Application of STING gene in skin injury repair

Abstract

This invention relates to the application of the STING gene in the preparation of formulations for rosacea. The formulations have one of the following functions: treatment of rosacea; evaluation of the efficacy of rosacea treatment. Another aspect of this application relates to the application of STING gene inhibitors in the preparation of drugs for treating rosacea. Based on relevant experimental data, it is known that downregulation of STING gene expression can alleviate the symptoms of rosacea, reduce the redness area, and LAPTM5 can maintain the stability of the STING protein and positively regulate the activation of the STING signaling pathway and the expression of inflammatory factors mediated by it. Therefore, LAPTM5 gene inhibitors can also be used to alleviate the symptoms of rosacea.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and further to the field of immunology, particularly to the application of the STING gene in skin damage repair. Background Technology

[0002] Rosacea is a chronic inflammatory skin disease that commonly affects the central face, primarily involving facial blood vessels and the peri-follicular and sebaceous gland units. Symptoms include facial flushing, erythema, papules, pustules, and telangiectasia. Histopathological examination of rosacea reveals various inflammatory cell infiltrations in the dermis, accompanied by increased sebaceous glands, enlarged glands, subcutaneous connective tissue hyperplasia, and vasodilation.

[0003] In 2002, the National Rosacea Society (NRS) classified rosacea into four subtypes: erythema telangiectatic rosacea (ETR), papulopustular rosacea (PPR), phymatous rosacea (PHR), and ocular rosacea. In the first three subtypes, a large number of macrophages were found to infiltrate the skin lesions of patients with clinical subtypes, which also indicates that rosacea is related to inflammation.

[0004] In the pathogenesis of rosacea, macrophage-mediated innate immune responses play a central role. Related studies have shown that abnormal activation of dermal macrophages was observed in skin lesions from rosacea patients and LL-37 model mice. Furthermore, these lesions exhibited high expression of pro-inflammatory factors, including Toll-like receptor 2 (TLR2), kallikrein-5 (KLK-5), and the antimicrobial peptide LL-37. This resulted in the production of large amounts of pro-inflammatory and chemokine-like factors, including interleukin 6 (IL6), tumor necrosis factor α (TNFα), interleukin 1β (IL1β), and inducible nitric oxide synthase (iNOS), leading to an inflammatory response. Currently, publicly available experimental data show that inhibiting the activation of the nuclear factor-κB (NF-κB) signaling pathway can effectively control the inflammatory response and improve the pathological symptoms of rosacea.

[0005] Even though there are clear reports that dermal fibroblasts, as the main cell type in the dermis, can produce various inflammatory factors through autocrine, paracrine, and intercellular interactions, regulating the function of keratinocytes and macrophages, and thus modulating the skin's inflammatory response, the mechanism by which the innate immune response, which plays a crucial role in the pathogenesis of rosacea, plays a role in rosacea remains undiscovered. This is because rosacea patients often present with multiple clinical features simultaneously, and there are no absolute boundaries between different subtypes. Consequently, the current technology has failed to find an effective treatment for rosacea plagued by inflammation that can inhibit the activation of T and B lymphocytes and macrophages.

[0006] Furthermore, 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 this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0007] This invention belongs to the field of immunology, specifically relating to the application of the STING gene (Gene ID: 72512, https: / / www.ncbi.nlm.nih.gov / gene / ?term=72512) as a biomarker in the preparation of formulations for rosacea. Preferably, the STING gene sequence is as shown in SEQ ID NO.1. Preferably, the transcript of the STING gene has an Accession Number of NM_028261.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_028261.1).

[0008] This application, in another respect, relates to the use of the STING gene (Gene ID: 340061, https: / / www.ncbi.nlm.nih.gov / gene / ?term=340061) as a biomarker in the preparation of formulations for rosacea. Preferably, the STING gene sequence is as shown in SEQ ID NO.3. Preferably, the transcript of the STING gene is Accession Number: NM_198282.4 (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_198282.4).

[0009] In this application, the STING gene sequence, the STING gene transcript sequence, the LAPTM5 gene sequence, and the LAPTM5 gene transcript sequence were all obtained from the NCBI data website.

[0010] According to a preferred embodiment, the above-mentioned preparation has one of the following functions: treating rosacea; evaluating the efficacy of rosacea treatment.

[0011] According to a preferred embodiment, the criterion for evaluating efficacy is that downregulation of the STING gene expression represents remission of rosacea.

[0012] Another aspect of this application relates to the use of STING gene inhibitors in the preparation of medicaments for treating rosacea.

[0013] This application, in another respect, relates to the use of H-151 in the preparation of a medicament for treating rosacea.

[0014] This application, in another aspect, relates to a medicament for treating rosacea. The medicament for treating rosacea comprises H-151 at a concentration of 1–100 mM. Preferably, the medicament for treating rosacea comprises H-151 at a concentration of 1–5 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 10–20 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 2 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 5 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 10 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 15 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 20 mM. More preferably, the medicament for treating rosacea comprises H-151 at a concentration of 3 mM. More preferably, the drug for treating rosacea contains H-151 at a concentration of 4 mM. More preferably, the drug for treating rosacea contains H-151 at a concentration of 3.75 mM. The dosage of the STING gene inhibitor is designed based on a specific analysis of the patient's condition.

[0015] According to a preferred embodiment, the dosage form of the STING gene inhibitor includes one or more of oral, injectable, or topical applications. Preferably, topical application includes a dressing or liquid spray. The method of administering a topical STING gene inhibitor involves applying the STING gene inhibitor to areas exhibiting the rosacea phenotype. The method of using or administering the STING gene inhibitor is designed based on a specific analysis of the patient's condition.

[0016] According to a preferred embodiment, the method of administering or using the STING gene inhibitor includes: administering or using the STING gene inhibitor after the patient develops erythema on the skin.

[0017] According to a preferred embodiment, the STING gene inhibitor comprises at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies.

[0018] According to a preferred embodiment, the STING gene inhibitor comprises shRNA for silencing the STING gene.

[0019] According to a preferred embodiment, the STING gene inhibitor comprises one or more of H-151, B-12019, C-176, C-178, and G150.

[0020] According to a preferred embodiment, the STING gene inhibitor comprises an agent that inhibits the expression of the LAPTM5 gene (Gene ID: 16792, https: / / www.ncbi.nlm.nih.gov / gene / ?term=16792). Preferably, the transcript of the LAPTM5 gene has an Accession Number of NM_010686 (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_010686).

[0021] According to a preferred embodiment, the STING gene inhibitor comprises an agent that inhibits the expression of the LAPTM5 gene (Gene ID: 7805, https: / / www.ncbi.nlm.nih.gov / gene / ?term=7805). Preferably, the transcript of the LAPTM5 gene has an Accession Number of NM_006762.3 (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_006762.3).

[0022] Another aspect of this application relates to the use of STING gene inhibitors in the preparation of drugs or skin care products for skin damage repair.

[0023] According to a preferred embodiment, the skin lesions include rashes and / or pustules caused by rosacea.

[0024] The beneficial effects of this technical solution are:

[0025] Inflammation is one of the main triggers for facial diseases. The facial condition, the type of disease, and the characteristics of the disease vary significantly depending on the skin environment and the underlying causes. For example, acne, which involves blockage of the pilosebaceous unit, excessive sebum production, bacterial infection (primarily Propionibacterium acnes), and inflammatory responses, utilizes the cGAS-STING pathway to exert its influence on the inflammatory response in acne (especially in sensing bacterial DNA and promoting the production of inflammatory factors).

[0026] This application primarily explores and provides the application of the STING gene and its inhibitors in alleviating the symptoms of rosacea. Rosacea is characterized by facial flushing, vasodilation, and the formation of papules and pustules. While the pathogenesis of rosacea is not fully understood, unlike acne which is influenced by the skin environment, abnormalities in the immune system, impaired skin barrier function, and alterations in the microbiome are considered key factors. During in-depth research into the cGAS-STING pathway, the inventors unexpectedly discovered that this pathway participates in the inflammatory response of rosacea. Distinguished from the pathogenesis of acne, it regulates the activity of inflammatory factors on the skin of patients with rosacea by forming a dynamically regulated signaling pathway with LAPTM5. Specifically, when the STING gene is inhibited, the release of inflammatory factors is reduced, the inflammatory response is alleviated, and it produces a significant alleviating effect on rosacea.

[0027] Currently, the pathogenesis and molecular pathways of rosacea are not fully understood. Therefore, this invention discovers, on the one hand, the signaling pathway involved in the STING gene and the activation of its synergistic gene LAPTM5, which induces rosacea. Specifically, when the skin is stimulated, it releases excessive amounts of LL-37, breaking the body's immune tolerance. LL-37 binds to the body's own DNA, activating the cGAS-STING-TBK1-IRF3 / NFκB signaling pathway in macrophages, producing a large number of inflammatory factors, leading to rosacea. Simultaneously, a large amount of the body's own DNA enters macrophages, promoting increased expression of LAPTM5 protein. LAPTM5 protein exacerbates the skin inflammatory response by maintaining the stability of STING protein and regulating the intracellular transport of STING (Example 3).

[0028] This invention explores the mechanism of action of the STING gene at the molecular level, discovering that its signaling pathway directly participates in the inflammatory response of rosacea (Example 1). Furthermore, this invention confirms through the administration of STING gene inhibitors to model mice that the inhibition of the STING gene can directly alleviate the symptoms / phenotype of rosacea (Example 2).

[0029] Furthermore, when enriching relevant genes in the skin lesions of rosacea patients, this invention found significant changes in both the LAPTM5 and STING signaling pathways. Through experimental analysis of the interaction between LAPTM5 and STING proteins (immunoprecipitation, co-localization), this application found that LAPTM5 can maintain the stability of the STING protein and positively regulate the activation of the STING signaling pathway and its mediated expression of inflammatory factors. Therefore, when rosacea symptoms worsen, the expression levels of both LAPTM5 and STING proteins increase (Example 3).

[0030] Based on the conclusion that the inhibition of the STING gene can directly alleviate the symptoms / phenotype of rosacea, this invention found that the inhibition of the LAPTM5 gene, or the reduction of LAPTM5 protein expression, can also alleviate the symptoms / phenotype of rosacea. Attached Figure Description

[0031] Figure 1 The expression of LAPTM5 mRNA and STING mRNA in rosacea lesions;

[0032] Figure 2 The expression of LAPTM5 and STING signaling pathway-related genes in different subtypes (ETR, PPR and PhR) of rosacea lesions in the GSE65914 dataset;

[0033] Figure 3 The expression levels of M1 macrophage markers CD86, LAPTM5, and STING in skin lesions of patients with rosacea;

[0034] Figure 4 TUNEL staining of skin lesions from patients with rosacea;

[0035] Figure 5 Phenotypic diagram of mice with the STING inhibitor H-151 involved;

[0036] Figure 6 HE staining results of skin lesions in LL-37 model mice;

[0037] Figure 7 The expression levels of LAPTM5, STING, and F4 / 80 in the skin lesion tissue of LL-37 model mice;

[0038] Figure 8 A schematic diagram of the protein expression of STING and LAPTM5 in LL-37-induced rosacea-like skin lesions in mice;

[0039] Figure 9This is a schematic diagram of the immunohistochemistry of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in rosacea-like rashes in mice treated with H-151.

[0040] Figure 10 TUNEL staining was used to investigate the expression levels of broken dsDNA fragments in mouse rosacea-like rashes.

[0041] Figure 11 A schematic diagram of co-expressed LAPTM5 and STING proteins; (A) Expression of LAPTM5 and STING proteins in RAW264.7 macrophages and L929 fibroblasts; (B) Western blot analysis of changes in the expression of STING, LAPTM5 and LC3B in RAW264.7 and L929 cells stimulated with HT-DNA in the presence of liposomes at different time points. Figure 11 The bottom figure shows the changes in the expression of phosphorylated STING, TBK1, IRF3, NFκBp65 and LAPTM5 proteins in RAW264.7 cells at different time points after LL-37 stimulation by Western blot.

[0042] Figure 12 The following figures illustrate the effect of LAPTM5 on STING protein expression: (A) Changes in STING protein expression were detected after RAW264.7 macrophages were infected with LAPTM5 shRNA lentivirus; (B) Changes in STING and LC3B protein expression were detected after L929 cells were infected with LAPTM5 overexpression lentiviruses with different MOI values; (C) RAW264.7 macrophages were infected with LAPTM5 shRNA lentivirus, treated overnight with lysosomal inhibitor BafA1 and proteasome inhibitor MG132, and then stimulated with DMXAA for 0h and 2h. Western blot was used to detect STING protein expression.

[0043] Figure 13 The graph shows the changes in type I interferon and inflammatory cytokine mRNA expression detected by RT-qPCR after LAPTM5 overexpression and silencing. (a, b) RAW264.7 macrophages were infected with LAPTM5 shRNA lentivirus and stimulated with HT-DNA and DMXAA for different time periods, respectively. The changes in type I interferon and inflammatory cytokine mRNA expression were detected by RT-qPCR. (c, d) L929 cells were infected with LAPTM5 overexpression lentivirus and stimulated with HT-DNA and DMXAA for 3 hours, respectively. The changes in type I interferon and inflammatory cytokine mRNA expression were detected by RT-qPCR.

[0044] Figure 14The images show Western blot analysis of the interaction between LAPTM5 and STING. (A, B) RAW264.7 macrophages were infected with LAPTM5 shRNA lentivirus and stimulated with HT-DNA and DMXAA for different time periods. Western blot analysis was used to detect changes in the phosphorylation levels of STING and its downstream signaling proteins. (C, D) L929 cells were infected with LAPTM5 overexpression lentivirus and stimulated with HT-DNA and DMXAA for different time periods. Western blot analysis was used to detect changes in the phosphorylation levels of STING and its downstream signaling proteins.

[0045] Figure 15 A 3D prediction image of the LAPTM5 and STING complex protein;

[0046] Figure 16 This is a schematic diagram of the interaction between LAPTM5 and STING observed by immunoprecipitation.

[0047] Figure 17 Immunofluorescence pattern showing co-localization of STING and LAPTM5;

[0048] Figure 18 A technical roadmap for investigating the role of the LAPTM5 and STING signaling pathways in the inflammatory response of rosacea;

[0049] Figure 19 A technical roadmap for investigating the effects of the STING gene inhibitor H-151 on the phenotype and molecular markers of rosacea-like skin inflammation in mice;

[0050] Figure 20 A technical roadmap for studying the mechanism and molecular pathway of LAPTM5 regulation of the STING signaling pathway;

[0051] Figure 21 This is a diagram showing the results of LAPTM5's ubiquitination modification of the STING protein. Detailed Implementation

[0052] The following is a detailed explanation with reference to the accompanying drawings.

[0053] The following examples are only used to further explain the beneficial effects of the STING gene in this application and should not be used to limit the scope of protection of this application. Those skilled in the art can understand the specific meaning of the terms in this invention according to the specific circumstances. Unless otherwise specified, the experimental procedures described in the following examples are conventional procedures. Unless otherwise specified, the reagents used in the following examples are commercially available.

[0054] This application relates to the mechanism of action of the STING gene in rosacea.

[0055] 1. Research on the role of LAPTM5 and STING signaling pathways in the inflammatory response of rosacea (technical route see [link]). Figure 18 )

[0056] (1) The mRNA expression levels of LAPTM5 and STING signaling pathway-related genes in the skin lesions of rosacea patients and rosacea-like lesion mice were analyzed using the GEO gene expression database (GSE65914, GSE147950) and the GSA genome sequence database (HRA000378). Related genes included: monocyte-macrophage marker molecules (CD68, CD11B, CD14 and F4 / 80), STING signaling pathway-related genes (CGAS, TBK1, IRF3, NFKB1, NFKB2, RELA, RELB and REL), and type I interferon and pro-inflammatory factors-related genes (IFNA1, IFNB1, IFIT1, IFIT2, IFIT3, MX1, TNF, IL6, IL1B and CXCL10).

[0057] (2) Correlation analysis was performed on the mRNA expression of LAPTM5 and STING, as well as the expression of both and monocyte-macrophage markers and inflammatory factors in the skin lesions of patients with rosacea. At the same time, GO / KEGG and GSEA enrichment analysis were performed on the dataset.

[0058] (3) In healthy controls and rosacea patients, nuclear DNA damage was observed under a fluorescence microscope by TUNEL staining, and the expression of broken dsDNA was observed.

[0059] 2. Analysis of the effects of the STING gene inhibitor H-151 on the phenotype and molecular markers of rosacea-like skin inflammation in mice (technical route see [link]). Figure 19 )

[0060] (1) Drug preparation:

[0061] LL-37: Add 1 mg of LL-37 to 695 μl of endotoxin-free water to prepare a 320 μM solution. Prepare fresh before use. H-151: Add 10 mg of H-151 to 358 μl of DMSO to prepare a 100 mM stock solution. Store frozen at -80°C. Take 7.5 μl (prepared with 0.9% sodium chloride injection + 5% Tween-80) and dilute to 750 nmol / 200 μl (3.75 mM). Prepare fresh before use.

[0062] (2) Mouse grouping and treatment

[0063] Female BALB / c mice aged 6-8 weeks were selected. The hair on the back of the mice was shaved with a razor 24 hours before the experiment. The mice were randomly divided into four groups: A (control group + Vehicle group), B (LL-37 model group + Vehicle group), C (control group + H-151 treatment group), and D (LL-37 + H-151 treatment group), with 6 mice in each group.

[0064] The same area on the back of the mouse was marked with a circle with a diameter of 1 cm using a marker pen, which was used as the test site.

[0065] Group A received 40 μl of endotoxin-free aqueous solution for preparing LL-37 via back injection, every 12 hours for a total of 4 times; and 200 μl of vehicle-based H-151 solution via abdominal injection, every 12 hours for a total of 4 times. Group B received 40 μl of LL-37 (320 μM) subcutaneously via intracircular injection in the back, every 12 hours for a total of 4 times; and 200 μl of vehicle-based H-151 solution via abdominal injection, every 12 hours for a total of 4 times. Group C received 40 μl of endotoxin-free aqueous solution for preparing LL-37 via back injection, every 12 hours for a total of 4 times; and 200 μl of 3.75 mM H-151 via intraperitoneal injection, every 12 hours for a total of 4 times. Group D received a subcutaneous injection of 40 μl LL-37 (320 μM) in the inner circle of the back, every 12 hours for a total of 4 times, and an intraperitoneal injection of 200 μl H-151 (3.75 mM), every 12 hours for a total of 4 times. 24 hours after the last LL-37 injection, photos were taken with a digital camera to evaluate the erythema score and area; after anesthesia, the skin was removed for subsequent monitoring.

[0066] (3) Detection of phenotypic and molecular markers at the lesion site

[0067] The severity of skin erythema in mice was observed and rated from 1 to 5 (5 being the highest). The area of ​​the erythema lesions in digital photographs was measured using ImageJ software and quantitatively analyzed.

[0068] Skin samples were harvested from mice after isoflurane inhalation anesthesia and divided into three aliquots: one aliquot was fixed in 10% formalin for HE staining to detect inflammatory cell infiltration in the skin lesions, and for immunohistochemistry and immunofluorescence to detect the expression levels and colocalization of LAPTM5, STING, and macrophage surface marker F4 / 80 proteins, as well as TUNEL assay to detect nuclear DNA damage and distribution in the tissue; one aliquot was frozen in liquid nitrogen for Western blot analysis to detect the expression of LAPTM5 and STING proteins and the activation of their downstream signaling proteins, namely the phosphorylation levels of TBK1, NFκB, and IRF3 proteins; and one aliquot was stored in RNAlater at -80°C for qRT-PCR analysis to detect the expression of inflammatory factors IFNβ, IL6, TNFα, IL1β, and CXCL10.

[0069] (4) Analysis of DNA damage caused by LL-37 in mouse skin tissue

[0070] In the control group and rosacea-like rash samples, nuclear DNA damage was observed under a fluorescence microscope using TUNEL staining, and the expression of fragmented dsDNA was investigated.

[0071] 3. Investigate the mechanism and molecular basis of LAPTM5 regulation of the STING signaling pathway (technical route see...) Figure 20 )

[0072] (1) Analysis of the effects of LAPTM5 on the activation of STING and its downstream signaling pathways and the expression of inflammatory factors.

[0073] a. Western blot analysis was conducted to detect the expression of LAPTM5 and STING in mouse macrophages and fibroblasts. Preliminary results showed that LAPTM5 was highly expressed in the macrophage line RAW264.7 and lowly expressed in the fibroblast line L929; STING was highly expressed in both cell lines. Therefore, macrophages were used for subsequent LAPTM5 knockdown experiments, while fibroblasts were used for LAPTM5 overexpression experiments.

[0074] b. A shRNA lentivirus of LAPTM5 was constructed (LAPTM5 Accession Number: NM_010686; shRNA sequence: gcTAGACTTCTGTTTGAGTAT), and infected mouse macrophages RAW264.7 to knock down LAPTM5 protein expression. Cells were stimulated with DMXAA (50 μg / ml), HT-DNA (2 μg / ml), or cGAMP (2 μM) for different time periods (0, 1, 2, 3, 4, and 6 h). Western blot was used to detect the effect of LAPTM5 knockdown on the phosphorylation level of STING and its downstream signaling proteins (TBK1, IRF3, and NFκB). qRT-PCR was used to detect the effect of LAPTM5 knockdown on the expression of IFNβ and interferon-stimulated genes (MX1, IFIT1, IFIT2, and IFIT3), as well as the effect on the expression of cytokines IL6, TNFα, IL1β, and CXCL10.

[0075] c. Construct a LAPTM5 overexpression lentivirus and infect it with the fibroblast cell line L929. Stimulate the cells with DMXAA (50 μg / ml), HT-DNA (2 μg / ml), or cGAMP (2 μM) for different time periods (0, 1, 2, 3, 4, and 6 h). Western blot was used to detect the effect of LAPTM5 overexpression on the activation and phosphorylation levels of STING and its downstream signaling proteins (TBK1, IRF3, and NFκB). qRT-PCR was used to detect the effect of LAPTM5 overexpression on the expression of IFNβ and interferon-stimulated genes (MX1, IFIT1, IFIT2, and IFIT3), as well as the effect on the expression of cytokines IL6, TNFα, IL1β, and CXCL10.

[0076] (2) Verify the interaction between LAPTM5 and STING protein

[0077] a. Immunoprecipitation assay to detect the interaction between endogenous STING and LAPTM5 in RAW264.7 cells

[0078] (A) After stimulation with DMXAA for different time periods, immunoprecipitation was performed with STING antibody and IgG was used as a negative control. Western blot was used to detect LAPTM5, TBK1 and pSTING.

[0079] (B) L929 cells were infected with FLAG-tagged LAPTM5 overexpressing lentivirus. After stimulation with cGAMP for different time periods, cells were collected and total protein was extracted. Immunoprecipitation was performed using STING antibody, with IgG as a negative control. Western blot was used to detect FLAG.

[0080] (C) L929 cells were infected with FLAG-tagged LAPTM5 overexpressing lentivirus. After stimulation with cGAMP for different time periods, cells were collected and total protein was extracted. Immunoprecipitation was performed using FLAG-tagged antibody, and Western blot was used to detect STING and TBK1.

[0081] b. Analyze the co-localization of LAPTM5 and STING in cells before and after STING activation.

[0082] Using confocal microscopy, we performed immunofluorescence staining on RAW264.7 cells to observe the colocalization of LAPTM5 (marked in red) and STING (marked in green) before and after treatment with the agonist DXMAA.

[0083] (3) The effects of LAPTM5 on the stability of STING protein, ubiquitination modification, and binding ability of STING to TBK1 were analyzed.

[0084] a. Analysis of the degradation pathways underlying STING protein degradation caused by the loss of LAPTM5 expression in both resting and activated states.

[0085] Mouse macrophage line RAW264.7 was infected with LAPTM5 shRNA lentivirus to inhibit LAPTM5 protein expression in knockdown cells. Cells were treated with 100 μg / ml actinomycin (CHX), a protein synthesis inhibitor, for different time periods (0, 2, 4, 8, 12, and 24 h). Total protein was extracted from the cells, and Western blot was used to detect changes in STING protein expression after LAPTM5 knockdown.

[0086] LAPTM5+ / + and LAPTM5- / - RAW264.7 cells were extracted and pretreated overnight with 10 μM proteasome inhibitor (MG132) and 50 nM lysosome inhibitor (BafA1). The next day, the cells were stimulated with HT-DNA (2 μg / ml) or cGAMP (2 μM) for different time periods (0, 2, 4, and 8 h). Total cellular protein was extracted and detected by Western blot to analyze whether the inhibitors could prevent the degradation of STING protein caused by LAPTM5 knockdown.

[0087] b. Analysis of the effect of LAPTM5 on ubiquitination modification of STING protein

[0088] Overexpression plasmids of Myc-STING, HA-Ub, HA-Ub-K48 / K63, and Flag-LAPTM5 were constructed. HEK293 cells were transiently transfected with these plasmids for 48 hours. Immunoprecipitation was used to detect changes in STING protein ubiquitination levels after LAPTM5 overexpression, as well as changes in ubiquitination levels at different linkages such as STING-K48 / K63. Cells were stimulated with htDNA (2 μg / ml) for different time periods (0, 2, 4, and 6 hours), and protein immunoprecipitation was used to verify the effect of LAPTM5 on ubiquitination modification of the STING-K63 linkage. Figure 21 As shown, the bar graph of immunoprecipitation (IP Myc) indicates that LAPTM5 and STING proteins interact and bind in vivo.

[0089] Example 1

[0090] Studies have shown that LAPTM5 and the STING signaling pathway are involved in the inflammatory response of rosacea.

[0091] 1. The mRNA expression of LAPTM5 and STING signaling pathway-related genes was significantly increased in the skin lesions of rosacea patients and LL-37-mediated rosacea lesions in mice, and the expression of LAPTM5 mRNA in rosacea patients was positively correlated with the expression of STING and inflammatory factor mRNA.

[0092] (1) Figure 1 The average expression level of LAPTM5 mRNA in the HC (control group) was 7.565, while the average expression level in skin samples from rosacea patients was 8.993. The average expression level of STING mRNA in the HC (control group) was 5.254, while the average expression level in skin samples from rosacea patients was 6.333. Analysis using the GEO (GSE65914) gene expression database showed high expression of both LAPTM5 mRNA and STING mRNA in rosacea lesions.

[0093] Figure 1 The results showed that LAPTM5 was positively correlated with STING mRNA expression.

[0094] Figure 1 The mean expression level of LAPTM5 mRNA in the HC (control group) was 10.258, while the mean expression level in skin samples from rosacea patients was 12.371. The mean expression level of STING mRNA in the HC (control group) was 10.232, while the mean expression level in skin samples from rosacea patients was 11.019. Analysis using the GSA (HRA000378) gene expression database showed high expression of both LAPTM5 mRNA and STING mRNA in rosacea lesions.

[0095] Figure 1 The average LAPTM5 mRNA expression level in the CON (control group) was 3.157, while the average LAPTM5 mRNA expression level in the skin lesions of LL-37-mediated rosacea mice was 3.546. The average STING mRNA expression level in the HC (control group) was 2.275, while the average STING mRNA expression level in the skin lesions of LL-37-mediated rosacea mice was 2.906. Analysis using the GEO (GSE147950) gene expression database showed that both LAPTM5 mRNA and STING mRNA expression levels were significantly increased in the skin lesions of LL-37-mediated rosacea mice.

[0096] (2) Table 1 shows the p-values ​​for differential expression of LAPTM5 and STING signaling pathway-related genes mRNA. The results showed that the expression of LAPTM5 and STING signaling pathway-related genes mRNA was upregulated in the GSE147950 and HRA000378 datasets. The following values ​​indicate the significance of the results, and ns indicates that the results are not significant. Significant values ​​indicate that the mRNA expression of the gene is upregulated.

[0097] Table 1

[0098]

[0099] (3) Figure 2 The expression levels of LAPTM5 and STING signaling pathway-related genes in rosacea lesions of different subtypes (ETR, PPR, and PhR) in the GSE65914 dataset are shown.

[0100] Figure 2 A shows the expression levels of monocyte-macrophage marker molecules. Figure 2 B shows the expression levels of LAPTM5 and genes related to the STING signaling pathway. Figure 2 C shows the expression levels of interferon-stimulated genes and pro-inflammatory factors.

[0101] The results showed that, compared with the control group (HC), the expression levels of LAPTM5 and STING signaling pathway-related genes were upregulated in rosacea lesions of different subtypes (ETR, PPR and PhR).

[0102] (4) Table 2 shows the correlations between LAPTM5 and STING in rosacea patients and macrophage surface markers, interferon-stimulated genes, and inflammatory factor expression, respectively (GSE65914). As shown in Table 2, LAPTM5 and STING in rosacea patients in the GSE65914 dataset were positively correlated with macrophage surface markers, interferon-stimulated genes, and inflammatory factor expression.

[0103] 2. Protein expression of LAPTM5 and STING in the skin tissues of rosacea patients and normal individuals.

[0104] like Figure 3As shown, the integrated optical density to area ratio of CD86, a marker of M1 macrophages, was 0.005 in the control group and 0.075 in the rosacea group. The integrated optical density to area ratio of LAPTM5 in the control group was 0.004, and the protein AOD of LAPTM5 in rosacea was 0.09. The integrated optical density to area ratio of STING in the control group was 0.01 and 0.075.

[0105] The results showed that the relative expression levels of M1 macrophage markers CD86, LAPTM5, and STING in the skin lesions of rosacea patients were significantly higher than those in the healthy control group. This result demonstrates that macrophages, LAPTM5, and STING are involved in the inflammatory response in a mouse rosacea model.

[0106] 3. Expression levels of broken dsDNA fragments in skin lesions of rosacea patients

[0107] like Figure 4 As shown, nuclear damage is increased in the skin lesions of patients with rosacea, and the expression level of broken dsDNA fragments is higher than that in the normal control group.

[0108] Example 2

[0109] Effects of the STING gene inhibitor H-151 on LL-37-induced rosacea-like rashes in mice.

[0110] 1. The STING gene inhibitor H-151 can significantly alleviate LL-37-induced rosacea-like rashes in mice.

[0111] (1) As Figure 5 As shown in the first column, the gross photographs of mice show that the LL-37 model group (LL37+Vehicle group) can form rosacea-like rashes with pustules on the local skin, while the group that was treated with the STING gene inhibitor before LL-37 modeling (LL37+H-151 group) reduced the area of ​​erythema and alleviated the degree of erythema. Figure 5 The dermoscopy images shown on the left in the second column illustrate the skin condition of mice under different treatments. Figure 5 The second column on the right shows the area and score of red skin in mice of each group.

[0112] In the Vehicle group (without exogenous drug treatment) and the H-151-treated normal mouse control group, no erythema was observed, and the erythema severity score was 0. The erythema area in the LL37-induced Vehicle group was 1.016 cm². 2 The erythema area in the H-151 treatment group, where LL37 modeling was successfully established, was 0.231 cm². 2The erythema severity score of the Vehicle group, where the LL37 model was successfully established, was 3.5. The erythema severity score of the H-151 treatment group, where the LL37 model was successfully established, was 1.5.

[0113] Based on the above experimental results, it can be seen that the STING gene inhibitor treatment group can alleviate rosacea-like rash erythema in mice.

[0114] (2) HE staining was performed on the skin areas of rosacea-like erythema in the above groups. For example... Figure 6 As shown, the LL-37 model group showed increased epidermal thickening and dermal inflammatory cell infiltration compared to the control group, while the group treated with STING gene inhibitor before LL-37 modeling (LL-37+H151 group) could significantly alleviate these manifestations.

[0115] (3) Immunohistochemical experiments were performed on the skin areas of rosacea-like erythema to evaluate the changes of STING protein and LAPTM5 protein in the skin areas of rosacea-like erythema.

[0116] like Figure 7 As shown, 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. The expression of F4 / 80 was downregulated in the group treated with STING gene inhibitor before LL-37 modeling. This result shows that STING gene inhibitor treatment reduced macrophage aggregation.

[0117] 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.

[0118] (4) Figure 8 As shown in Figure A, LL-37 induces increased protein expression of STING and LAPTM5 in rosacea-like lesions (the band corresponding to LL-37 is darker than that of the control group).

[0119] like Figure 8 As shown in B, the phosphorylation level of STING and its downstream pathways is increased (the band corresponding to LL-37+H-151 is lighter than the band corresponding to LL-37).

[0120] Western blot results demonstrated that LL-37 induced increased protein expression of STING and LAPTM5 in rosacea-like lesions, and that H-151 significantly inhibited STING pathway activation.

[0121] (5) Immunohistochemical results showed that LL-37 induced increased expression of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in rosacea-like lesions, and H-151 inhibited the expression of IFN-β, IL-1β, IL-6, TNF-α, and CXCL-10 in LL-37 induced rosacea-like rashes in mice, as shown in the figure. Figure 9 As shown.

[0122] (6) TUNEL staining showed increased nuclear damage in the skin lesions of mice in the LL-37 model group, and an increased number of dsDNA-positive cells compared to the control group. Figure 10 As shown.

[0123] Example 3

[0124] To explore the molecular mechanism by which LAPTM5 regulates the activation of the STING signaling pathway, the mechanisms of action of STING and LAPTM5 at the cellular level were investigated.

[0125] 1. Extracellular DNA can induce upregulation of LAPTM5 protein expression.

[0126] Figure 11 Figure A shows the expression of STING and LAPTM5 in RAW264.7 macrophages and L929 fibroblasts. The results showed that STING was highly expressed in both RAW264.7 macrophages and L929 fibroblasts, while LAPTM5 was highly expressed in RAW264.7 macrophages and lowly expressed in L929 fibroblasts.

[0127] Figure 11 B shows the expression of STING and LAPTM5 after transfection with HT-DNA into RAW264.7 and L929 cells. Western blot results showed that with increasing treatment time, STING protein expression decreased and LC3B expression increased in both RAW264.7 and L929 cells. This result indicates that HT-DNA activation of STING induces autophagy. Simultaneously, HT-DNA can induce upregulation of LAPTM5 protein expression.

[0128] Figure 11 The expression of STING and LAPTM5 in RAW264.7 cells treated with LL-37 at different time points is shown. Western blot results showed that the expression of phosphorylated STING, TBK1, IRF3, NFκBp65, and LAPTM5 proteins increased with increasing treatment time.

[0129] 2. LAPTM5 can positively regulate the expression of STING protein, promote the stability of STING protein, and enhance its induction of autophagy.

[0130] like Figure 12 As shown in (A, B), knocking down LAPTM5 reduces STING protein expression, while overexpressing LAPTM5 increases STING protein expression, and the expression of the autophagy marker LC3B protein also increases. Figure 12 (C) The results showed that in RAW264.7 without DMXAA stimulation, the proteasome inhibitor MG132 could restore STING protein expression, while in the DMXAA-stimulated group, the lysosomal inhibitor BafA1 could restore STING protein expression. This suggests that the degradation of STING protein caused by the downregulation of LAPTM5 expression before and after STING activation depends on different degradation pathways, with the former depending on the proteasome degradation pathway and the latter depending on the lysosomal degradation pathway.

[0131] 3. LAPTM5 positively regulates the production of type I interferon and inflammatory factors mediated by STING.

[0132] like Figure 13 As shown in (a, b), knocking down LAPTM5 in RAW264.7 cells led to a decrease in the expression of type I interferon and inflammatory factors induced by HT-DNA and DMXAA.

[0133] like Figure 13 As shown in (c, d), L929 cells overexpress LAPTM5, leading to an increase in the expression of type I interferon and inflammatory factors induced by HT-DNA and DMXAA.

[0134] 4. LAPTM5 can positively regulate STING and its downstream signaling pathways.

[0135] like Figure 14 As shown in (A, B), knocking down LAPTM5 in RAW264.7 inhibits the phosphorylation levels of STING and its downstream TBK1 and IRF3.

[0136] like Figure 14 As shown in (C, D), L929 cells overexpress LAPTM5, which enhances the phosphorylation level of STING and its downstream TBK1 and IRF3, and enhances the induction of autophagy by STING.

[0137] 5. LAPTM5 and STING are interacting proteins.

[0138] a. Interaction prediction between LAPTM5 and STING proteins

[0139] AlphaFold2 was used to predict the crystal structures of the two proteins. The obtained protein crystals were then processed using the Protein Preparation Wizard module of Schrödinger software for protein preprocessing, regenerate states of native ligand, H-bond assignment optimization, protein energy minimization, and water removal.

[0140] Perform protein-protein interaction simulation on the processed protein (using the protein-protein docking module), setting the Nubmer of ligand rotations to probe to 70000 and the Maximum poses to return to 30.

[0141] Based on the protein-protein interaction simulation results, pose 2 ranked first, with a PIPER posescore of -930.222 and a PIPER pose energy of -1774.039 kcal / mol. Both of these binding free energies are lower than other poses, indicating that this interaction pose represents the most stable binding between the two proteins in 70,000 calculations. The PIPER pose energy is below -500 kcal / mol, suggesting a relatively tight binding between LAPTM5 and the STING protein.

[0142] Figure 15 The 3D stereoscopic images shown demonstrate that LAPTM5 (yellow) and STING (blue) can be stably bonded together, with LAPTM5's GLU176 forming a hydrogen bond with STING's GLN334, LAPTM5's PHE74 forming a π-π bond and 11 van der Waals forces with STING's HIS94, and LAPTM5's GLU42 forming a hydrogen bond and a salt bridge with STING's LYS150.

[0143] b. LAPTM5 and STING can interact to form a complex before and after DMXAA stimulation.

[0144] The complex of LAPTM5 and STING was verified by immunoprecipitation.

[0145] like Figure 16As shown in a, immunoprecipitation was used to detect the interaction between endogenous STING and LAPTM5 in RAW264.7 cells. After stimulation with DMXAA for different time periods, immunoprecipitation was performed using STING antibody, with IgG as a negative control. Western blot was used to detect the expression of LAPTM5, TBK1 and pSTING.

[0146] like Figure 16 As shown in b, L929 cells were infected with a FLAG-tagged LAPTM5 overexpressing lentivirus. After stimulation with cGAMP for different time periods, the cells were collected and total protein was extracted. Immunoprecipitation was performed using STING antibody, with IgG as a negative control. Western blot was used to detect FLAG expression.

[0147] like Figure 16 As shown in c, L929 cells were infected with a FLAG-tagged LAPTM5 overexpressing lentivirus. After stimulation with cGAMP for different time periods, cells were collected to extract total protein. Immunoprecipitation was performed using a FLAG-tagged antibody, and Western blot was used to detect the expression of STING and TBK1.

[0148] Furthermore, STING and LAPTM5 were co-localized using immunofluorescence. For example... Figure 17 As shown, RAW264.7 cells were stimulated with DMXAA (50 μg / ml). The co-localization of LAPTM5 and STING before and after stimulation was observed in the figure. The co-localization of the two cells was increased by laser confocal microscopy.

[0149] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; terms such as "preferredly," "according to a preferred embodiment," or "optionally" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, features introduced by "preferredly" are merely optional and should not be construed as mandatory. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. STING The application of gene inhibitors in the preparation of drugs for treating rosacea, characterized in that, The STING gene inhibitor is H-151.

2. The application according to claim 1, characterized in that, The treatment also includes rashes and / or pustules caused by rosacea.

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