Marker and therapeutic drug for skin injury caused by ultraviolet rays

Abstract

The invention relates to the field of diagnosis and treatment of skin injury caused by ultraviolet rays, in particular to succinate dehydrogenase (SDH) serving as a marker of the skin injury caused by the ultraviolet rays and application of the succinate dehydrogenase (SDH) to preparation of a treatment medicine. The activity of SDH in skin tissues and cells is remarkably reduced due to ultraviolet irradiation, so that macrophages are promoted to be polarized to M1 type, and inflammatory response is aggravated. The SDH activity is detected, so that the SDH can be used as an effective marker for diagnosing and monitoring skin injury caused by ultraviolet rays. Through overexpression of SDH subunit A or use of a TRAP1 inhibitor DN401, SDH activity can be recovered, macrophage M1 polarization can be inhibited, and skin erythema, edema and the like caused by ultraviolet rays can be significantly relieved. The invention provides a new diagnosis and treatment strategy, effectively prevents and treats skin injury caused by ultraviolet rays by intervening SDH activity, and has a remarkable clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of diagnosis and treatment of skin damage caused by ultraviolet radiation, and specifically to biomarkers and therapeutic drugs for skin damage caused by ultraviolet radiation. Background Technology

[0002] Ultraviolet radiation, especially UVB (ultraviolet-medium wave), is a major environmental factor causing acute skin damage (sunburn) and even skin tumors. According to the consensus in the dermatological community, UV-induced skin damage is a pathological spectrum, encompassing everything from acute sunburn to chronic cumulative damage (collectively known as photodamage), represented by photoaging and photocarcinogenesis. The root cause lies in the direct and indirect damage of ultraviolet radiation to skin cell DNA, the oxidative stress system, and connective tissue. Histopathologically, sunburn is characterized by the appearance of sunspot cells (i.e., apoptotic keratinocytes) accompanied by infiltration of acute inflammatory cells in the dermis; chronic photodamage manifests as irregular epidermal proliferation and atrophy, chronic dermal inflammation, and elastic fiber degeneration.

[0003] Recent studies have revealed that the innate immune system plays a central role in ultraviolet (UV)-induced skin damage. Macrophages, as key inflammatory regulatory cells, are recruited after UV exposure and polarized into the pro-inflammatory M1 phenotype. They maintain and amplify the inflammatory cascade by releasing factors such as TNF-α and IL-1β, which is crucial for the persistence of clinical symptoms. Therefore, UV-induced skin damage is a dynamic inflammatory process dominated by immune cells.

[0004] Therefore, inhibiting the excessive polarization of macrophages towards the M1 type is considered a promising new therapeutic strategy for alleviating UV-induced skin damage.

[0005] However, current mainstream clinical treatments—topical corticosteroids—primarily work by broadly inhibiting inflammatory mediators, acting as a downstream interceptor. While they can relieve symptoms, long-term use can easily lead to side effects such as skin atrophy and telangiectasia, failing to intervene in the immune process of M1 polarization at its source or within macrophages. Therefore, there is an urgent need in this field to develop more efficient and safer sunburn prevention and treatment methods that can precisely regulate the skin's immune microenvironment and inhibit macrophage M1 polarization and subsequent inflammatory outbreaks from the upstream source.

[0006] The innovation of this treatment strategy relies on a deep understanding of the cutting-edge field of "immunometabolic metabolism." The tricarboxylic acid cycle (TCA cycle) is the core of eukaryotic cell energy metabolism, and its function is a decisive factor in the activation and differentiation of immune cells. Specifically, the activation of M1 macrophages is closely related to their unique metabolic pattern, including the disruption of the TCA cycle and the accumulation of specific metabolites. Among them, succinate dehydrogenase (SDH), as a key enzyme in the TCA cycle, is responsible for catalyzing succinate to fumarate. Its activity directly affects the intracellular levels of key metabolites such as succinate. These metabolites themselves can act as signaling molecules, directly participating in the regulation of the expression of inflammation-related genes and the production of cytokines, making key TCA enzymes such as SDH a ​​hub connecting cellular metabolism and immune function.

[0007] Despite the growing importance of SDH in immune metabolism, no research has yet revealed its role in the process of UV-induced skin damage by regulating macrophage polarization. Furthermore, no proposed technology has been developed to specifically inhibit macrophage M1 polarization by intervening in the activity or function of this enzyme, thereby reversing UV-induced skin damage and inflammatory responses, and to utilize it for the prevention or treatment of photodamage. Summary of the Invention

[0008] One of the objectives of this invention is to provide a new application for succinate dehydrogenase, which offers new insights into the prevention and treatment of skin damage caused by ultraviolet radiation.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] Applications of succinate dehydrogenase in the preparation of biomarkers for UV-induced skin damage, or as a target for drug regulation of the TRAP1 signaling pathway.

[0011] Succinate dehydrogenase (SDH) plays an important biomarker role in UV-induced skin damage. SDH is a key enzyme in the tricarboxylic acid cycle, responsible for catalyzing the conversion of succinate to fumarate. Following UV irradiation, SDH activity in skin tissue significantly decreases, which is closely related to the severity of skin damage. Therefore, SDH activity can serve as an important biomarker for diagnosing and monitoring UV-induced skin damage.

[0012] Furthermore, using SDH (succinate dehydrogenase) as a target for screening drugs that regulate the TRAP1 signaling pathway includes two aspects: first, using SDH (succinate dehydrogenase) as a target for drugs or formulations targeting the TRAP1 signaling pathway for screening TRAP1 signaling pathway inhibitors; second, using SDH (succinate dehydrogenase) as a target for drugs or formulations targeting the TRAP1 signaling pathway for screening TRAP1 signaling pathway agonists.

[0013] Furthermore, the application of SDH (succinate dehydrogenase) as a target of drugs or formulations targeting the TRAP1 signaling pathway in the screening of TRAP1 signaling pathway inhibitors specifically refers to: evaluating the efficacy of TRAP1 signaling pathway inhibitors by detecting changes in SDH activity; the application of SDH (succinate dehydrogenase) as a target of drugs or formulations targeting the TRAP1 signaling pathway in the screening of TRAP1 signaling pathway agonists specifically refers to: evaluating the efficacy of TRAP1 signaling pathway agonists by detecting changes in SDH activity.

[0014] Furthermore, the TRAP1 signaling pathway inhibitor binds to TRAP1, preventing TRAP1 from inhibiting the activity of SDH (succinate dehydrogenase), thereby maintaining the normal activity and physiological function of SDH.

[0015] Furthermore, the drug is selected from any of the following:

[0016] Drugs for the prevention or treatment of diseases or disorders caused by abnormalities in the TRAP1 signaling pathway;

[0017] Drugs that specifically activate or inhibit the TRAP1 signaling pathway are needed for diseases or disorders.

[0018] Furthermore, the aforementioned diseases or disorders include, but are not limited to: photoaging of the skin, skin inflammation, and skin damage.

[0019] Furthermore, in the skin damage caused by ultraviolet radiation, the increased expression of TRAP1 led to a decrease in SDH activity.

[0020] In the skin damage caused by the ultraviolet radiation, TRAP1 expression increased by at least 1.2 times, and SDH activity decreased by at least 25%.

[0021] Furthermore, in the aforementioned application, the proportion of macrophage M1 polarization and / or the expression level of TRAP1 are increased in the skin damage caused by ultraviolet radiation.

[0022] In UV-induced skin damage, the M1 polarization ratio of macrophages is significantly increased, along with TRAP1 expression. The increased M1 polarization ratio indicates a shift towards a pro-inflammatory phenotype in macrophages, while the increased TRAP1 expression further inhibits SDH activity, exacerbating skin damage. Therefore, increased macrophage M1 polarization ratio and TRAP1 expression are important biomarkers of UV-induced skin damage.

[0023] Furthermore, in the skin damage caused by ultraviolet radiation in the application described above, the M1 polarization rate of macrophages increases to at least 20% and / or the expression level of TRAP1 increases to at least 1.2 times.

[0024] Experimental results showed that after UVB irradiation, the M1 polarization rate of macrophages significantly increased to at least 20%, and TRAP1 expression increased by at least 1.2-fold. These specific numerical changes can serve as quantitative standards for diagnosing and monitoring UV-induced skin damage. By detecting the specific range of changes in these markers, the degree of skin damage can be assessed more accurately.

[0025] Furthermore, in the aforementioned applications, the biomarkers are used for the qualitative and quantitative diagnosis, monitoring, or assessment of skin lesions.

[0026] Specifically, in diagnosis, UV-induced skin damage can be diagnosed by detecting SDH activity, macrophage M1 polarization ratio, and TRAP1 expression levels. In monitoring, changes in these markers can be periodically monitored during treatment to track skin damage recovery. In evaluation, the degree of skin damage and treatment effectiveness can be assessed by quantitatively analyzing changes in SDH activity, macrophage M1 polarization ratio, and TRAP1 expression levels.

[0027] A second objective of this invention is to provide an in vitro model for diagnosing, monitoring, or assessing skin damage, which can accurately determine whether the keratinocytes to be tested have been damaged by ultraviolet radiation.

[0028] To achieve the above objectives, the technical solution of the present invention is as follows:

[0029] A cell model for diagnosing, monitoring, or evaluating skin lesions, wherein the cell model is a co-culture model of human keratinocyte line HaCaT cells and human mononuclear / macrophage line THP-1 cells;

[0030] Control group: HaCaT cells were seeded and irradiated with UVB. THP-1 cells were added and co-cultured. THP-1 cells were collected and intracellular SDH activity was detected.

[0031] Test group: Keratinocytes to be tested were seeded, THP-1 cells were added and co-cultured, THP-1 cells were collected, and intracellular SDH activity was detected.

[0032] Furthermore, in the control group, HaCaT cells were seeded with 50 mJ / cm². 2 THP-1 cells were irradiated with UVB, then added to Transwell chambers and co-cultured for 24 hours. THP-1 cells were collected, and intracellular SDH activity was measured. SDH activity was significantly reduced in THP-1 cells co-cultured with UVB-irradiated HaCaT cells. SDH activity remained normal in THP-1 cells co-cultured with unirradiated HaCaT cells.

[0033] From another perspective, this can be understood as a Transwell chamber containing two types of co-cultured cells. In UVB-induced skin damage, the Transwell chamber co-culture model containing HaCaT cells and THP-1 cells can more accurately simulate the intercellular interactions during the skin damage process. Specifically, HaCaT cells, as a model of keratinocytes, can simulate epidermal damage after UVB irradiation, while THP-1 cells, as a model of mononuclear macrophages, can simulate the response of macrophages in the dermis during skin damage. Experimental results showed that after UVB irradiation of HaCaT cells, the SDH activity in THP-1 cells was significantly reduced. This finding indicates that harmful substances or signals produced by HaCaT cells after UVB irradiation can affect the metabolic activity of THP-1 cells, leading to a decrease in SDH activity.

[0034] From a diagnostic perspective, changes in SDH activity in THP-1 cells can be detected using a co-culture model of HaCaT cells and THP-1 cells to diagnose skin damage caused by ultraviolet radiation. Specific methods include enzyme histochemical staining combined with micro-methods to detect intracellular SDH activity.

[0035] From a monitoring perspective, during treatment, a co-culture model of HaCaT cells and THP-1 cells / keratinocytes to be tested is used. Regular monitoring of SDH activity changes in THP-1 cells / keratinocytes to be tested allows for monitoring of skin damage recovery. Regular monitoring of SDH activity in THP-1 cells can also assess treatment efficacy.

[0036] A third objective of this invention is to provide a pharmaceutical use for an accelerator of succinate dehydrogenase. This use offers a new therapeutic approach for treating skin damage caused by ultraviolet radiation.

[0037] To achieve the above solution, the technical solution of the present invention is as follows:

[0038] The use of succinate dehydrogenase promoters in the preparation of drugs for treating skin damage caused by ultraviolet radiation.

[0039] A pharmaceutical composition comprising an SDH promoter and one or more other active ingredients for treating UVB-induced skin damage. Preferably, the other active ingredients include anti-inflammatory agents, antioxidants, or cytoprotective agents.

[0040] Furthermore, in the application described, the promoter can enhance SDH activity in THP-1 cells / macrophages; and / or thereby inhibit the increase in macrophage M1 polarization ratio and TRAP1 expression.

[0041] Furthermore, the promoter is a small molecule inhibitor that promotes THP-1 cells / macrophages to enhance SDH activity, a TRAP1 inhibitor, DN401 (MedChemExpress, HY-115781).

[0042] Furthermore, the promoter is a gene editing reagent, specifically a gene editing reagent for overexpressing SDH subunit A. The full-length cDNA of human SDH subunit A (NCBI reference sequence: NM_004168.4) was inserted into an LV5 (EF-1a / GFP & Puro) lentiviral vector (Shanghai Jima Pharmaceutical Technology Co., Ltd.) using molecular cloning, and subsequently infected THP-1 cells. The cells were then screened with puromycin for several days to construct THP-1 cells overexpressing SDH.

[0043] Furthermore, the promoter can significantly improve skin erythema, edema and damage caused by ultraviolet radiation.

[0044] Furthermore, the promoter can significantly reduce skin inflammatory cell infiltration induced by ultraviolet radiation.

[0045] Application of DN401 in the preparation of drugs for treating skin damage caused by ultraviolet radiation.

[0046] Furthermore, the DN401 is an intradermal injection.

[0047] The three sections above all involve methods for detecting SDH activity.

[0048] Specifically, SDH activity in skin tissue or cells can be detected by enzyme histochemical staining (tetrazole salt method). SDH activity in skin tissue or THP-1 cells can also be detected by micro-methods.

[0049] Beneficial effects

[0050] This invention provides a novel method for treating UV-induced skin damage by intervening in the activity or function of succinate dehydrogenase (SDH). Experimental results show that decreased SDH activity is closely related to UV-induced skin damage, and that overexpression of SDH subunit A or the use of the TRAP1 inhibitor DN401 can significantly restore SDH activity, inhibit M1 polarization of macrophages, and thus reduce skin erythema, edema, and damage. Furthermore, intradermal injection of DN401 significantly improves UV-induced skin damage and exhibits good safety and efficacy at lower doses. These results indicate that the methods and compositions provided by this invention can not only effectively prevent and treat UV-induced skin damage but also possess high safety and clinical application potential, offering new strategies and means for the prevention and treatment of sunburn. Attached Figure Description

[0051] Appendix Figure 1 This study investigated the changes in SDH activity in skin under light exposure and after UVB irradiation. 1-A compared SDH activity in human skin tissue from exposed and unexposed areas; 1-B showed the effect of chronic UVB irradiation on SDH activity in mouse skin tissue (*p<0.05, 6 mice per group); 1-C showed the effect of UVB irradiation on SDH activity in keratinocytes (**p<0.01, 3 biological replicates); and 1-D showed the effect of a UVB-irradiated keratinocyte co-culture system on SDH activity in macrophages (*p<0.05, 3 biological replicates).

[0052] Appendix Figure 2 This study investigated the regulatory effects of SDH subunit A overexpression on substrate metabolism and phenotype in macrophages. 2-A represents the change in succinic acid content after SDH subunit A overexpression in macrophages (*p<0.05, **p<0.01, nsp>0.05, 3 biological replicates). 2-B represents the proportion of CD86-positive macrophages after SDH subunit A overexpression (the horizontal axis in the flow cytometry results represents the relative intensity of CD86 fluorescence or scattered light signals, with values ​​of 0, 10, etc.). 3 10 4 10 5 10 6 The vertical axis SSC represents the granularity of the cell. The larger the intracellular organelles and granularity, the larger the SSC. In the figure, the values ​​are 0, 50K, 100K, 150K, 200K, and 250K, respectively.

[0053] Appendix Figure 3This study investigated the changes in TRAP1 expression and the regulatory effect of its inhibitor DN401 on SDH activity in light-exposed and cell co-culture systems. 3-A shows the immunohistochemical (IHC) staining results of TRAP1 protein expression levels in skin tissues from exposed and unexposed areas. 3-B presents the statistical analysis of the average gray values ​​of the IHC staining results in 3-A (**p<0.01, 8 samples from unexposed areas and 5 samples from exposed areas). 3-C shows the macrophages co-cultured with UVB-irradiated keratinocytes and macrophages co-cultured with unirradiated keratinocytes. The results of TRAP1 expression level detection; 3-D ​​shows the changes in SDH activity after tumor necrosis factor α (TNF-α) stimulation of macrophages, and the results of the regulatory effect of TRAP1 inhibitor DN401 on SDH (*p<0.05, 3 biological replicates); 3-E shows the changes in the proportion of CD86-positive macrophages after TNF-α stimulation of macrophages, and the results of the effect of TRAP1 inhibitor DN401 on the proportion of CD86-positive macrophages (the horizontal axis in the flow cytometry results represents the relative intensity of CD86 fluorescence signal or scattered light signal, in the figure, -10 is the value of the relative intensity ... 3 ,0,10 3 10 4 The vertical axis SSC represents the granularity of the cell. The larger the intracellular organelles and granularity, the larger the SSC. In the figure, the values ​​are 0, 50K, 100K, 150K, 200K, and 250K, respectively.

[0054] Appendix Figure 4 The study investigated the inhibitory effect of intradermal injection of DN401 (1 mg / kg) on ​​UVB-induced skin damage in mice. Figure 4-A shows representative gross results from four animal experiments; Figure 4-B shows the quantitative evaluation statistics of skin damage in the four animal experiments (*p<0.05, **p<0.01, ***p<0.001, nsp>0.05, with 4 mice in the UVB group and 6 mice in the other two groups); and Figure 4-C shows the HE staining results of mouse skin tissue in the four animal experiments.

[0055] Appendix Figure 5 The study showed the inhibitory effect of intradermal injection of DN401 (5 mg / kg) on ​​UVB-induced skin damage in mice. Figure 5-A shows the representative gross results of the animal experiment, and Figure 5-B shows the HE staining results of mouse skin tissue in the animal experiment.

[0056] Appendix Figure 6 The effect of topical application of DN401 (1 mg / kg) on ​​UVB-induced skin damage in mice is shown in Figure 6-A, which is a representative gross result of the animal experiment; Figure 6-B shows the HE staining results of mouse skin tissue in the animal experiment. Detailed Implementation

[0057] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0058] To enhance understanding of the present invention, certain key technologies and scientific terms will be clearly defined below. Unless specifically defined herein, all other technologies and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be emphasized that the scope of the present invention is not limited to the described technical features and terms, but allows for reasonable variations and adjustments in these aspects. Furthermore, please understand that the terminology used herein is intended to describe specific embodiments and not to be construed as restrictive.

[0059] Furthermore, all references cited in this patent, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are considered to be incorporated into this document in their entirety through citation, unless directly cited, as a reference. If there are any inconsistencies or conflicts between the content of these cited documents or similar materials and this application, particularly in terms of terminology definitions, usage of terms, and technical descriptions, the content of this application shall prevail.

[0060] the term

[0061] Skin damage caused by ultraviolet radiation: Skin damage caused by ultraviolet radiation refers to skin damage caused by excessive ultraviolet radiation, including pathological features such as sunburn, erythema, edema, and dermal inflammatory cell infiltration.

[0062] Erythema, edema, and lesions: Erythema, edema, and lesions refer to the typical pathological features of the skin after ultraviolet radiation, including skin erythema, telangiectasia, edema, skin thickening, and tissue damage such as exudation and crusting.

[0063] Cell models: Cell models are experimental systems used to simulate the skin damage process. For example, a model in which HaCaT cells and THP-1 cells are co-cultured in Transwell chambers, where HaCaT cells simulate keratinocytes and THP-1 cells simulate mononuclear macrophages.

[0064] Succinate dehydrogenase (SDH): Succinate dehydrogenase (SDH) is an enzyme located on the inner mitochondrial membrane that is responsible for catalyzing the conversion of succinate in the tricarboxylic acid cycle to fumarate, and also participates in the mitochondrial respiratory electron transport chain.

[0065] SDH activity: SDH activity refers to the ability of succinate dehydrogenase to catalyze the conversion of succinate to fumarate, which can be detected by methods such as enzyme histochemical staining (tetrazole salt method) and micro-method. SDH activity threshold: The SDH activity threshold refers to the level at which SDH activity decreases and can significantly affect the degree of skin damage, such as below 50% or below 70%.

[0066] Gene editing reagents: Gene editing reagents are reagents used to alter gene expression within cells, such as increasing SDH activity by overexpressing SDH subunit A.

[0067] Macrophage M1 polarization: M1 polarization refers to the polarization of macrophages towards a pro-inflammatory phenotype. Macrophages in this polarized state release a large number of pro-inflammatory factors, such as TNF-α, IL-1β and IL-6, leading to an exacerbation of the inflammatory response.

[0068] M1 polarization ratio of macrophages: The M1 polarization ratio refers to the proportion of M1 phenotype cells in macrophages, which is usually assessed by flow cytometry to detect the proportion of CD86 positive cells to CD68 positive cells.

[0069] TRAP1: TRAP1 (Tumor Receptor-Associated Protein 1) is a molecular chaperone protein located in mitochondria that can competitively bind to SDH, inhibiting its activity and thus affecting cellular metabolism and inflammatory responses.

[0070] TRAP1 expression level: TRAP1 expression level refers to the expression level of TRAP1 protein in cells or tissues, which can be detected by methods such as Western blotting or immunohistochemical staining.

[0071] TRAP1 inhibitors: TRAP1 inhibitors are compounds that can inhibit TRAP1 activity, thereby restoring SDH activity and reducing inflammatory responses and damage processes.

[0072] DN401: DN401 is a TRAP1 inhibitor that can restore SDH activity by inhibiting TRAP1 activity, thereby reducing UV-induced skin damage.

[0073] Intradermal injections: Intradermal injections are drug preparations administered via intradermal injection for the local treatment of skin lesions.

[0074] Topical preparations: Topical preparations are drug formulations that are applied topically to treat skin lesions.

[0075] Oral preparations: Oral preparations are drug formulations that are administered orally for the systemic treatment of skin lesions.

[0076] Example 1

[0077] 1. Changes in SDH activity under skin light exposure and after UVB irradiation.

[0078] 1.1 Fresh skin tissue from exposed areas (such as the face and outer arm) and non-exposed areas (such as the abdomen and buttocks) of the patient was collected after surgery (from the Biobank of the Institute of Dermatology, Chinese Academy of Medical Sciences). After being frozen in liquid nitrogen, succinate dehydrogenase was subjected to enzyme histochemical staining (tetrazole salt method, Beijing Solarbio Science & Technology Co., Ltd., #G2000). High activity of succinate dehydrogenase was manifested as blue-purple crystalline precipitate.

[0079] like Figure 1 As shown in -A, compared with unexposed skin tissue, SDH activity in exposed human skin tissue was significantly reduced, demonstrating the inhibitory effect of the exposure environment on SDH activity in skin tissue.

[0080] 1.2 A UVB-induced chronic skin injury model was constructed in mice to simulate long-term human sun exposure. The procedure was as follows: One day after hair removal on the backs of ICR mice, the mice were irradiated with gradient doses of UVB, with a dose of 100 mJ / cm² in the first week. 2 Subsequently, it increased by 100 mJ / cm² per week. 2 It increased to 400 mJ / cm until the fourth week. 2 The dosage was maintained until week eight, at which time mice were sacrificed and their tissues collected. The experiment was divided into a non-irradiated group (Sham) and a chronic UVB irradiation group. Sham meant that, except for actions not subject to UVB irradiation, the mice underwent similar hair removal, anesthesia, and grasping behaviors to the irradiated group. Approximately 20 mg of skin tissue was collected, fat was removed, and 200 μL of physiological saline was added. The tissue was then homogenized and centrifuged at 13300 rpm for 15 minutes at 4°C. The supernatant was collected, and a portion was analyzed for protein concentration using the BCA method (Thermo Fisher Scientific, #23227). The remaining portion was analyzed for SDH activity using a micro-method (Beijing Solarbio Science & Technology Co., Ltd., #BC0955). Finally, the SDH activity was divided by the protein concentration for correction.

[0081] like Figure 1 As shown in Figure B, compared with the unirradiated group, the SDH activity of the back skin of mice decreased after chronic UVB irradiation, indicating that ultraviolet exposure reduces SDH activity in mouse skin tissue.

[0082] 1.3 Cell Culture Procedure: The human keratinocyte cell line HaCaT was obtained from the China Collection of Culture Collections (CCTCC). Maintenance culture conditions were 5% CO2, 37℃ in a cell culture incubator, and the maintenance medium was DMEM containing 10% fetal bovine serum. One 25cm flask... 2 HaCaT cells that had grown stably in cell culture flasks were digested with trypsin and then seeded into a six-well plate. Once the cells reached 70% adhesion and growth density, they were treated with 50 mJ / cm² of liquid nitrogen. 2 Cells were collected 24 h after UVB irradiation. After sonication, the cells were centrifuged at 13,300 rpm for 10 minutes at 4°C. The supernatant was used for intracellular SDH activity detection using the micro-method, and protein concentration was determined by the BCA method to correct for SDH activity.

[0083] like Figure 1 As shown in -C, we found that UVB irradiation of HaCaT cells led to a decrease in intracellular SDH activity.

[0084] 1.4 The monocyte / macrophage cell line THP-1 was co-cultured with UVB-irradiated HaCaT cells and unirradiated HaCaT cells, respectively. The culture conditions were 5% CO2, 37℃ in a cell culture incubator, and the co-culture medium was RPMI 1640 medium containing 10% fetal bovine serum. The co-culture procedure was as follows: HaCaT cells were seeded into six-well plates and cultured for 24 h. The medium was replaced with fresh medium in the Sham group, and the UVB group was irradiated with 50 mJ / cm². 2 After UVB exposure, replace the culture medium with fresh medium. Insert a Transwell chamber (0.4 μm pore size) into the top of the six-well plate, and add THP-1 cells (1~2×10⁶ cells) to the chamber. 6 After co-culturing cells / wells for 24 h, THP-1 cells were collected from the chambers, lysed by sonication, and centrifuged at 13300 rpm for 10 minutes at 4°C. The supernatant was then collected for intracellular SDH activity (micro-method) detection and protein concentration determination to correct for SDH activity.

[0085] like Figure 1 As shown in Figure -D, we found that UVB irradiation of the HaCaT cell microenvironment reduced SDH activity in THP-1 cells.

[0086] Example 2

[0087] 1. Overexpression of SDH subunit A can reduce succinate levels and inhibit macrophage M1 polarization.

[0088] 1.1. Human full-length SDHA cDNA (NCBI reference sequence: NM_004168.4) cloned and inserted into an LV5 (EF-1a / GFP & Puro) lentiviral vector (Shanghai Jima Pharmaceutical Technology Co., Ltd.) to construct a lentivirus overexpressing (OE) human SDH subunit A (SDHA). Construction of SDHA-overexpressing macrophages: 2 × 10⁶ cells were seeded in 12-well plates. 5 THP-1 cells / well were cultured in 1 mL of RPMI 1640 medium containing 10% fetal bovine serum, with 1 μL of Polybrene (Shanghai Jima Pharmaceutical Technology Co., Ltd.). Lentiviral overexpressing SDHA was added according to an average number of viral particles (MOI) of 100 per cell, resulting in empty vector cells (containing meaningless sequences, NC) and overexpressing cells (OE-SDHA). After 48 h, the cells were cultured in normal medium for 7 days, and puromycin was added to select for stable culture. Once the cells were stable, SDHA gene expression in THP-1 cells was detected to confirm the successful construction of THP-1 cells overexpressing SDHA.

[0089] NC THP-1 cells and OE-SDHA THP-1 cells were divided according to... Figure 1 THP-1 cells were co-cultured with HaCaT using the -D method, and the following groups were established: NC THP-1 cells co-cultured with Sham HaCaT, OE-SDHA THP-1 cells co-cultured with Sham HaCaT, NC THP-1 cells co-cultured with UVB HaCaT, and OE-SDHA THP-1 cells co-cultured with UVB HaCaT. Intracellular succinate content and protein concentration were measured in all four groups of THP-1 cells to correct for succinate content.

[0090] like Figure 2 As shown in Figure -B, compared with NC THP-1 cells co-cultured with Sham HaCaT cells, NC THP-1 cells co-cultured with UVB HaCaT cells accumulated succinic acid. The succinic acid content in OE SDHA THP-1 cells decreased significantly after being affected by UVB irradiation (compared with NC THP-1 cells), indicating that increased SDHA expression in macrophages can reduce the accumulation of substrate succinic acid induced by UVB irradiation.

[0091] 1.2. Cell grouping and Figure 2Consistent with -A, NC THP-1 and OE SDHA THP-1 cells were collected for flow cytometry analysis. The procedure was as follows: after cell collection, FcR blocker (BD biosciences, #564219) was added for blocking, and CD68 FITC (eBioscience #11-0689-41) and CD86 Percpcy 5.5 (BioLegend, #374215) were incubated at 4°C in the dark for 20 minutes. After washing once with PBS, flow cytometry analysis was performed, and the proportion of CD86 positive cells to CD68 positive cells was used to represent macrophages with pro-inflammatory M1 polarization.

[0092] like Figure 2 As shown in Figure -B, NC THP-1 cells underwent pro-inflammatory M1 polarization after being influenced by UVB-irradiated HaCaT cells (compared to NC THP-1 cells co-cultured with unirradiated HaCaT cells). The proportion of M1 polarization in OE SDHA THP-1 cells was significantly reduced after being influenced by UVB-irradiated HaCaT cells (compared to NC THP-1 cells), indicating that macrophage overexpression of SDHA can reverse the increase in the proportion of CD86-positive macrophages caused by the UVB irradiation microenvironment.

[0093] Example 3

[0094] 1. TRAP1 competitively binds to SDH, inhibiting SDH activity. This study investigated the changes in TRAP1 expression and the regulatory effect of its inhibitor DN401 on SDH activity in light-exposed and cell co-culture systems.

[0095] 1.1 Fresh skin tissue from exposed areas (e.g., face, lateral arm) and non-exposed areas (e.g., abdomen, buttocks) of the human body was collected after surgery (from the Biobank of the Institute of Dermatology, Chinese Academy of Medical Sciences). The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with TRAP1 antibody (Wuhan Aibotek Biotechnology Co., Ltd., #A2748) using immunohistochemistry (IHC) to demonstrate the difference in TRAP1 expression between exposed and non-exposed skin tissues. Brown DAB staining in the cytoplasm indicated a positive result. After mounting, 400x images were acquired under a microscope, with at least three fields of view for each sample. The average grayscale was measured using ImageJ software (average grayscale is positively correlated with protein expression). The average grayscale of all fields of view for each sample was taken as the mean. Figure 3 The value of a sample in -B is calculated with 8 samples in the non-exposed area and 5 samples in the exposed area. Statistical analysis is performed using Graphpad software. Figure 3 -A represents the image. Figure 3As shown in Figure -B, the staining intensity of TRAP1 in human exposed skin tissue is higher than that in unexposed skin tissue, indicating that the expression level of TRAP1 is increased in human exposed skin tissue.

[0096] 1.2 After co-culturing THP-1 cells and HaCaT cells according to the 1-D method, THP-1 cells were collected. Cells were sonicated using RIPA lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd., #P0013C). The cells were then centrifuged at 13300 rpm for 10 minutes at 4°C, and the supernatant was collected for BCA protein quantification. 20 μg of protein was prepared, and 5× loading buffer was added. The mixture was then heated at 100°C for denaturation for 10 minutes, followed by SDS-PAGE gel electrophoresis at 160 V. The gel was transferred to a 0.2 μm PVDF membrane at a constant current of 0.25 A and the membrane was incubated for 1 hour. The membrane was then blocked for 5 minutes with rapid blocking buffer (BioRad, #12010020). The membrane was then blocked with TRAP1 antibody (Wuhan Aibote Biotechnology Co., Ltd., #A2748) and ACTB antibody (Cell Signaling Antibody). Technology, #4970) 4. After overnight incubation and TBST washing for 30 minutes, goat anti-rabbit antibody was incubated at room temperature for 2 hours, followed by TBST washing for 30 minutes and chemiluminescence development.

[0097] The results are as follows Figure 3 As shown in Figure C, compared with the THP-1 cell group co-cultured with unirradiated HaCaT cells, the expression level of TRAP1 in THP-1 cells co-cultured with UVB-irradiated HaCaT cells was significantly increased, indicating that UVB irradiation of keratinocytes can promote the expression of TRAP1 protein in co-cultured macrophages.

[0098] 1.3 TNF-α can promote macrophage polarization towards the pro-inflammatory M1 type, and TRAP1 can bind to the TNF receptor, mediating the downstream signaling pathway of TNF-α. The experimental groups included a control group, a TNF-α group, a TNF-α + 2.5 μM TRAP1 inhibitor DN401 group (MedChemExpress, HY-115781), and a TNF-α + 5 μM DN401 group. The experimental procedure was as follows: THP-1 cells were incubated at 10... 6 Cells were seeded at a density of 10 ng / mL in six-well plates, and 10 ng / mL of TNF-α and / or different concentrations of the TRAP1 inhibitor DN401 were added. After 24 h of maintenance culture, THP-1 cells were collected, and intracellular SDH activity was detected (micro-method). Protein concentration was determined using the BCA method to correct for SDH activity.

[0099] As shown in 3-D, TNF-α leads to a decrease in intracellular SDH activity in THP-1 cells, and 2.5 μM DN401 can increase the TNF-α-induced decrease in SDH activity. This indicates that TNF-α can inhibit SDH activity in macrophages by affecting TRAP1, and the TRAP1 inhibitor DN401 can reverse this inhibitory effect.

[0100] 1.4 We performed flow cytometry on the four groups of THP-1 cells described in 3-D, following the method described in 2-B, to analyze the proportion of CD86-positive cells to CD68-positive cells. For example... Figure 3 As shown in Figure -E, compared to the control group, the proportion of M1 pro-inflammatory macrophages increased after TNF-α stimulation of THP-1 cells. In the 2.5 μM DN401 group, the proportion of M1 pro-inflammatory macrophages decreased compared to the TNF-α group, indicating that TNF-α can promote macrophage polarization towards the pro-inflammatory M1 type by affecting TRAP1, and that the TRAP1 inhibitor DN401 can reverse this pro-inflammatory effect.

[0101] Example 4

[0102] 1. Intradermal injection of DN401 (1 mg / kg) can inhibit UVB-induced skin damage.

[0103] 1.1. Figure 4 -A shows representative results from animal experiments, illustrating changes in skin appearance in mice under different treatment groups. The experimental procedure was as follows: DN401 was dissolved in DMSO and diluted with corn oil to a concentration of 1 mg / kg. The matrix group consisted of a mixture of DMSO and corn oil. One day after shaving the backs of C57BL / 6 mice, they were administered 430 mJ / cm² of medication. 2 Mice were exposed to UVB radiation, and intradermal injections were administered at two points on the shaved back of mice one day before and the day after UVB radiation. Mice were sacrificed 48 hours after UVB radiation for imaging and tissue sampling. The experiment was divided into four groups: no irradiation (Sham), irradiation group (UVB), matrix group (UVB + matrix), and DN401 (1 mg / kg) intervention group (UVB + DN401). The skin responses of the four groups of mice to UVB radiation were observed and compared. Figure 4 As shown in -A, the UVB group mice showed obvious erythema and damage on their back skin, while the matrix group showed no significant relief. The DN401 intervention group showed a significant reduction in the degree of UVB-induced skin damage (such as erythema and lesions) compared to the matrix group, demonstrating that 1 mg / kg of DN401 can effectively prevent and inhibit UVB-induced skin damage.

[0104] 1.2.4-B is a quantitative evaluation statistical chart. A standardized scoring method (0-3 graded scores, with higher scores indicating more severe damage, assessed by two blind researchers and the average score used for statistical analysis) was employed to evaluate the degree of skin damage in mice from different treatment groups. The horizontal axis represents the area of ​​skin damage. For example... Figure 4 As shown in -B, DN401 prevents and improves skin erythema, edema and damage caused by UVB.

[0105] 1.3.4-C shows a histopathological image. Mouse skin samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for observation. Figure 4 - Microstructural changes in skin tissue of mice in different treatment groups (A). For example... Figure 4 As shown in -C, the unirradiated group (Sham) showed an intact dermis with regular cell arrangement and no obvious inflammatory cell infiltration. The UVB group mice showed thickened skin tissue, unclear dermal-epidermal boundary, disordered stratum corneum, and inflammatory cell infiltration in the dermis, indicating that UVB caused significant skin tissue damage and inflammatory response. The DN401 (1 mg / kg) intervention group mice showed reduced epidermal structure disorder, reduced inflammatory cell infiltration, and histopathological changes that were closer to normal skin, which is consistent with the gross observation results of 4-A and the quantitative scoring results of 4-B.

[0106] Example 5

[0107] 1. Intradermal injection of DN401 (5 mg / kg) can inhibit UVB-induced skin damage.

[0108] 1.1. A UVB-induced mouse skin damage model was constructed as described in Example 4. DN401 (5 mg / kg) or a matrix was injected intradermally one day before and the day after UVB irradiation. Mice were sacrificed 48 hours after UVB irradiation for imaging and tissue collection. The experiment was set up as a matrix injection group (UVB + matrix) and a DN401 (5 mg / kg) injection group (UVB + DN401). The skin responses of the two groups of mice to UVB irradiation were observed and compared. Figure 5 As shown in AB, compared to the matrix group, the DN401 (5 mg / kg) group significantly improved UVB-induced skin damage. Figure 5 As shown in -A, the mice in the matrix group showed obvious erythema and damage on their backs. The mice in the DN401 intervention group had less damage to their skin caused by UVB (such as erythema and lesions) compared to the mice in the matrix group, which proved that 5 mg / kg of DN401 can effectively prevent and alleviate skin damage caused by UVB.

[0109] 1.2 5-B is a histopathological image of the mice in 5-A. The mouse skin was stained with hematoxylin-eosin (HE) as described in Example 4, and the results were observed. Figure 5 -Microstructural changes in the skin tissue of mice in groups A. For example... Figure 5 As shown in -B, the skin tissue of mice in the matrix group showed thickening, unclear dermal-epidermal boundary, disordered epidermal structure, disappearance of the basal layer, and infiltration of inflammatory cells in the dermis, indicating that the matrix could not improve the skin tissue damage and inflammatory response caused by UVB. The degree of disordered epidermal structure, clear dermal-epidermal boundary, and reduced infiltration of inflammatory cells in the skin tissue of mice in the DN401 (5 mg / kg) intervention group were reduced, and the histopathological changes were closer to those of normal skin, which is consistent with the gross observation results of 5-A.

[0110] Example 6

[0111] 1. Topical application of DN401 (1 mg / kg) to the back skin failed to improve UVB-induced skin damage.

[0112] 1.1. A UVB-induced mouse skin damage model was constructed as described in Example 4. DN401 (1 mg / kg) or a matrix was applied to the back of the mouse the day before and daily after irradiation. Mice were sacrificed 48 hours after UVB irradiation, and images and tissue samples were collected. The experiment was set up as a matrix application group (UVB + matrix) and a DN401 (1 mg / kg) application group (UVB + DN401). The skin responses of the two groups of mice to UVB irradiation were observed and compared. Figure 5 As shown in -A, the matrix group mice showed obvious erythema and damage on their backs. Compared with the matrix group, the DN401 (1 mg / kg) topical administration group did not improve the skin damage caused by UVB.

[0113] 1.2 6-B is a histopathological image of the mice in 6-A. The mouse skin was stained with hematoxylin-eosin (HE) as described in Example 4. Figure 6 -Microstructural changes in the skin tissue of mice in groups A. For example... Figure 6 As shown in -B, the matrix group mice showed pathological changes such as thickening of the skin tissue, unclear dermal-epidermal boundary, disordered epidermal structure, and disappearance of the basal layer, indicating that the matrix could not improve the skin tissue damage and inflammatory response caused by UVB; the pathological changes of the skin tissue of the mice in the DN401 (1 mg / kg) application group were not improved, proving that the local application of 1 mg / kg DN401 on the back could not significantly improve the skin damage and inflammatory response caused by UVB.

Claims

1. The application of succinate dehydrogenase as a biomarker for preparing ultraviolet-induced skin damage, or the application of succinate dehydrogenase as a target for drug regulation of the TRAP1 signaling pathway.

2. The application according to claim 1, characterized in that, In the skin damage caused by ultraviolet radiation, the proportion of macrophage M1 polarization increased and / or the expression level of TRAP1 in skin tissue increased.

3. The application according to claim 2, characterized in that, In UV-induced skin damage, the proportion of macrophage M1 polarization is increased by at least 20% and / or TRAP1 expression is increased by at least 1.2-fold.

4. The application according to claim 1, characterized in that, The biomarkers are used for the qualitative and quantitative diagnosis, monitoring, or assessment of skin lesions.

5. A cell model for diagnosing, monitoring, or evaluating skin lesions, characterized in that, The cell model described above is used as a co-culture model of HaCaT cells and THP-1 cells; Control group: HaCaT cells were seeded and irradiated with ultraviolet light. THP-1 cells were added and co-cultured. THP-1 cells were collected and intracellular SDH activity was detected. Test group: Keratinocytes to be tested were seeded, THP-1 cells were added and co-cultured, THP-1 cells were collected, and intracellular SDH activity was detected.

6. The application of succinate dehydrogenase promoters in the preparation of drugs for the prevention and treatment of skin damage caused by ultraviolet radiation.

7. The application according to claim 6, characterized in that, The promoter can increase SDH activity in THP-1 cells / macrophages; and / or thereby inhibit the increase in M1 polarization ratio and TRAP1 expression.

8. The application according to claim 6, characterized in that, The promoters are: gene editing reagents that promote THP-1 cells / macrophages to enhance SDH activity, TRAP1 inhibitors, and DN401.

9. Application of DN401 in the preparation of drugs for treating skin damage caused by ultraviolet radiation.

10. The application according to claim 9, characterized in that, The DN401 is an intradermal injection, a topical ointment, or an oral preparation.

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