Application of LAPTM5 molecule as target spot in developing or screening or preparing medicine for preventing and / or treating inflammatory skin diseases

Abstract

The invention relates to application of an LAPTM5 molecule as a target spot in developing or screening or preparing a medicine for preventing and / or treating inflammatory skin diseases. The invention discloses the application of the LAPTM5 as a target spot in the aspect of treating rosacea for the first time, and particularly, the LAPTM5 realizes a treatment effect by positively regulating the activity of STING protein. Although the research of the LAPTM5 in the aspect of treating cancers exists, the research of the LAPTM5 on the STING-mediated inflammation regulation is still blank, and the participation of LAPTM5 and STING signal pathways in inflammatory dermatosis is not reported, especially in the aspect of inflammation such as rosacea and the like. The molecular mechanism of the LAPTM5 for regulating and controlling the STING signal channel is clarified, and a new thought is provided for researching the pathogenesis of the skin inflammation reaction. Aiming at the defect of limited treatment effect of rosacea in the past, the treatment target provided by the invention can bring a more effective treatment scheme so as to improve symptoms and life quality of patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the use of LAPTM5 as a target in the development, screening or preparation of drugs for preventing and / or treating inflammatory skin diseases, and in particular to the use of LAPTM5 as a target in the development, screening or preparation of drugs for preventing and / or treating inflammatory skin diseases such as rosacea. Background Art

[0002] Rosacea is a common chronic inflammatory skin disease characterized by facial erythema, papules, pustules and telangiectasia. The exact cause of rosacea has not yet been fully elucidated, but it is known that a variety of factors may be related to its pathogenesis, including genetic predisposition, skin barrier dysfunction, increased vascular reactivity, microbial infection and abnormal immune response. In 2002, the National Rosacea Society (NRS) proposed a classification method that divides rosacea into four main forms, as follows: Erythematotelangiectatic type: This type of rosacea is mainly manifested by persistent erythema and vasodilation on the face, that is, the skin appears persistently red with increased visibility of small blood vessels; Papulopustular type: This subtype of rosacea is characterized by the appearance of papules and pustules on the face. These symptoms are clinically similar to acne, but usually do not cause scars; Hypertrophic type: This subtype of rosacea is characterized by thickening of the skin, which is common in the nose, leading to hyperplasia of nasal tissue, forming the so-called "rosacea"; Ocular type: This type of rosacea affects the eyes and may cause dry eyes, redness, itching or other eye discomfort. Studies have shown that patients with erythematotelangiectatic type (ETR), papule-pustular type (PPR), and hypertrophic type (PHR) have some common cell infiltration characteristics in pathology - a large number of macrophages are found infiltrating the skin lesions. Currently, commonly used treatments include topical medications (such as metronidazole, sulfonamides), oral antibiotics, laser treatment, etc. Although these methods can control inflammation and vasodilation to a certain extent, they may be accompanied by side effects and are not effective for all patients (there are significant individual differences in patients' responses to treatment, and some patients may not respond to specific treatments or have limited effects).

[0003] The pathogenesis of rosacea involves the central role of macrophages in the innate immune response. In diseased individuals and LL-37-induced mouse models, abnormal activation of dermal macrophages is accompanied by high expression of inflammatory mediators such as Toll-like receptor 2 (TLR2), kallikrein 5 (KLK-5), and antimicrobial peptide LL-37. These mediators trigger the massive production of proinflammatory factors and chemokines including IL-6, TNF-α, IL-1β, and iNOS, thereby promoting the development of inflammation (Yuan, X.; Li, J.; Li, Y.; Deng, Z.; Zhou, L.; Long, J.; Tang, Y.; Zuo, Z.; Zhang, Y.; Xie, H. Artemisinin, a Potential Option to Inhibit Inflammation and Angiogenesis in Rosacea. Biomed. Pharmacother. 2019, 117, 109-181).

[0004] Patent document with publication number CN114113630A discloses the application of SERPINB3 / B4 as a target in the treatment of inflammatory skin diseases such as rosacea. It clarifies the expression of SERPINB3 / B4 in the skin lesions of patients with rosacea and psoriasis, and the expression of Serpinb3a in the skin lesions of LL-37-induced rosacea-like mouse model and imiquimod-induced psoriasis-like mouse model by means of ELISA, immunohistochemistry, qRT-PCR, immunofluorescence and other means. The expression of Serpinb3a in mouse epidermal cells is locally knocked down by intradermal injection of siRNA, and its effect on the phenotype and histopathology of skin lesions in the inflammatory model is observed. The results show that the loss of Serpinb3a can significantly improve the development of rosacea in the LL-37-induced rosacea-like mouse model in terms of skin inflammatory response manifestations, histopathological conditions and mRNA expression. In this patent document, SERPINB3 / B4 promotes the occurrence of inflammatory response through the NF-κB signaling pathway, and does not involve the LAPTM5 and STING (Stimulator of Interferon Genes) signaling pathways at all.

[0005] The cGAS-STING signaling pathway plays a vital role in maintaining the stability of the intracellular environment, pathogen defense, and the regulation of immune responses. This pathway consists of two core proteins: cyclic guanosine monophosphate-adenylate synthetase (cGAS) and interferon gene stimulator protein (STING). When the cell is not stimulated, STING is usually located in the endoplasmic reticulum region; after activation, it moves to the Golgi apparatus and activates the autophagy mechanism. The final step of this process is to send STING to the lysosome for degradation to prevent the excessive production of inflammatory factors. Although some studies have suggested that interferon-induced transmembrane protein 3 (IFITM3) and STING have an interaction relationship identified in macrophages RAW264.7 (Motani, K., & Kosako, H. (2020). BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING). The Journal of biological chemistry, 295 (32), 11174-11183), the specific molecular mechanism is not clear.

[0006] Lysosomal-associated transmembrane protein 5 (LAPTM5) is a member of the lysosomal membrane protein family. In macrophages, LAPTM5 plays an important regulatory role. It positively regulates the NF-κB signaling pathway mediated by TLR and tumor necrosis factor receptor (TNF receptor, TNFR), promoting the production of various inflammatory factors. At present, although there are studies on LAPTM5 in the treatment of cancer, there is still a lack of research on LAPTM5's regulation of STING-mediated inflammation. There are no reports on the involvement of LAPTM5 and the STING signaling pathway in inflammatory skin diseases, especially in rosacea and other inflammatory diseases. Summary of the invention

[0007] Activation of the STING signaling pathway plays an important role in skin inflammation. It participates in the pathogenesis of a variety of skin inflammatory diseases by regulating the innate immune response. At present, skin inflammation induced by activation of the STING signaling pathway includes lupus, psoriasis, dermatomyositis and acne. There are no reports on the involvement of LAPTM5 and the STING signaling pathway in the inflammatory response of rosacea. It is unclear how LAPTM5 regulates the STING signaling pathway. The relationship between the pathway action mode and molecular mechanism involved and the inflammatory response of rosacea needs further study.

[0008] In view of the deficiencies of the prior art, the first aspect of the present invention provides the use of LAPTM5 molecules as targets in the development, screening or preparation of drugs for the prevention and / or treatment of inflammatory skin diseases.

[0009] According to a preferred embodiment, the LAPTM5 molecule participates in the inflammatory response by positively regulating the STING signaling pathway.

[0010] According to a preferred embodiment, the inflammatory skin disease can include rosacea, lupus, psoriasis and dermatomyositis.

[0011] According to a preferred embodiment, the LAPTM5 molecule can be a LAPTM5 gene or a LAPTM5 protein.

[0012] The second aspect of the present invention provides the use of a LAPTM5 antagonist or inhibitor in the preparation of a medicament for preventing and / or treating inflammatory skin diseases.

[0013] According to a preferred embodiment, the inflammatory skin disease can include rosacea, lupus, psoriasis and dermatomyositis.

[0014] According to a preferred embodiment, the LAPTM5 antagonist or inhibitor includes one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses that can inhibit the expression of LAPTM5.

[0015] According to a preferred embodiment, LAPTM5 antagonists or inhibitors alleviate and / or treat rosacea by inhibiting or reducing type I interferon and inflammatory factors mediated by STING. Preferably, LAPTM5 antagonists or inhibitors reduce the expression of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10.

[0016] According to a preferred embodiment, LAPTM5 antagonists or inhibitors relieve and / or treat the inflammatory response of rosacea by inhibiting the phosphorylation level of STING and its downstream pathways. Preferably, LAPTM5 antagonists or inhibitors relieve and / or treat the inflammatory response of rosacea by inhibiting the phosphorylation level of STING and its downstream TBK1 and IRF3.

[0017] The third aspect of the present invention provides a preparation for treating rosacea, which comprises one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses that inhibit or reduce the expression of LAPTM5 molecules, and pharmaceutically acceptable carriers thereof.

[0018] A fourth aspect of the present invention provides a carrier for delivering a LAPTM5 antagonist or inhibitor, and the carrier is preferably a liposome, a nanoparticle or a polymer matrix.

[0019] A fifth aspect of the present invention provides a method for preparing a drug containing a LAPTM5 antagonist or inhibitor, the method comprising: mixing the LAPTM5 antagonist or inhibitor with at least one excipient to form a preparation or a pharmaceutical composition; and performing appropriate physical or chemical treatment to enhance its pharmacological activity.

[0020] A sixth aspect of the present invention provides a method for treating rosacea, comprising administering to a subject an effective amount of a preparation or pharmaceutical composition containing a LAPTM5 antagonist or inhibitor.

[0021] Technical effect: The present invention reveals for the first time the application of LAPTM5 as a target in the treatment of rosacea, especially that LAPTM5 achieves the therapeutic effect by positively regulating the activity of STING protein. The present invention used the GEO gene expression database (GSE65914, GSE147950) and the GSA genome sequence database (HRA000378) to conduct preliminary research, and performed gene expression analysis on patients with rosacea and mouse models showing rosacea-like lesions, with particular attention paid to mRNA levels related to LAPTM5 and STING signaling pathways and inflammatory responses. The genes involved in the study include: monocyte-macrophage surface markers, such as CD68, CD11B, CD14 and F4 / 80, which help to identify and distinguish monocytes and macrophages; key genes in the STING signaling pathway, including CGAS, TBK1, IRF3, and NFKB1, NFKB2, RELA, RELB and REL in the NF-κB family, which play a core role in regulating immune responses and cellular stress; genes related to type I interferon signaling and inflammatory processes, such as IFNA1, IFNB1, and interferon-induced proteins IFIT1, IFIT2, IFIT3 and MX1. In addition, major pro-inflammatory cytokines such as tumor necrosis factor TNF, interleukin IL6, IL1B and CXCL10 are included, which play a key role in promoting the inflammatory process.

[0022] In the skin lesion areas of patients with rosacea, the present invention conducted an in-depth gene expression correlation analysis to explore the interaction between LAPTM5 and STING, and their association with the mRNA expression levels of monocyte-macrophage surface markers and inflammatory factors, including gene expression correlation analysis, inflammatory factor analysis, bioinformatics analysis, GSEA analysis and data set integration, aiming to evaluate the functions and pathways behind the changes in LAPTM5 and STING expression, and further reveal their potential role in the pathogenesis of rosacea.

[0023] Although the skin inflammations induced by the activation of the STING signaling pathway that have been reported so far include lupus, psoriasis, dermatomyositis and acne, whether this pathway is involved in rosacea skin inflammation has not been reported. The present invention studies the effects of HT-DNA-induced upregulation of LAPTM5 protein expression on STING protein stability and signaling pathway activation, and how these changes jointly promote the expression of inflammatory factors and inhibit inflammatory negative regulatory factors. The results of the present invention indicate that a large amount of self-DNA influx into macrophages may activate LAPTM5 protein, thereby aggravating skin inflammation by maintaining the stability of STING protein and promoting its intracellular transport. This discovery provides a new perspective for understanding the molecular pathological mechanisms of rosacea, and may provide a potential target for the development of new diagnostic and therapeutic strategies.

[0024] In particular, the interaction between LAPTM5 and STING proteins studied in the present invention provides a potential target for the development of new rosacea therapeutic drugs. In view of the shortcomings of limited treatment effects of rosacea in the past, the therapeutic targets provided by the present invention can bring more effective treatment options to improve patients' symptoms and quality of life. The treatment of specific molecules or signaling pathways in the present invention may have fewer side effects and be safer than the current extensive treatment methods. The new LAPTM5 target may help develop personalized medical strategies and customize treatment plans according to the molecular characteristics of patients. The new LAPTM5 target provides a new direction for drug development and promotes the pharmaceutical industry to develop new drugs for the treatment of rosacea. The LAPTM5 target may help with early diagnosis and intervention to prevent further aggravation of the disease; the LAPTM5 target may also help develop strategies to prevent the recurrence of rosacea and reduce the burden of long-term treatment for patients. Since rosacea is related to the immune system, the new target may help regulate the immune response and balance inflammation and immunosuppression.

[0025] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a macroscopic photo of a mouse showing that the STING inhibitor H-151 can significantly alleviate the LL-37-induced rosacea-like rash in mice;

[0027] Figure 2 HE staining results;

[0028] Figure 3 The results are immunohistochemical results;

[0029] Figure 4 The results are as follows: The protein expressions of STING and LAPTM5 in LL-37-induced rosacea-like lesions from different mice in each group and the effects of STING inhibitor H-151 on downstream pathway proteins;

[0030] Figure 5 The immunohistochemical results of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in LL-37-induced rosacea-like lesions;

[0031] Figure 6 This is the result of nuclear damage in the skin lesions of mice in the LL-37 model group;

[0032] Figure 7 The results of extracellular DNA-induced LAPTM5 protein expression and phosphorylation levels of STING, TBK1, IRF3, NF-κB p65, and LAPTM5 protein expression after LL-37 treatment;

[0033] Figure 8 Results of immunoblot analysis of LAPTM5 in skin biopsy samples of BALB / c mice injected intradermally with AAV-shLaptm5 (shRNA1, shRNA2, shRNA3) and AAV-shControl;

[0034] Fig. 9 AAV-mediated knockdown of LAPTM5 reduced STING activation and alleviated LL-37-induced rosacea-like inflammation;

[0035] Fig.10 The results show the effect of LAPTM5 on STING protein expression;

[0036] Fig.11 The result is that LAPTM5 positively regulates STING-mediated production of type I interferons and inflammatory factors;

[0037] Fig.12 The result is that LAPTM5 positively regulates STING and its downstream signaling pathways;

[0038] Fig.13 This is a 3D picture of the interaction between LAPTM5 protein and STING protein;

[0039] Fig.14 The results of co-immunoprecipitation detection of the interaction between LAPTM5 and STING to form a complex;

[0040] Fig.15 The results of immunofluorescence detection of co-localization of STING and LAPTM5. DETAILED DESCRIPTION

[0041] The following is a detailed description in conjunction with the accompanying drawings. In the accompanying drawings, "hrs" is equivalent to "hours". In the accompanying drawings, vehicle represents an empty vector, control represents a control group, and EV refers to an empty vector (Empty Vector) packaged by a lentivirus. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. In the drawings of the present invention, NS represents no statistical significance, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0042] In the LL-37 model mice, LL-37 stimulated inflammasomes in macrophages, and this activation process triggered a series of inflammatory events. The cumulative effect of these inflammatory events led to pathological changes in the mouse skin similar to those of rosacea. The presence of LL-37 in the skin lesions of patients with rosacea is the result of the combined action of multiple cell types. In addition to the keratinocytes of the epidermis, fibroblasts in the dermis and monocytes and macrophages involved in the inflammatory response are also involved in the synthesis of LL-37. The mechanism of action of LL-37 in inducing interferon production includes binding to double-stranded RNA, and this combined effect is manifested in multiple cell types, such as keratinocytes, endothelial cells, and macrophages. At the same time, the increase in LL-37 levels in the skin lesion area is accompanied by damage to nuclear DNA, and the released DNA fragments further intensify the inflammatory response.

[0043] The present invention selects 6-8 week old female BALB / c mice and randomly divides them into a control group, an LL-37 modeling group, an H-151 treatment group, and an LL-37+H-151 treatment group, with 6 mice in each group. 24 hours before the test, the hair on the back of the mice was shaved with a razor. A circle with a diameter of 1 cm was marked with a marker pen on the same part of the back of the mice as the test site. The LL-37 modeling group was subcutaneously injected with 40 μl of LL-37 (320 μM) in the circle on the back to produce a rosacea animal model, once every 12 hours, for a total of 4 times. The mice in the H-151 treatment group were intraperitoneally injected with 200 μl of H-151 (3.75 mM) (STING inhibitor), once every 12 hours, for a total of 4 times. The LL-37+H-151 treatment group was intraperitoneally injected with 200 μl of H-151 (3.75 mM) 30 min before each injection of LL-37 (320 μM). The control group was injected with PBS once every 12 h, for a total of 4 times. After the last injection of LL-37, a digital camera was used to take pictures and evaluate the erythema score and area 24 h later. After anesthesia, the skin was peeled and subsequent indicators were observed.

[0044] Example 1

[0045] This example is a method and result of detecting the phenotype and molecular indexes of mouse skin lesions.

[0046] Detection method:

[0047] 1. Mouse skin erythema scoring method: The severity of the erythema was scored from 1 to 5 points (5 points being the highest); the area of ​​erythema in the digital photos was measured using ImageJ software and then quantitatively analyzed.

[0048] 2. HE staining: After the mice were anesthetized with isoflurane, tissue blocks were taken and fixed in 10% formalin, and then gradient dehydration, wax immersion, embedding and sectioning, hydration, and staining were performed. After drying, the tissue blocks were observed under a microscope to detect the infiltration of inflammatory cells in the skin lesions.

[0049] 3. Immunohistochemistry: sample processing, permeabilization, blocking, primary antibody incubation, secondary antibody incubation, color development or fluorescence detection, microscopic observation after sealing. Immunofluorescence: sample processing, permeabilization, blocking, antibody incubation, washing and sealing, fluorescence microscopic observation. Immunohistochemistry and immunofluorescence detection of LAPTM5, STING and macrophage surface marker molecule F4 / 80 protein expression levels and co-localization.

[0050] 4. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) detection: sample preparation, fixation, permeabilization, washing, incubation, termination reaction, washing, nuclear staining, sealing, fluorescence microscopy observation and analysis of nuclear DNA damage and distribution in tissues.

[0051] 5.Western blot was used to detect the expression of LAPTM5 and STING proteins and the activation of their downstream signaling proteins, specifically the phosphorylation levels of TBK1, NF-κB and IRF3 proteins.

[0052] 6.qRT-PCR was used to detect the expression of inflammatory factors IFNβ, IL6, TNFα, IL1β and CXCL10.

[0053] Test results:

[0054] 1. Effect of STING antagonist H-151 on LL-37-induced rosacea-like rash in mice Figure 1 As shown, Figure 1 A is a general photo of mice; Figure 1 B is a dermatoscopic photo of mice; Figure 1 C is the statistical result of erythema area; Figure 1 D is the erythema score result. p < 0.05 indicates statistical significance.

[0055] Compared with the control group, the local skin of the LL-37 modeling group formed rosacea-like rashes accompanied by pustules, while the erythema area of ​​the LL-37+H-151 treatment group was significantly smaller than that of the LL-37 modeling group, the pustular symptoms were improved, and the severity of the erythema was reduced ( Figure 1 A~ Figure 1 D). The results suggest that the STING antagonist treatment group (LL-37+H-151 treatment group) can alleviate the rosacea-like rash erythema in mice, which may work by inhibiting the release of inflammatory mediators or reducing the activity of inflammatory cells. At the same time, STING antagonists may help restore or enhance the skin barrier, thereby alleviating erythema and pustule symptoms.

[0056] 2. HE staining results are as follows Figure 2 As shown, Figure 2 A is a HE staining picture; Figure 2 B is the statistical result of cell infiltration number / HPF (High Power Field); Figure 2 C is the statistical result of epidermal thickness. Figure 2 In the figure, scale bar = 100 μm.

[0057] Compared with the control group, the epidermal thickness of the LL-37 model group was significantly thickened, and the dermal inflammatory cell infiltration was significantly increased ( Figure 2 A~ Figure 2 C), however, compared with the LL-37 modeling group, the epidermal thickness of the LL-37+H-151 treatment group was significantly reduced and the dermal inflammatory cell infiltration was improved, indicating that STING antagonists may reduce skin inflammation, promote healing or reduce cell proliferation, help reduce the severity of skin lesions, and promote the recovery of skin structure.

[0058] 3. Immunohistochemical results of the expression of macrophage marker molecule F4 / 80 Figure 3 shown.

[0059] Compared with the control group, the expression level of macrophage marker molecule F4 / 80 in the skin lesions of mice in the LL-37 model group was significantly higher than that in the control group, and the expression levels of LAPTM5 and STING in the skin lesions of mice in the LL-37 model group were significantly higher than those in the control group, indicating that LAPTM5 and STING are involved in the inflammatory response in the mouse rosacea model.

[0060] 4. Western blot results are as follows Figure 4 As shown, Figure 4 A shows the expression changes of STING and LAPTM5 in the skin lesions of mice in the LL-37 model group; Figure 4 B shows the phosphorylation levels of STING and its downstream pathways.

[0061] Compared with the control group, the protein expression of STING and LAPTM5 in the skin lesions of mice in the LL-37 model group was increased ( Figure 4 A). Compared with the LL-37 model group, the LL-37+H-151 treatment group showed increased phosphorylation levels of STING and its downstream pathways, specifically increased expression of pSTING, pIRF3, IRF3, pNF-κB p65, NF-κB p65, and LAPTM5 ( Figure 4 B).

[0062] 5. Immunohistochemistry results Figure 5 As shown, Figure 5 A shows the expression changes of IFN-β and IL-1β in rosacea-like lesions; Figure 5 B shows the expression changes of IL-6, TNF-α, and CXCL-10 in rosacea-like lesions.

[0063] Compared with the control group, the expression levels of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in the skin lesions of mice in the LL-37 modeling group were significantly increased, while the expression levels of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in the skin lesions of mice in the LL-37+H-151 treatment group were significantly decreased compared with the LL-37 modeling group ( Figure 5 A and Figure 5B), H-151 inhibits the expression of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in LL-37-induced rosacea-like rash in mice. IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 are important signaling molecules in the immune system, involved in regulating immune responses and inflammatory processes. The changes in the expression of these cytokines and chemokines prove that STING antagonists (H-151) help reduce inflammation, control the inflammatory process, and may also participate in the immunosuppressive process.

[0064] 6. Figure 6 The results are for the expression of broken dsDNA fragments in rosacea-like rash in mice.

[0065] Compared with the control group, the positive signal intensity in the skin lesions of mice in the LL-37 model group was greater, and the dsDNA positive cell count was significantly increased compared with the control group. This result indicates that the response to DNA damage after LL-37 treatment is more significant, and the level of cell apoptosis is increased.

[0066] Example 2

[0067] In this example, relevant research was conducted at the cellular level to obtain the molecular mechanism by which LAPTM5 regulates the activation of the STING signaling pathway.

[0068] 1. Figure 7 is the expression of LAPTM5 protein induced by extracellular DNA, among which, Figure 7 A is the expression results of STING and LAPTM5 in macrophages RAW264.7 and fibroblasts L929; Figure 7 B is the expression levels of STING, LAPTM5 and LC3B after HT-DNA and liposomes acted together on macrophages RAW264.7 and fibroblasts L929.

[0069] Figure 7 A shows that STING is highly expressed in macrophages RAW264.7 and fibroblasts L929; LAPTM5 is highly expressed in macrophages RAW264.7 and lowly expressed in fibroblasts L929. Figure 7B The results showed that as the co-action time of HT-DNA and liposomes increased (0 to 7 hours), the expression of STING protein decreased in macrophages RAW264.7 and fibroblasts L929, the expression of LAPTM5 increased, and the expression of LC3B gradually increased, reaching a peak and then decreasing. LC3B is a key protein in the autophagy process, and its increased expression is usually associated with the activation of autophagy. After activation, STING may be transformed into other forms (such as phosphorylated form) to activate the autophagy pathway, and STING protein is degraded, suggesting that HT-DNA activates STING and induces autophagy.

[0070] Ht-DNA (extracellular DNA) can induce upregulation of LAPTM5 protein expression ( Figure 7 B). Extracellular DNA may induce a stress response in cells, and this stress state may increase the expression of LAPTM5 through stress-related signaling pathways. On the other hand, extracellular DNA may indirectly lead to the upregulation of LAPTM5 protein expression by affecting the expression of autophagy-related genes. In addition, extracellular DNA may also activate specific signal transduction pathways by binding to receptors on the cell surface, thereby affecting the transcriptional activity of the LAPTM5 gene and leading to the upregulation of its protein expression level.

[0071] Mouse fibroblasts L929 and macrophages RAW264.7 were treated with different concentrations of LL-37, and the phosphorylated levels of STING (pSTING), TBK1 (pTBK1), IRF3 (pIRF3), NF-κB p65 (pNF-κB p65) and LAPTM5 protein expression were detected at 24 hours.

[0072] 2. The experimental method and test results of knocking down LAPTM5 are as follows.

[0073] The preparation of LAPTM5 knockdown animal model is as follows:

[0074] Female BALB / c mice, 5 weeks old, were selected; AAV (adeno-associated virus) was injected locally at a single point on the back to establish a BALB / c mouse knockout LAPTM5 mouse model; a circle with a diameter of 1 cm was marked with a marker on the same part of the back of the mouse as the test site. The mice were randomly divided into 4 groups (6 mice in each group), including: PBS+empty vector group, PBS+rAAV-shLAPTM5 (rAAV-shLAPTM5 means knockout LAPTM5) group, LL-37+empty vector group and LL-37+rAAV-shLAPTM5 group. The LL-37+empty vector group and the LL-37+rAAV-shLAPTM5 group were subcutaneously injected with 40μl of LL-37 (320μM) in the back circle, once every 12h, for a total of 4 times. The following tests were performed 24 hours after the last injection: ① A digital camera was used to take gross photos of the skin to observe the clinical manifestations of the area and degree of erythema; ② A dermatoscope was used to observe clinical manifestations such as skin erythema, capillary dilation, pustules, and crusting; ③ HE staining was used to observe the thickness of the epidermis and the infiltration of inflammatory cells in the dermis; ④ Immunohistochemistry was performed to observe the expression changes of F4 / 80, IL-1β, TNF-α, IFN-β, IL-6, and CXCL10. This example uses an adeno-associated virus type 9 (AAV9) vector carrying a short hairpin RNA (shRNA) targeting LAPTM5. The Laptm5 shRNA sequence was synthesized and cloned into the pHBAAV-U6-MCS vector (see Table 1 for the shRNA sequence). 21 days after AAV injection, immunoblotting analysis was used to evaluate the effect of Laptm5 gene knockdown, as shown in Figure 1. Figure 8 Laptm5shRNA1 (SEQ.ID.NO: 1) was used in subsequent experiments.

[0075] Table 1 shRNA sequences

[0076]

[0077] Fig. 9 The results show that AAV-mediated knockdown of LAPTM5 gene reduces STING activation and alleviates LL-37-induced rosacea-like inflammation. n = 6 per group. All data are expressed as mean ± standard error. Two-tailed unpaired Student's t test was used. Fig. 9 A is a schematic diagram of intradermal injection of AAV-shLaptm5 in BALB / c mice 21 days before LL-37 injection; Fig. 9 B is a representative image of the macroscopic appearance of the skin of mice in different groups 12 hours after the last LL-37 injection; Fig. 9 C is a representative polarized dermatoscopic image of the mouse skin in different groups; Fig. 9 D is the erythema area of ​​mice in different groups; Fig. 9E is the severity score of rosacea phenotype in different groups of mice; Fig. 9 F is HE staining of lesion skin sections of mice in different groups (the lower panel is an enlarged view of the boxed area. Scale bar: 100 μm); Fig. 9 G is the quantitative result of HE staining; Fig. 9 H is IHC staining of pSTING and STING in skin sections (the right panel is an enlarged view of the boxed area. Scale bar: 100 μm); Fig. 9 I is the quantitative analysis result of IHC staining intensity (IOD / area) of pSTING in skin sections; Fig. 9 J is the quantitative analysis result of IHC staining intensity (IOD / area) of STING in skin sections; Fig. 9 K is the IHC staining results of TNF-α, IL-6, IL-1β, IFN-β, and CXCL-10 in skin sections (the right panel is an enlarged view of the boxed area. Scale bar: 100 μm); Fig. 9 L is the quantitative analysis result of IHC staining intensity (IOD / area) of TNF-α in skin sections; Fig. 9 M is the quantitative analysis result of IHC staining intensity (IOD / area) of IL-6 in skin sections; Fig. 9 N is the quantitative analysis result of the IHC staining intensity (IOD / area) of IL-1β in skin sections; FIG. 90 is the quantitative analysis result of the IHC staining intensity (IOD / area) of IFN-β in skin sections; Fig. 9 P is the quantitative analysis result of IHC staining intensity (IOD / area) of CXCL-10 in skin sections.

[0078] Compared with the control group, the LAPTM5 gene knockdown group (AAV-shLAPTM5) showed a significant decrease in the erythema area and severity of the erythema score on the mouse skin ( Fig. 9 B~9E). Compared with the control group, the number of dermal infiltrating cells (the number of inflammatory cells infiltrating the dermis) in the AAV-shLAPTM5 group was significantly reduced ( Fig. 9 G). Compared with the control group, the IHC staining intensity (IOD / area) of pSTING and STING in the AAV-shLAPTM5 group was significantly reduced ( Fig. 9 H~9J). Compared with the control group, the skin section IHC staining of TNF-α, IL-6, IL-1β, IFN-β, and CXCL-10 and the IHC staining intensity (IOD / area) in the AAV-shLAPTM5 group were significantly reduced ( Fig. 9 K, Fig. 9 L~9P).

[0079] Therefore, AAV-mediated knockdown of LAPTM5 reduced STING activation and alleviated LL-37-induced rosacea-like inflammation.

[0080] Fig.10 The results show the effect of LAPTM5 on STING protein expression, among which, Fig.10 A is the change of STING protein expression detected after macrophage RAW264.7 cells were infected with LAPTM5shRNA lentivirus; Fig.10 B shows the changes in STING and LC3B protein expression detected after fibroblast L929 cells were infected with LAPTM5 overexpression lentivirus at different MOI values; Fig.10 C shows the expression of STING protein by Western blot after RAW264.7 macrophages were infected with LAPTM5shRNA lentivirus and the cells were treated with lysosomal inhibitor BafA1 and proteasome inhibitor MG132 overnight. The cells were then stimulated with DMXAA at 0h and 2h.

[0081] After knocking down LAPTM5, STING protein expression decreased ( Fig.10 A). The MOI value indicates the average number of virus particles that each host cell is exposed to during the infection process. Cells infected with viruses of different MOI values ​​can result in different levels of LAPTM5 overexpression. After overexpression of LAPTM5, STING protein expression increased, suggesting that LAPTM5 can promote STING protein stability. At the same time, the expression of the autophagy marker LC3B protein also increased ( Fig.10 B), indicating that the STING pathway is activated and autophagy is initiated. DMXAA, as a mouse STING agonist, can activate the STING pathway and has an effect on mouse STING. When macrophage RAW264.7 is not stimulated with DMXAA, the proteasome inhibitor MG132 can restore STING protein expression; after macrophage RAW264.7 is stimulated with DMXAA, the lysosome inhibitor BafA1 can restore STING protein expression ( Fig.10C). This result indicates that before and after STING activation, the degradation of STING protein caused by down-regulation of LAPTM5 expression depends on different degradation pathways. Specifically, before STING activation, the degradation of STING protein caused by down-regulation of LAPTM5 expression depends on the proteasome degradation pathway; after STING activation, the degradation of STING protein caused by down-regulation of LAPTM5 expression depends on the lysosomal degradation pathway. STING may have undergone structural or functional changes before and after activation, resulting in its transition from proteasome-dependent degradation to lysosomal-dependent degradation. STING activation may be accompanied by its relocalization in the cell, such as transfer from the cytoplasm to the endoplasmic reticulum or Golgi apparatus, and then transported to the lysosome for degradation. The activated STING protein may have acquired new modifications (such as phosphorylation, ubiquitination or glycosylation), which may instruct the cell to degrade through different pathways. The activation of STING may trigger specific cell signaling pathways, which may promote the interaction of STING protein with lysosomes, thereby promoting its degradation. The activated STING protein may be encapsulated in the autophagosome, and then fuse with the lysosome to form an autophagolysosome for degradation.

[0082] This dual degradation mechanism of STING protein provides cells with a fine-grained regulatory method to ensure that STING protein can be effectively and appropriately processed in different states. Cells can precisely regulate the stability and function of STING protein according to its activation state to ensure the accuracy of signal transduction. Cells respond adaptively to external stimuli or changes in internal demand, and achieve functional changes by adjusting the degradation pathway of STING protein. Understanding the degradation mechanism of STING protein can reveal new targets for disease treatment, especially when STING protein is closely related to disease development. By targeting specific degradation pathways of STING protein, new drugs can be developed to treat related diseases, such as regulating the level of STING protein by enhancing or inhibiting specific degradation pathways. The degradation mechanism of STING not only reflects the complexity of internal cell regulation, but also provides new perspectives and strategies for biomedical research and the development of rosacea treatment drugs.

[0083] 3. The results of LAPTM5 positively regulating STING-mediated production of type I interferons and inflammatory factors are as follows Fig.11 As shown, Fig.11 A: Macrophage RAW264.7 cells were infected with LAPTM5shRNA lentivirus, and the cells were stimulated with Ht-DNA for 3 hours. The mRNA expression changes of type I interferons (IFN-β, IFIT 1) and inflammatory factors (TNF-α, IL-6, CXCL10) were detected by RT-qPCR; Fig.11B: Macrophage RAW264.7 cells were infected with LAPTM5shRNA lentivirus, and the cells were stimulated with DMXAA for different time periods to detect the mRNA expression changes of type I interferons (IFN-β, IFIT 1) and inflammatory factors (TNF-α, IL-6, CXCL10) by RT-qPCR; Fig.11 C is the fibroblast L929 cells infected with LAPTM5 overexpressing lentivirus, and the cells were stimulated with HT-DNA for 3 hours, and the mRNA expression changes of type I interferons (IFN-β, IFIT 1) and inflammatory factors (TNF-α, IL-6, CXCL10) were detected by RT-qPCR; Fig.11 D is the fibroblast L929 cells infected with LAPTM5 overexpressing lentivirus, and the cells were stimulated with DMXAA for 3 hours, and the mRNA expression changes of type I interferons (IFN-β, IFIT 1) and inflammatory factors (TNF-α, IL-6, Cxcl 10) were detected by RT-qPCR. Rel.ifnβmRNA level refers to the relative quantitative mRNA level of interferon β. Rel.Ifit 1 mRNA level refers to the relative quantitative mRNA level of Ifit 1. Rel.TNF-α mRNA level refers to the relative quantitative mRNA level of TNF-α. Rel.IL-6 mRNA level refers to the relative quantitative mRNA level of IL-6. Rel.Cxcl 10 mRNA level refers to the relative quantitative mRNA level of Cxcl 10. After knockdown of LAPTM5 in RAW264.7 macrophages, the expression of type I interferons (IFN-β, IFIT 1) and inflammatory factors (TNF-α, IL-6, CXCL10) induced by HT-DNA and DMXAA was significantly decreased ( Fig.11 A and Fig.11 B). Overexpression of LAPTM5 in L929 fibroblasts significantly increased the expression of type I interferons (IFN-β, IFIT 1) and inflammatory factors (TNF-α, IL-6, CXCL10) induced by T-DNA and DMXAA ( Fig.11 C and Fig.11C). These results indicate that LAPTM5 positively regulates STING-mediated production of type I interferon and inflammatory factors. Specifically, LAPTM5 may encode a molecule that positively regulates the expression of type I interferon and inflammatory factors. After knocking down the LAPTM5 gene, signal transduction was weakened, resulting in reduced expression of these factors. Conversely, overexpression of the LAPTM5 gene may enhance signal transduction, thereby increasing the expression of these factors. LAPTM5 may affect the stability of type I interferon and inflammatory factor mRNA. Knocking down the LAPTM5 gene may reduce the stability of mRNA, while overexpression may increase the stability of mRNA. LAPTM5 may also directly affect the transcription of type I interferon and inflammatory factor genes. Knocking down the LAPTM5 gene may reduce the transcriptional activation of these genes, while overexpression of the LAPTM5 gene may increase transcriptional activation. LAPTM5 plays a key role in regulating the expression of type I interferon and inflammatory factors mediated by STING.

[0084] 4. The results of LAPTM5 positively regulating STING and its downstream signaling pathways are as follows Fig.12 As shown, Fig.12 A: Macrophage RAW264.7 cells were infected with LAPTM5shRNA lentivirus, and the cells were stimulated with DMXAA for different time periods. Western blot was used to detect the expression changes of STING and its downstream signaling protein phosphorylation levels; Fig.12 B: Macrophage RAW264.7 cells were infected with LAPTM5shRNA lentivirus, and the cells were stimulated with HT-DNA for different time periods. Western blot was used to detect the expression changes of STING and its downstream signaling protein phosphorylation levels; Fig.12 C is L929 cells infected with LAPTM5 overexpressing lentivirus, and the cells were stimulated with DMXAA for different time periods, and Western blot was used to detect the expression changes of STING and its downstream signaling protein phosphorylation levels; Fig.12 D: L929 cells were infected with LAPTM5 overexpression lentivirus, and the cells were stimulated with HT-DNA for different time periods. Western blot was used to detect the expression changes of STING and its downstream signaling protein phosphorylation levels.

[0085] After knocking down LAPTM5 in macrophage RAW264.7 cells, the phosphorylation levels of STING and its downstream TBK1 and IRF3 were inhibited, as shown in the following Fig.12 A and Fig.12 The expression levels of pSTING, pTBK1, and pIRF3 in B were reduced. After LAPTM5 was overexpressed in fibroblast L929, the phosphorylation levels of STING and its downstream TBK1 and IRF3 were enhanced, as shown in Fig.12 C and Fig.12The expression of pSTING, pTBK1, and pIRF3 in D increased. In addition, overexpression of LAPTM5 enhanced STING's induction of autophagy. Enhanced autophagy induction can help cells remove damaged or aged organelles and protein aggregates and maintain the cleanliness of the internal cell environment. Autophagy can remove intracellular pathogens, such as certain viruses and bacteria, thereby enhancing the cell's ability to resist infection. Autophagy is involved in regulating the activity of immune cells, including the function of antigen-presenting cells, and contributes to the stability and development of the immune system.

[0086] 5. The results that LAPTM5 protein and STING protein are interacting proteins are as follows Fig.11 shown.

[0087] AlphaFold2 was used to predict the crystal structures of LAPTM5 and STING proteins in the validation process. The protein crystals were processed by Protein Preparation Wizard of Schrödinger software for protein preprocessing, regenerate states of native ligand, H-bond assignment optimization, protein energy minimization, and remove waters. Next, protein-protein interaction simulation was performed using the protein-protein docking module of the software. During this simulation, the "Number of ligand rotations to probe" was set to 70,000, and the "Maximum poses to return" was set to 30. According to the simulation results, the top-ranked pose is the second pose (pose 2), with a PIPER pose score of -930.222 and a PIPER pose energy of -1774.039 kcal / mol. The binding free energy of this pose is lower than all other poses, indicating that this is the most stable configuration for the two proteins to bind in 70,000 calculations. The PIPER pose energy is less than -500 kcal / mol, indicating that the binding between LAPTM5 and STING protein is relatively strong and tight.

[0088] Fig.13This is a 3D stereoscopic picture of LAPTM5 (yellow) and STING (blue) combined. The picture shows that LAPTM5 protein and STING protein can be stably combined, and LAPTM5's GLU176 forms a hydrogen bond with STING's GLN334, LAPTM5's PHE74 forms a π-π bond and 11 van der Waals forces with STING's HIS94, and LAPTM5's GLU42 forms a hydrogen bond and a salt bridge with STING's LYS150. Therefore, LAPTM5 and STING are interacting proteins.

[0089] 6. The results of immunoprecipitation detection of the interaction between LAPTM5 and STING to form a complex are as follows Fig.14 shown. Fig.14 A is the immunoprecipitation detection of the interaction between endogenous STING and LAPTM5 in macrophage RAW264.7. This result is the Western blot detection of the expression of LAPTM5, TBK1 and pSTING after DMXAA stimulated macrophage RAW264.7 for different action time. Fig.14 In A, STING antibody was used for immunoprecipitation, and IgG was used as a negative control. Fig.14 B is the fibroblast L929 cells infected with lentivirus overexpressing LAPTM5 with FLAG tag. After stimulation with cGAMP for different time periods, the cells were collected to extract total protein and FLAG was detected by Western blot. Fig.14 In B, STING antibody was used for immunoprecipitation, and IgG was used as a negative control. Fig.14 C is the fibroblast L929 cells infected with FLAG-tagged LAPTM5 overexpression lentivirus. After stimulation with cGAMP for different time periods, the cells were collected to extract total protein and the expression levels of STING and TBK1 were detected by Western blot. Fig.14 In C, immunoprecipitation was performed using a FLAG tag antibody.

[0090] Before and after DMXAA stimulation and cGAMP stimulation, STING and LAPTM5 can form an interactive complex ( Fig.14 A~ Fig.14 C). No co-precipitation of LAPTM5 was observed in the IgG control group, demonstrating the specificity of the interaction between STING and LAPTM5. STING pathway activation and the interaction between STING and LAPTM5 proteins are time-dependent ( Fig.14 C), TBK1 is a kinase associated with the STING pathway and can be used to assess the activation of the STING pathway. Fig.14In C, TBK1 expression increased over time, and enhanced interaction between STING and LAPTM5 was observed within the time window of STING pathway activation, which may indicate that activation of the STING pathway promotes this interaction.

[0091] 7. The results of co-localization of STING and LAPTM5 using immunofluorescence are as follows Fig.15 As shown, Fig.15 A is an immunofluorescence image of co-localization of STING and LAPTM5; Fig.15 B shows the co-localization of LAPTM5 and STING in the perinuclear region, quantified by Pearson's correlation coefficient. Fig.15 In the figure, scale bar = 10 μm.

[0092] Macrophage RAW264.7 cells were stimulated with DMXAA (50 μg / ml), and the co-localization of LAPTM5 and STING before and after stimulation was observed using a laser confocal microscope. Compared with the control group, after DMXAA stimulation of macrophage RAW264.7 cells, the co-localization of LAPTM5 and STING in the cells increased ( Fig.15 A), indicating that LAPTM5 and STING have a stronger correlation in spatial distribution in cells, that is, the two have synergistic biological functions or participate in common cellular processes. The increase in the Pearson correlation coefficient shows that the correlation between the distribution of LAPTM5 and STING proteins in cells has increased. As the expression or distribution of one protein increases, the other protein also shows a similar trend ( Fig.15 B). DMXAA stimulation increased the co-localization of LAPTM5 and STING, indicating that the two proteins interacted directly or indirectly in the cell, and this interaction was activated or enhanced after DMXAA stimulation. The increase in the co-localization of LAPTM5 and STING may reveal the molecular mechanism of action of drugs used to treat rosacea, and provide a basis for the evaluation and optimization of drug effects.

[0093] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", both of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as being required. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. Use of LAPTM5 molecule as a target in the development, screening or preparation of drugs for the prevention and / or treatment of inflammatory skin diseases.

2. The use according to claim 1, characterized in that: LAPTM5 molecules participate in inflammatory response by positively regulating the STING signaling pathway.

3. The use according to claim 1 or 2, characterized in that: Inflammatory skin diseases can include rosacea, lupus, psoriasis, and dermatomyositis.

4. Use of LAPTM5 antagonists or inhibitors in the preparation of drugs for preventing and / or treating inflammatory skin diseases.

5. The use according to claim 4, characterized in that: LAPTM5 antagonists or inhibitors include one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses that can inhibit the expression of LAPTM5.

6. The use according to claim 4 or 5, characterized in that: LAPTM5 antagonists or inhibitors alleviate and / or treat rosacea by inhibiting or reducing STING-mediated production of type I interferon and inflammatory factors.

7. The use according to claim 4 or 5, characterized in that: LAPTM5 antagonists or inhibitors alleviate and / or treat the inflammatory response of rosacea by inhibiting the phosphorylation level of STING and its downstream pathways.

8. A preparation for treating rosacea, characterized in that: The preparation includes one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses that inhibit or reduce the expression of LAPTM5 molecules, and pharmaceutically acceptable carriers thereof.

9. A carrier, characterized in that The carrier is used to deliver the LAPTM5 antagonist or inhibitor for preventing and / or treating inflammatory skin diseases, and the carrier is preferably a liposome, nanoparticle or polymer matrix.

10. A method for preparing a medicament containing a LAPTM5 antagonist or inhibitor for preventing and / or treating inflammatory skin diseases, characterized in that: The method comprises: mixing the LAPTM5 antagonist or inhibitor with at least one excipient to form a preparation or a pharmaceutical composition; and performing appropriate physical or chemical treatment to enhance its pharmacological activity.

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