients for at least 3 weeks prior to biopsy.

[0600] At the same time, 30 normal skin tissue samples and corresponding serum samples from healthy subjects were collected as controls.

[0601] This study protocol was approved by the Ethics Committee of Shenyang Seventh People's Hospital, with ethics approval reference number AF / SC-08 / 03.2. All participants signed written informed consent forms.

[0602] The collected skin tissue was used for subsequent detection of LYN, p-STAT3, LRG1, Ki-67, and macrophage-related markers; serum samples were used for the determination of LRG1 and inflammatory factor levels.

[0603] 9.2 Detection of LYN, p-STAT3 and LRG1 in skin tissue of psoriasis patients 9.2.1 Immunohistochemical detection The following proteins were detected in skin tissues of psoriasis patients and healthy controls using the aforementioned immunohistochemical methods: (1) LYN; (2) Phosphorylated STAT3, i.e. p-STAT3; (3) LRG1.

[0604] Tissue samples were fixed, paraffin-embedded, and cut into 5 μm thick sections. After dewaxing, rehydration, and antigen retrieval, primary antibodies LYN, p-STAT3, and LRG1 were added and incubated. Then, the corresponding secondary antibodies were added and staining and counterstaining were completed.

[0605] The expression of corresponding proteins in the skin tissue of each group was observed under a microscope and quantitatively compared.

[0606] See relevant results Figure 15 A~C and Figure 15 E~G.

[0607] 9.2.2 Experimental Results The results showed that, compared with healthy control skin tissue, LYN, p-STAT3 and LRG1 all showed higher detection signals in the skin lesions of psoriasis patients.

[0608] Elevated p-STAT3 signaling indicates enhanced STAT3 activation in the skin lesions of psoriasis patients; meanwhile, elevated LRG1 expression is consistent with the findings in the aforementioned psoriasis-like animal model and keratinocyte model.

[0609] Therefore, the clinical sample results further indicate that: Elevated LYN levels, enhanced STAT3 phosphorylation, and elevated LRG1 expression can coexist in the skin lesions of psoriasis patients.

[0610] This result is consistent with the changes in the LYN / STAT3 / LRG1 related signals established in the previous embodiments.

[0611] It should be noted that the clinical tissue detection in this embodiment is mainly used to verify the expression characteristics of the above signaling molecules in the skin lesions of psoriasis patients; the causal regulatory relationship between LYN, STAT3 and LRG1 is mainly demonstrated by the aforementioned cell function experiments and molecular mechanism experiments.

[0612] 9.3 Detection of Ki-67 in skin tissue of psoriasis patients Abnormal proliferation of keratinocytes is an important pathological feature of psoriasis. To evaluate the cell proliferation status in the skin lesions of psoriasis patients, this example uses immunofluorescence to detect Ki-67.

[0613] Following the aforementioned immunofluorescence detection method, skin tissue sections from psoriasis patients and healthy controls were stained with Ki-67, and observed and quantitatively analyzed using a fluorescence microscope. The relevant results are shown below. Figure 15 D and Figure 15 H.

[0614] The results showed that, compared with the healthy control group, the Ki-67 positive signal in the skin lesions of psoriasis patients was increased, suggesting that there was a more obvious active cell proliferation in the psoriasis lesions.

[0615] This result is consistent with the increase in Ki-67 in psoriasis-like mouse skin lesions induced by IMQ in Example 2, and also corroborates the aforementioned experimental results of DST inhibiting abnormal proliferation of keratinocytes by regulating LYN / STAT3 / LRG1 related signals.

[0616] Therefore, the Ki-67 assay in clinical tissues further demonstrates that abnormal proliferation of keratinocytes is an important pathological phenotype observable in the clinical psoriasis samples of this study.

[0617] 9.4 Detection of Pro-inflammatory Phenotype of Macrophages in Skin Lesions of Psoriasis Patients To evaluate changes in macrophages and their pro-inflammatory phenotypes in the skin lesions of psoriasis patients, this embodiment uses CD68 and CD80 for immunofluorescence co-localization detection.

[0618] CD68 was used to label macrophages; CD80 served as a marker for M1 type pro-inflammatory macrophages.

[0619] CD68 and CD80 staining was performed on skin tissues from psoriasis patients and healthy controls using the aforementioned immunofluorescence method, and the co-localization of the two substances in the tissues was observed. The relevant results are shown in [link to results]. Figure 15 I~J.

[0620] The results showed that the colocalization signal of CD80 and CD68 in the skin lesions of psoriasis patients was enhanced compared with that in the healthy control group, suggesting an increase in pro-inflammatory macrophage-related phenotypes in psoriasis lesions.

[0621] This result is consistent with the increase in co-localization of F4 / 80 and iNOS in mouse skin lesions induced by IMQ in Example 6, and the results of LRG1-related signals from keratinocytes promoting M1 polarization of macrophages in Examples 6 and 7.

[0622] Therefore, clinical samples further support the presence of an immune microenvironment characterized by enhanced pro-inflammatory polarization of macrophages in psoriatic lesions.

[0623] 9.5 Detection of LRG1 and inflammatory-related factors in serum of psoriasis patients 9.5.1 Detection Indicators The following indicators in the serum of psoriasis patients and healthy controls were detected using methods such as ELISA: (1) LRG1; (2) TNF-α; (3) IL-1β; (4) IL-6; (5) IL-17A; (6) IL-22; (7) IL-23.

[0624] See relevant results Figure 15 K~Q.

[0625] 9.5.2 Experimental Results The results showed that, compared with the healthy control group, patients with psoriasis had elevated levels of LRG1 and various inflammatory-related factors in their serum.

[0626] The inflammatory markers detected included TNF-α, IL-1β, IL-6, IL-17A, IL-22, and IL-23.

[0627] The above results indicate that psoriasis patients not only exhibit enhanced LYN / p-STAT3 / LRG1-related signals and pro-inflammatory macrophage-related changes in the local skin lesions, but also show elevated levels of LRG1 and various inflammatory mediators in peripheral serum.

[0628] This result corresponds to the findings in the aforementioned animal experiments where IL-17A, IL-22, IL-6, IL-1β, and TNF-α were elevated, and the corresponding inflammatory factors decreased after DST treatment.

[0629] 9.6 Correspondence between clinical sample results and the aforementioned mechanistic experiments Based on the clinical sample test results of this embodiment, the following characteristics can be observed: (1) LYN expression is elevated in the skin lesions of psoriasis patients; (2) Increased p-STAT3 expression suggests enhanced STAT3 phosphorylation activation; (3) Increased LRG1 expression; (4) Increased Ki-67 positive signal indicates abnormal and active cell proliferation in the skin lesions; (5) Enhanced co-localization of CD80 and CD68 suggests an increase in pro-inflammatory macrophage-related phenotypes; (6) Serum levels of LRG1, TNF-α, IL-1β, IL-6, IL-17A, IL-22 and IL-23 were elevated.

[0630] The above clinical results are consistent with the direction of molecular and cellular phenotypic changes observed in the aforementioned animal and cell experiments.

[0631] Combining the upstream mechanisms established in Examples 3-5, the following can be formed: Enhanced LYN-related signaling → Enhanced phosphorylation of STAT3 at Y705 site → Increased LRG1 expression → Abnormal activation and enhanced proliferation of keratinocytes; Based on the intercellular communication mechanisms established in Examples 6-8, the following can be further developed: Enhanced LRG1-related signaling in keratinocytes → Activation of ERK / CREB1 signaling in macrophages → Macrophage shift towards the M1 pro-inflammatory phenotype → Enhanced inflammatory paracrine feedback → Further promoting abnormal proliferation, migration, and inflammatory response of keratinocytes.

[0632] The synchronous changes in LYN, p-STAT3, LRG1, Ki-67, CD80 / CD68, and multiple inflammatory factors in the clinical samples of this embodiment provide further support for the relevance of the above mechanisms in the clinical state of psoriasis.

[0633] 9.7 Analysis of Results in This Example This embodiment validates the results obtained from the aforementioned cell and animal experiments at the clinical sample level by testing skin tissue and serum samples from 30 psoriasis patients and 30 healthy controls.

[0634] The results showed that the following were present in the skin lesions of psoriasis patients: Increased LYN, p-STAT3, LRG1, Ki-67 positive cells, and enhanced CD80 / CD68 colocalization; Meanwhile, psoriasis patients have elevated levels of LRG1 and various pro-inflammatory factors in their serum.

[0635] These results demonstrate that the abnormal LYN / STAT3 / LRG1 signaling, abnormal keratinocyte proliferation, pro-inflammatory macrophage polarization, and elevated inflammatory mediators observed in the aforementioned examples are not only present in in vitro cell models or IMQ-induced animal models, but can also be observed in clinical psoriasis patient samples with the same molecular and inflammatory phenotypes.

[0636] Based on the experimental results of Examples 1 to 9, the following relatively complete mechanism of action can be formed: DST → Regulates LYN-related signaling → Reduces STAT3 Y705 phosphorylation → Downregulates LRG1 expression in keratinocytes → Inhibits abnormal proliferation and migration of keratinocytes; Simultaneously, DST → reduces LRG1-related exosome signaling from keratinocytes → inhibits macrophage ERK / CREB1 signaling activation → inhibits macrophage polarization towards the M1 pro-inflammatory phenotype → weakens the pro-inflammatory, pro-proliferative, and pro-migration feedback effects of macrophages on keratinocytes → improves the psoriasis-related immune-inflammatory microenvironment.

[0637] Therefore, this embodiment validated the abnormal LYN / STAT3 / LRG1 signaling and its related inflammatory phenotypes at the clinical sample level, and further enhanced the overall technical solution of DST to exert anti-psoriasis effects by regulating the LYN / STAT3 / LRG1 signaling axis and keratinocyte-macrophage inflammatory communication.

[0638] In summary, this invention, through in vivo and in vitro experiments and clinical sample verification, demonstrates that the active ingredient DST derived from Dioscorea hypoglauca can improve psoriasis-related skin lesions and inflammatory responses. It can inhibit abnormal proliferation and migration of keratinocytes by regulating the LYN / STAT3 / LRG1 signaling axis, reducing phosphorylation at the Y705 site of STAT3 protein, and decreasing LRG1 expression in keratinocytes. Simultaneously, DST can reduce LRG1-related exosome signaling from keratinocytes, inhibit macrophage ERK / CREB1 signaling activation and M1-type pro-inflammatory polarization, thereby weakening pro-inflammatory cell communication between keratinocytes and macrophages and improving the psoriasis-related immune-inflammatory microenvironment. These experimental results collectively provide experimental evidence for the use of DST in the preparation of drugs for treating psoriasis and its related mechanisms of action.

[0639] The above description is merely a preferred embodiment and specific example of the present invention, used to illustrate the technical solution and its effects, and is not intended to limit the scope of protection of the present invention. After reading this specification, those skilled in the art can make conventional adjustments, substitutions, or equivalent transformations to the experimental conditions, processing methods, detection methods, reagent types, administration methods, and other technical parameters in the embodiments according to actual needs, without departing from the basic concept and technical principles of the present invention.

[0640] For the experimental methods involved in this specification, such as cell culture, protein expression detection, nucleic acid expression detection, immunohistochemistry, immunofluorescence, flow cytometry, cell migration and invasion detection, exosome isolation and identification, omics analysis, and animal models, those skilled in the art can use conventional methods with the same or substantially the same technical purpose and detection effect to replace them, as long as the resulting technical solution is still based on the technical concept disclosed in this invention and can achieve the corresponding technical effect.

[0641] The specific cell lines, animal models, reagents, instruments, detection indicators, dosages, and treatment conditions used in this specification are primarily for illustrating the feasibility of the technical solution of this invention. Unless otherwise expressly defined in the claims, this invention should not be limited to the specific experimental materials, detection platforms, or experimental parameters listed in the embodiments.

[0642] The terms "comprising," "including," and "having" used in this specification are open-ended expressions and, unless the context explicitly specifies otherwise, do not exclude the inclusion of other components, steps, or technical features not explicitly listed. "And / or" indicates any one or any combination of two or more of the listed technical features. "At least one" indicates one or more of the listed objects.

[0643] The numerical values, concentrations, dosages, times, temperatures, and other experimental parameters mentioned in this specification should be understood as allowing for deviations within a reasonable range, provided that they conform to the usual experimental and detection errors in the art. Unless a specific numerical range is explicitly defined in the claims, a single numerical value in the embodiments should not be construed as an absolute limitation on the scope of protection of this invention.

[0644] The descriptions of the mechanisms of action in this specification, including the LYN / STAT3 / LRG1 signaling axis, STAT3 Y705 phosphorylation, LRG1-related exosomes, ERK / CREB1 signaling, and macrophage M1 polarization, serve to illustrate the experimental basis for the technical effects produced by the present invention. Equivalent regulatory methods that achieve the same or substantially the same technical effects and do not depart from the core technical concept of this invention may be determined to fall within the scope of protection of this invention based on the specific claims.

[0645] The embodiments, accompanying drawings, and experimental results listed in this specification are interconnected and serve to illustrate the technical solutions of the present invention from different perspectives. Those skilled in the art can reasonably combine the technical features described in different embodiments without creating technical contradictions, and the resulting technical solutions can also serve as specific implementations of the present invention.

[0646] It should be noted that the scope of protection of this invention is not limited to the specific embodiments described above. Any equivalent substitutions, simple transformations, conventional selections, or combinations made based on the technical content disclosed in this specification and drawings, and on the basis of the technical concept of this invention, as long as they do not substantially depart from the technical solution defined by the claims of this invention, should be determined according to the content of the relevant claims to determine whether they fall within the scope of protection of this invention.

[0647] The final scope of protection of this invention shall be determined by the scope defined in the claims. The specification and drawings may be used to interpret the meaning of the relevant technical terms and technical features in the claims.

Claims

1. The use of DST, an active ingredient derived from Dioscorea hypoglauca, in the preparation of drugs for treating psoriasis.

2. The use according to claim 1, characterized in that, The drug exerts its anti-psoriasis effect by regulating the LYN / STAT3 / LRG1 signaling axis.

3. The use according to claim 2, characterized in that, The DST reduces the phosphorylation level of tyrosine residue 705 of the STAT3 protein by inhibiting LYN-mediated signal transduction and downregulates the transcription and / or protein expression of LRG1 in keratinocytes.

4. The use according to claim 3, characterized in that, The DST reduces the level of phosphorylated STAT3 without reducing the expression level of total STAT3 protein.

5. The use according to claim 3, characterized in that, The drug inhibits the abnormal proliferation and / or migration of psoriasis-associated keratinocytes by reducing the phosphorylation level of tyrosine residue 705 of the STAT3 protein and downregulating the expression of LRG1 in keratinocytes.

6. The use according to claim 1, characterized in that, The drug reduces the epidermal thickness and / or the number of Ki-67 positive cells in psoriatic lesions, and reduces the expression and / or secretion of at least one of the pro-inflammatory factors among TNF-α, IL-1β, IL-6, IL-17A and IL-22.

7. The use according to claim 3, characterized in that, The drug reduces the LRG1 content in extracellular vesicles derived from keratinocytes by downregulating the expression of LRG1 in keratinocytes, the extracellular vesicles including exosomes.

8. The use according to claim 7, characterized in that, The drug inhibits macrophage polarization toward the M1 pro-inflammatory phenotype by reducing the content of LRG1 in keratinocyte-derived exosomes and decreasing the phosphorylation levels of ERK and CREB1 in macrophages.

9. The use according to claim 8, characterized in that, The drug reduces the expression of at least one M1 polarization-related marker among CD80, iNOS, and CD86 in macrophages, and reduces the expression of at least one pro-inflammatory factor among TNF-α and IL-6.

10. The use according to claim 8, characterized in that, The drug reduces the promoting effect of M1 macrophages on abnormal proliferation, migration and / or inflammatory response of keratinocytes by inhibiting macrophage polarization to the M1 pro-inflammatory phenotype.

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