Use of METTL3 enzyme inhibitor in the preparation of a drug for vitiligo and the drug

Abstract

The present invention discloses the use of METTL3 enzyme inhibitors in the preparation of drugs for vitiligo and the drugs thereof, belonging to the technical field of vitiligo treatment. The present invention discloses the use of METTL3 enzyme inhibitors in the preparation of drugs for vitiligo, and the use of METTL3 enzyme inhibitors in the preparation of drugs for treating vitiligo, seeking a new treatment approach and drugs for treating vitiligo, thereby delaying the progression of vitiligo white patches and treating vitiligo.

Description

Technical Field

[0001] The present invention relates to the technical field of vitiligo treatment, and particularly to the application of METTL3 enzyme inhibitors in the preparation of vitiligo drugs and the drugs thereof. Background Art

[0002] Vitiligo is a depigmenting skin disease caused by the destruction of epidermal and mucosal melanocytes, which has a serious impact on the appearance and mental health of patients. In recent years, domestic and foreign studies have confirmed that the local immune microenvironment of the skin initiated by oxidative stress in environmental factors is disordered, thereby mediating CD8 + The key link in the pathogenesis of vitiligo is that T cells secrete toxic substances such as perforin, IFN-γ, and granzyme B to kill melanocytes. In the process of oxidative stress initiating autoimmunity, other resident cells in the local skin, such as keratinocytes and fibroblasts, also play an important role. However, when the functions of keratinocytes are abnormal due to various factors, they disrupt the immune homeostasis by paracrine of various pro-inflammatory factors, resulting in local epidermal immune microenvironment disorder, and then participate in the occurrence and progression of various skin diseases such as vitiligo. However, the specific pathogenesis of keratinocyte dysfunction and its subsequent involvement in vitiligo under the action of harmful external environmental stimuli is not fully understood.

[0003] Under the long-term harmful stimulation of external oxidative stress and other factors, epigenetic reprogramming occurs in the internal genetic material of cells, which can affect cell functions by regulating the expression of gene products and participate in the occurrence and development of various diseases, such as leukemia, melanoma, lung cancer, psoriasis, etc. Epigenetic modifications produce phenotypes that do not depend on the DNA sequence, the process is reversible and easily remodeled, and it plays an important role in different physiological and pathological states such as external stimuli, disease states, and developmental processes. Currently known epigenetic modification methods in eukaryotes mainly include DNA methylation, histone acetylation, and ubiquitination, etc. The METTL3 enzyme belongs to methyltransferase. Currently, STM2457 can inhibit the activity of the methyltransferase of METTL3 by binding to the SAM (S-adenosyl methionine) binding site of METTL3, thereby significantly reducing the growth and proliferation of cell lines from leukemia patients and inducing apoptosis to kill cancer cells. However, the application of this inhibitor in the treatment of vitiligo has not been found yet. Therefore, it is promising to become a new prospect for vitiligo treatment by studying the specific mechanism of epigenetic modification in oxidative stress-mediated abnormal immune response in vitiligo and combining METTL3 enzyme inhibition of vitiligo, providing a new strategy for future targeted treatment of vitiligo. Summary of the Invention

[0004] Therefore, the objective of the present invention is to find a new treatment approach and drug for vitiligo, inhibit the onset of vitiligo by inhibiting the METTL3 enzyme, and further delay the progression of vitiligo white patches so as to treat vitiligo.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] Use of a METTL3 enzyme inhibitor in the preparation of a drug for vitiligo.

[0007] Furthermore, the METTL3 enzyme inhibitor is STM2457.

[0008] Furthermore, the METTL3 enzyme inhibitor treats vitiligo by inhibiting the combined expression of METTL3 enzyme and YTHDC1, thereby reducing the expression of S100A9.

[0009] Experiments verified that the expression of METTL3 in different skin affected areas of vitiligo patients was significantly up-regulated compared with that of healthy human skin, and it was mainly expressed in keratinocytes. At the same time, keratinocytes enhanced the killing effect function of vitiligo CD8 + T cells and promoted the maturation of DC cells through paracrine S100A9, thereby promoting the onset of vitiligo. The present invention discovered a new use of the STM2457 inhibitor, which can inhibit the combined expression of METTL3 enzyme and YTHDC1 and thereby reduce the expression of S100A9 to treat vitiligo.

[0010] The present invention also discloses a drug for treating vitiligo, and the drug includes a METTL3 enzyme inhibitor.

[0011] Furthermore, the METTL3 enzyme inhibitor is STM2457.

[0012] Furthermore, the drug can be made into an injection, cream, ointment, or liniment for use.

[0013] Furthermore, STM2457 is made into a drug together with pharmaceutically acceptable excipients. When made into a cream, STM2457 can be used in combination with stearic acid, cetostearyl alcohol, liquid paraffin, triethanolamine, glycerol, disodium edetate, and petrolatum.

[0014] Furthermore, the concentration of STM2457 in the drug is 0.01 - 0.1%.

[0015] Beneficial effects:

[0016] The present invention discovers a new use of STM2457 inhibitor, which can inhibit the combined expression of METTL3 enzyme and YTHDC1, thereby reducing the expression of S100A9 to treat vitiligo. Moreover, through mouse experiments, it is known that after the STM2457 inhibitor is made into a cream, it can inhibit the onset of vitiligo, thereby delaying the progression of vitiligo white patches and treating vitiligo. Description of the Drawings

[0017] Figure 1 : METTL3 expression and statistical chart in the skin lesions of vitiligo patients and the epidermis of healthy people (A. Immunofluorescence detection of METTL3 expression in the skin lesions of vitiligo patients and the epidermis of healthy people; B. Statistical analysis of immunofluorescence intensity);

[0018] Figure 2 : Statistical chart of METTL3 expression in vitiligo skin lesions and normal blister skin (A. qRT-PCR detection of the expression level of METTL3 mRNA in vitiligo skin lesions and normal skin blister skin; B. Western-blot detection of the expression level of METTL3 protein in vitiligo skin lesions (L) and normal skin (N) blister skin);

[0019] Figure 3 : Statistical chart of METTL3 expression in keratinocytes under hydrogen peroxide stimulation (A. qRT-PCR detection of the expression level of METTL3 mRNA; B. Western blot detection of the expression level of METTL3 protein);

[0020] Figure 4 : Overall m6A level map of keratinocytes induced by oxidative stress (Colorimetric method for detecting the overall modification level of m6A);

[0021] Figure 5 : Map of interfering with METTL3 to down-regulate the overall m6A modification level in keratinocytes under oxidative stress (A-B. Western blot detection of the expression level of METTL3 protein; C. qRT-PCR detection of the expression level of METTL3 mRNA; D. Colorimetric method for detecting the overall m6A modification level in HaCaT);

[0022] Figure 6 : Comparison chart of S100A9 and CCL5 expression in the sera of vitiligo patients and healthy people (A-D. ELISA detection of the expression of SAA1, S100A9, CCL5, and LCN2 in the sera of rapidly progressive vitiligo patients and healthy controls);

[0023] Figure 7: Comparison charts of S100A9, SAA1, and CCL5 expressions in the blister fluid of vitiligo lesions and normal skin (A-D. ELISA was used to detect the expressions of SAA1, CCL5, S100A9, and LCN2 in the blister fluid of vitiligo lesions and normal skin);

[0024] Figure 8 : Comparison charts of S100A9 and SAA1 mRNA expressions in the blister epidermis of vitiligo lesions and normal skin (A-D. qRT-PCR was used to detect the expressions of SAA1, CCL5, S100A9, and LCN2 mRNA in the blister epidermis of vitiligo lesions and normal skin);

[0025] Figure 9 : Diagrams showing the downregulation of SAA1 and S100A9 expressions in HaCaT cells by interfering with METTL3 under oxidative stress (A C. qRt-PCR was used to detect the expression levels of SAA1 and S100A9 mRNA; B D. Western blot was used to detect the expression levels of SAA1 and S100A9 proteins);

[0026] Figure 10 : Construction of a lentivirus stable transfection HaCaT cell line with overexpressed METTL3 (Western blot was used to detect the expression level of METTL3 protein);

[0027] Figure 11 : Diagrams showing that METTL3 cooperates with YTHDC1 to promote S100A9 expression under oxidative stress (A C. qRT-PCR was used to detect the expression levels of SAA1 and S100A9 mRNA; B D. Western blot was used to detect the expression levels of SAA1 and S100A9 proteins);

[0028] Figure 12 : Diagrams showing that YTHDC1 improves the stability of S100A9 mRNA (A B. qRT-PCR was used to detect the expression levels of SAA1 and S100A9 mRNA);

[0029] Figure 13 : S100A9 significantly enhances the effector function of cytotoxic CD8 + T cells (A B. Flow cytometry was used to detect IFN-γ + CD8 + T cells, Perforin + CD8 + T cell ratio, ns indicates no significant difference);

[0030] Figure 14 : METTL3 in keratinocytes promotes the killing function of cytotoxic CD8 + T cells (A B. Flow cytometry was used to detect IFN-γ +CD8 + T cells, Perforin + CD8 + T cell ratio);

[0031] S100A9 significantly promotes the maturation of DC cells in vitiligo patients (detection of the ratios of CD80+CD11C+, CD86+CD11C+, HLA-DR+CD11C+ cells by flow cytometry)

[0032] METTL3 in keratinocytes promotes the maturation of DC cells (detection of the ratios of CD80+CD11C+, CD86+CD11C+, HLA-DR+CD11C+ cells by flow cytometry);

[0033] Figure 17 : Experimental diagram of vitiligo mice. Specific implementation manners

[0034] The present invention will be described in detail below in conjunction with specific embodiments and drawings:

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0036] Unless otherwise specified, all materials, reagents, etc. in the following examples can be obtained from commercial channels.

[0037] Example 1: Expression of METTL3 enzyme in vitiligo skin lesions

[0038] In order to clarify whether the expression of METTL3 enzyme in vitiligo skin lesions is abnormal compared with that in healthy people, vitiligo skin lesion samples should be obtained first.

[0039] Inclusion criteria for patients with progressive vitiligo: 1) Skin lesions in non-exposed areas; 2) Without other autoimmune diseases. Skin lesions of 8 patients with progressive vitiligo were collected in the vitiligo specialty clinic, excluding those with other autoimmune diseases; at the same time, skin tissues of 8 patients undergoing chest and abdominal plastic surgery with matched age and gender were collected in the plastic surgery department of our hospital. After obtaining the vitiligo and healthy control skin tissues, they were paraffin-embedded for subsequent immunofluorescence experiments. The experimental purpose was informed to the enrolled vitiligo patients and healthy volunteers, and it was certified by the ethics committee of our hospital.

[0040] Take paraffin sections of skin tissues from healthy people (n = 8) and skin lesion tissues of patients with progressive vitiligo (n = 8), and perform METTL3 immunofluorescence staining. The results showed that compared with healthy people, the expression of METTL3 in epidermal keratinocytes of patients with progressive vitiligo was up-regulated by 2.625 ± 0.873 times (P = 0.0037, n = 8, vs healthy controls) (the obtained results are as Figure 1-A), 10 field pictures were selected from each tissue section. The results of fluorescence intensity analysis showed that the expression of METTL3 in the epidermis of vitiligo patients was significantly higher than that in the healthy control group, and it was mainly expressed in keratinocytes( Figure 1 -B).

[0041] It can be seen from this that the expression of METTL3 in vitiligo lesions is significantly higher than that in healthy controls.

[0042] Example 2: Expression of METTL3 in the blister skin of vitiligo lesions

[0043] Since there are significant differences in m6A methylation modification among different individuals, we further analyzed the lesional skin and the suction blister skin of normal skin of 12 patients with stable vitiligo in autologous epidermal transplantation surgery. The blister skin samples of vitiligo should be obtained first.

[0044] Inclusion criteria for stable vitiligo: 1) The area of the original white patch has not expanded or new lesions have appeared in the past year; 2) And no Koebner phenomenon has occurred; 3) The vitiligo disease activity score (VIDA) is 0. 18 cases of lesional and non-lesional blister skins of patients with stable vitiligo who received autologous epidermal transplantation surgery were collected in the Department of Dermatologic Surgery. The suction blister skin tissues of normal skin and lesional skin during autologous epidermal transplantation surgery were collected and placed in 1.5 mL EP tubes containing tissue cryopreservation solution, and then frozen at -80 °C for subsequent experiments. All enrolled vitiligo patients were informed of the purpose of the experiment and certified by the Ethics Committee of our hospital.

[0045] After sample collection, proteins and total RNA were extracted from the blister skin respectively. The expression level of METTL3 mRNA was detected by qRT-PCR; the expression level of METTL3 protein was detected by Western blot.

[0046] The results showed that: (1) The expression level of METTL3 mRNA in the lesional blister skin of vitiligo patients was significantly up-regulated by 1.805 ± 0.5007 times compared with that in normal skin (P = 0.0022, n = 12, vs Non-Lesional skins, Figure 2 -A); (2) Western blot also showed that the protein expression level of METTL3 in the lesional blister skin of vitiligo patients was significantly increased compared with that in normal skin( Figure 2 -B).

[0047] Example 3: Effects of oxidative stress on the overall levels of METTL3 and m6A in keratinocytes

[0048] HaCaT cells (human immortalized keratinocytes) were cultured in DMEM medium supplemented with 10% fetal bovine serum, and routine cell culture was carried out in a cell incubator (5% CO2, 37 °C). Fresh medium containing serum was used when the cells were stimulated.

[0049] 1. Externally administer H2O2 to HaCaT cells to simulate and construct a vitiligo oxidative stress model. After treating HaCaT cells with different concentrations of H2O2 (100 μM, 200 μM, 300 μM) for 24 h, extract total cellular RNA and protein, and detect the expression level of METTL3 mRNA by qRT-PCR; detect the expression level of METTL3 protein by Western blot.

[0050] The results showed that: (1) After treatment with different concentrations of H2O2, the expression level of METTL3 mRNA in HaCaT cells was significantly upregulated ( Figure 3 -A); (2) The Western blot results showed that after treatment with different concentrations of H2O2, the expression level of METTL3 protein in HaCaT cells was significantly upregulated, and the change trend was consistent with that of mRNA ( Figure 3 -B). (*P < 0.05, ***P < 0.001, vs Control)

[0051] 2. After treating HaCaT cells with different concentrations of H2O2 (100 μM, 200 μM, 300 μM) for 24 h, determine the overall m6A modification level by colorimetry. The results showed that the overall m6A modification level in HaCaT cells was slightly increased after treatment with 100 μM and 200 μM H2O2, and the increase was most significant under the treatment condition of 300 μM H2O2 ( Figure 4 )(***P < 0.001, ****P < 0.0001, vs Control). Therefore, under the condition of treating HaCaT cells with 300 μM H2O2, the levels of METTL3 and overall m6A modification were significantly increased, and this concentration can be continuously used for subsequent experiments.

[0052] 3. Transfect five METTL3-siRNA interference fragments with different sequences and one control NC-siRNA interference fragment into HaCaT cells, and detect the METTL3-siRNA interference efficiency by Western blot. The subsequent experiments were carried out using the two interference fragments with the best interference efficiency. Transfect the METTL3-siRNA and NC-siRNA interference fragments into HaCaT cells. After 48 h, continue to treat HaCaT cells with 300 μM H2O2 for 24 h, and detect the expression levels of METTL3 protein and mRNA by qRT-PCR and Western blot respectively; determine the overall m6A modification level by colorimetry. Among them, five METTL3-siRNAs and two YTHDC1-siRNAs were designed and synthesized by Suzhou GenePharma Co., Ltd., and the sequences are shown in Table 1:

[0053] Table 1 siRNA (interference fragment) sequences

[0054]

[0055]

[0056] Results showed that: (1) Compared with the NC-siRNA group, the expression levels of METTL3 protein in HaCaT cells in the five different METTL3-siRNA interference groups were significantly decreased, indicating good interference efficiency ( Figure 5 -A); (2) Under the treatment condition of H2O2, the levels of METTL3 protein and mRNA in the METTL3-siRNA interference group were significantly decreased ( Figure 5 -B-C); (3) Under the treatment condition of H2O2, the overall m6A modification level increased, and the overall m6A modification level in the METTL3-siRNA interference group decreased ( Figure 5 -D). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs Control).

[0057] Example 4: Changes in protein expression levels in vitiligo patients

[0058] 1. Changes in the expression levels of S100A9 and CCL5 in serum

[0059] Collect serum samples: Inclusion criteria for patients with progressive vitiligo: 1) The area of the original white patch has expanded within the past 3 months; 2) Or there are new white patches; 3) Vitiligo disease activity score (VIDA) ≥ 4 points. 20 cases of blood from patients with rapidly progressive vitiligo and healthy individuals were collected at the vitiligo specialty clinic. 4 mL of venous blood from the above-mentioned patients with rapidly progressive vitiligo and healthy controls was collected in yellow clot activator tubes, centrifuged at 1300 rpm for 5 minutes, and the supernatant plasma was taken into 1.5 mL EP tubes and stored at -80 °C for later use; another 20 mL of peripheral venous blood from vitiligo patients was collected in EDTA anticoagulant purple tubes, and peripheral blood mononuclear cells (PBMCs) were isolated, and then immediately given different treatments for subsequent experiments. The vitiligo patients and healthy volunteers included in the study were informed of the experimental purpose and approved by the ethics committee of our hospital.

[0060] Serum from healthy individuals (n = 20) and patients with rapidly progressive vitiligo (n = 20) was taken respectively, and the expression levels of four secreted proteins, SAA1, S100A9, LCN2, and CCL5, were detected by ELISA.

[0061] Results showed that: (1) Compared with healthy individuals, there were no differences in the expression levels of SAA1 and LCN2 in the serum of patients with rapidly progressive vitiligo (ns, no significant difference, P = 0.5570 or 0.9828, Figure 6-A-C); (2) Compared with healthy individuals, the expression levels of S100A9 and CCL5 in the sera of patients with rapidly progressive vitiligo were significantly increased (*P < 0.05, **P < 0.01, vs Healthy Controls, Figure 6 -B-D). The above results suggest that the expressions of S100A9 and SAA1 in the sera of patients with rapidly progressive vitiligo are significantly higher than those of healthy controls.

[0062] 2. Changes in the expression levels of S100A9, SAA1, and CCL5 in blister fluids

[0063] Collection of vitiligo blister fluid samples: Inclusion criteria for patients with stable vitiligo: 1) The area of the original white patch has not expanded or new skin lesions have not appeared in the past year; 2) Koebner's phenomenon has not occurred; 3) The vitiligo disease activity score (VIDA) is 0. Twenty cases of blister fluids from lesional and normal skins of patients with stable vitiligo who underwent autologous epidermal transplantation were collected in the Department of Dermatologic Surgery. Ten milliliters of disposable syringes were used to aspirate the negative pressure blister fluids from normal and lesional skins during autologous epidermal transplantation. After centrifugation at 1000 rpm for 20 minutes to remove impurities, the samples were stored in 1.5 mL EP tubes and frozen at -80 °C for subsequent experiments. All enrolled vitiligo patients were informed of the purpose of the experiment and approved by the Ethics Committee of our hospital.

[0064] Due to the large methylation differences among different individuals, we further performed ELISA on 20 cases of lesional and 20 cases of normal skin negative pressure blister fluids from vitiligo patients during autologous epidermal transplantation. The results showed that: (1) There was no difference in the expression level of LCN2 in the blister fluids of vitiligo lesions compared with that of non-lesional skins (ns, no significant difference, P = 0.8022, vs Non-Lesional skins, Figure 7 -B); (2) Compared with non-lesional skins, the expression levels of SAA1, S100A9, and CCL5 in the blister fluids of vitiligo lesions were significantly increased ( Figure 7 -A-C-D) (**P < 0.01, ****P < 0.0001, vs Non-Lesional skins). The above results confirmed that the expressions of S100A9 and SAA1 in the blister fluids of lesions in patients with rapidly progressive vitiligo were significantly higher than those of non-lesional skins.

[0065] 3. Increased mRNA expressions of SAA1 and S100A9 in blister skins

[0066] The mRNA expression levels of four cytokines in the blister skins of lesional and normal skins from 12 pairs of vitiligo patients during autologous epidermal transplantation were detected by qRT-PCR. The results showed that: (1) Compared with non-lesional skins, the mRNA expressions of SAA1 and S100A9 in the blister skins of vitiligo lesions were significantly increased ( Figure 8-A-B); (2) Compared with the autologous normal skin blister skin, there was no difference in the expression level of LCN2 mRNA in the vitiligo lesional blister skin (ns, no significant difference, P = 0.9060, vs Non-Lesional skins, Figure 8 -D), the expression of CCL5 mRNA in the blister skin was too low to be detected ( Figure 8 -C) (**P < 0.01, ***P < 0.001, vs Non-Lesional skins). The above results confirmed that the expressions of S100A9 and SAA1 mRNA in the lesional blister skin of patients with rapidly progressive vitiligo were significantly higher than those in their own normal skin.

[0067] 4. Interfering with METTL3 in keratinocytes inhibits the expression of SAA1 and S100A9

[0068] It was found through Experiments 1 - 3 that the downstream target genes SAA1 and S100A9 of METTL3 were elevated in the serum, blister fluid, and blister skin of vitiligo patients. To verify the regulatory effect of METTL3 on SAA1 and S100A9, METTL3-siRNA and NC-siRNA were transfected into HaCaT cells, and after 48 h, 300 μM H2O2 was given for continuous treatment for 24 h. The expressions of SAA1 and S100A9 under hydrogen peroxide conditions were detected by qRT-PCR and Western blot. The results showed that in the METTL3-siRNA interference group, the mRNA levels of SAA1 and S100A9 decreased significantly, and the protein levels also decreased significantly ( Figure 9 )(*P < 0.05, **P < 0.01, ****P < 0.0001, vs Control).

[0069] Example 5: Effect of METTL3 co-regulated with YTHDC1 on the expression of S100A9

[0070] 1. Transfect METTL3-LV lentivirus into HaCaT cells, and after screening with 5 μg / ml puromycin, a stable HaCaT cell line overexpressing METTL3-LV lentivirus was obtained. The overexpression efficiency of METTL3-LV lentivirus was verified by Western blot ( Figure 10 ), and the results showed that the expression of METTL3 in HaCaT cells increased significantly after transfection with METTL3-LV, indicating that the overexpression efficiency was good and could be used for subsequent experiments.

[0071] In the METTL3-LV lentiviral stable transfected cell line, YTHDC1-siRNA and NC-siRNA interference fragments were transfected. After 48 h, 300 μM H2O2 was continued to be added for culturing for 24 h, and the mRNA and protein levels of SAA1 and S100A9 were detected by qRT-PCR and Western blot respectively. The results showed that: (1) In the OE-NC and OE-METTL3 transfection groups, interfering with YTHDC1 could reduce the mRNA expression of SAA1 and S100A9 ( Figure 11 -A-C); (2) In the OE-NC transfection group, interfering with YTHDC1 could reduce the protein levels of SAA1 and S100A9, but in the OE-METTL3 group, interfering with YTHDC1 reduced the expression of S100A9 but did not reduce the expression of SAA1 ( Figure 11 -B-D) (*P < 0.05, **P < 0.01, ****P < 0.0001, vs Control). The above results confirmed that the regulation of S100A9 by METTL3 depends on YTHDC1, while the regulation of SAA1 does not depend on YTHDC1.

[0072] 2. NC-siRNA, METTL3-siRNA, and YTHDC1-siRNA interference fragments were transfected into HaCaT cells. After 48 h, 5 μg / ml actinomycin D was continuously added and stimulated for 0, 3, 6, and 9 h respectively, and then total RNA was collected. The relative contents of the remaining mRNAs of SAA1 and S100A9 at different time periods were detected by qRT-PCR, and their stability was represented by the mRNA degradation rate. The results showed that: (1) The stability of both SAA1 and S100A9 mRNAs decreased in the METTL3-siRNA transfection group; (2) The stability of S100A9 mRNA decreased in the YTHDC1-siRNA transfection group, but the stability of SAA1 mRNA increased ( Figure 12 -A-B) (**P < 0.01, ***P < 0.001, vs Control). The above results confirmed that after METTL3 performs m6A modification on SAA1 and S100A9 mRNAs, it can improve their mRNA stability, and YTHDC1 can participate in the regulation of S100A9 mRNA stability, but does not affect the stability of SAA1 mRNA.

[0073] Example 6: Effect of S100A9 on the expression of perforin in CD8 + T cells of vitiligo patients

[0074] CD8 +T cells secrete toxic substances such as IFN-γ, granzyme B, and perforin, which can cause specific killing of melanocytes and then trigger vitiligo. Previous studies have verified that in keratinocytes of vitiligo patients, METTL3 combined with YTHDC1 promotes the expression of S100A9 by affecting the stability of S100A9 mRNA. To clarify the effect of S100A9 on the killing function of CD8 + T cells, next, peripheral blood of vitiligo patients was collected. After separating PBMCs, they were stimulated with CD3 / CD28 antibody-conjugated magnetic beads. At the same time, different concentrations of recombinant human S100A9 protein at 0, 5, 10, and 15 μg / ml were co-stimulated for 24 h. The proportions of IFN-γ + CD8 + T cells and Perforin+CD8+T cells in PBMCs were detected by flow cytometry. The results showed that 10 and 15 μg / ml of recombinant human S100A9 protein could significantly reduce the proportion of Perforin + CD8 + T cells in PBMCs, while having no effect on the proportion of IFN-γ + CD8 + T cells, indicating that the downstream target gene S100A9 of METTL3 affects the expression of perforin in CD8 + T cells ( Figure 13 -A-B) (ns means no significant difference, **P < 0.01, vs Control).

[0075] Example 7: High expression of METTL3 in keratinocytes promotes the killing function of CD8 + T cells

[0076] The above experiments confirmed that the downstream target gene S100A9 of METTL3 can stimulate CD8+T cells to secrete perforin. To explore whether the abnormality of METTL3 in keratinocytes directly affects the killing function of CD8 + T cells, we transfected NC-siRNA and NC-siMETTL3 interference fragments into HaCaT cells. After 48 h, 300 μM H2O2 was added and the cells were cultured for another 24 h, and then the supernatant was collected. Next, peripheral blood of vitiligo patients was collected and PBMCs were separated. The PBMCs were co-cultured with the supernatant collected in the previous step and stimulated with CD3 / CD28 antibody-conjugated magnetic beads. After 24 h, the proportions of IFN-γ + CD8 + T cells, Perforin + CD8 + T cells in PBMCs were detected by flow cytometry. The results showed that compared with the NC-siRNA group, after interfering with METTL3 in HaCaT cells, Perforin + CD8+ The proportion of T cells was significantly decreased, while IFN-γ + CD8 + The proportion of T cells showed no significant change in expression. Combining the above experiments further confirmed that abnormal METTL3 in keratinocytes can regulate the cytotoxic CD8 + T cell killing effect by regulating the secretion of S100A9.( Figure 14 -A-B) (ns no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, vs Control).

[0077] Example 8: S100A9 promotes the maturation of vitiligo DC cells

[0078] As a DAMPs, S100A9 can activate the innate immune response by binding to TLR4 and participate in various immune inflammatory processes. To further clarify the effect of S100A9 on DC maturation and antigen presentation, peripheral blood of vitiligo patients was drawn, and PBMCs were isolated and co-incubated with the supernatant of keratinocytes in the state of oxidative stress for 24 h, and stimulated with the S100A9 inhibitor Paquinimode for 24 h. The proportions of CD80 + CD11C + 、CD86 + CD11C + 、HLA-DR + CD11C + cells in PBMCs were detected by flow cytometry. The results showed that the S100A9 inhibitor Paquinimode under oxidative stress could significantly reduce the proportions of CD80 + CD11C + 、CD86 + CD11C + cells in PBMCs, but had no effect on the proportion of HLA-DR + CD11C + cells, indicating that the METTL3 downstream target gene S100A9 affects the DC cell maturation process( Figure 15 )(ns no significant difference, *P < 0.05 vs Control).

[0079] Example 9: High expression of METTL3 in keratinocytes promotes the maturation of DC cells

[0080] The above experiments confirmed that the downstream target gene S100A9 of METTL3 could promote the maturation of DC cells. To explore whether the abnormality of METTL3 in keratinocytes directly affects the maturation process of DC cells, we transfected NC-siRNA and NC-siMETTL3 interference fragments into HaCaT cells. After 48 h, 300 μM H2O2 was added and the cells were cultured for another 24 h, and then the supernatant was collected. Next, we extracted peripheral blood from vitiligo patients and isolated PBMCs, which were co-cultured with the supernatant collected in the previous step. After 24 h, the proportions of CD80 + CD11C + , CD86 + CD11C + , HLA-DR + CD11C + cells in PBMCs were detected by flow cytometry. The results showed that compared with the NC-siRNA group, the proportions of CD80 + CD11C + , CD86 + CD11C + cells, and HLA-DR + CD11C + cells in HaCaT cells with METTL3 interference were significantly decreased. The above experiments confirmed that the abnormality of METTL3 in keratinocytes could regulate DC maturation and thus participate in the innate immune response.( Figure 16 )(**P < 0.01 vs Control).

[0081] Based on comprehensive analysis, the above examples show that:

[0082] 1. Under oxidative stress, the abnormal m6A methylation modification mediated by METTL3 enzyme in keratinocytes of vitiligo patients can regulate the expression of pro-inflammatory factors such as S100A9 and SAA1. Keratinocytes enhance the killing effect of CD8 + T cells in vitiligo through paracrine S100A9 and promote the maturation of DC cells.

[0083] 2. The expression of METTL3 in the lesional epidermis and the blister skin of leukoderma patches in vitiligo patients was significantly up-regulated compared with that in the skin of healthy people, and it was mainly expressed in keratinocytes, suggesting that the expression level of the enzyme METTL3 was different in different skin affected parts of the same individual, that is, oxidative stress regulated the function of vitiligo keratinocytes by mediating the expression of methyltransferase METTL3.

[0084] 3. Analyze the expression of METTL3 downstream target genes in vitiligo. The results show that the secretion of S100A9 and CCL5 in the serum of patients with acute progressive vitiligo is significantly higher than that in healthy people. Moreover, the levels of CCL5, S100A9, and SAA1 are significantly increased in the lesional blister fluid. However, only the expressions of S100A9 and SAA1 are increased in the lesional blister epidermis, while the expression level of CCL5 in the epidermis is too low to be detected. Therefore, it is confirmed that the expressions of SAA1 and S100A9 in the local keratinocytes of vitiligo lesions are increased, mainly regulated by METTL3, and oxidative stress is the initiating factor mediating this result.

[0085] 4. Oxidative stress induces abnormal expression of METTL3 in keratinocytes and jointly promotes the expression of S100A9 mRNA with YTHDC1.

[0086] 5. S100A9 in vitiligo white patches regulates the immune response of CD8 + T cells, a key effector link in the pathogenesis of vitiligo, that is, S100A9 participates in the pathogenesis and development of vitiligo by regulating the local immune microenvironment of the epidermis. Under oxidative stress, the expression of the m6A modification enzyme METTL3 in the epidermal keratinocytes of vitiligo lesions is significantly increased, regulating the killing effect function of CD8 + T cells and the maturation process of DCs, and thus participating in the occurrence and development of vitiligo.

[0087] Based on the above conclusions, the present invention conducts experiments on the use of the METTL3 inhibitor STM2457 for the treatment of vitiligo.

[0088] Example 10: Preparation of STM2457 cream

[0089] Take 100 mg of STM2457 inhibitor, 36 g of stearic acid, 36 g of octadecanol, 72 g of liquid paraffin, 28 g of triethanolamine, 100 g of glycerol, 0.4 g of disodium ethylenediaminetetraacetate, 36 g of petrolatum, and 600 g of distilled water. The concentration of the STM2457 inhibitor is about 0.011%;

[0090] Dissolve the STM2457 inhibitor in an appropriate amount of dimethyl sulfoxide for later use;

[0091] Mix disodium ethylenediaminetetraacetate, triethanolamine, glycerol, and distilled water, and heat to 70 °C to melt. After melting, a water phase is prepared. Heat petrolatum, octadecanol, stearic acid, and liquid paraffin to 80 °C, and stir evenly to prepare an oil phase;

[0092] Slowly add the dissolved STM2457 inhibitor and the water phase to the oil phase, stop heating, and stir evenly by ultrasonic wave to obtain STM2457 cream.

[0093] Example 11: Application of METTL3 Enzyme Inhibitor in Treating Vitiligo Mice

[0094] 1. Experimental Materials

[0095] 1.1 Vitiligo mice: C57BL / 6 mice, female, raised under conventional laboratory conditions;

[0096] 1.1 Reagents: STM2457 cream prepared in Example 10;

[0097] 1.3 Establishment of vitiligo-like model:

[0098] Wild-type C57BL / 6J mice at 8-10 weeks of age and weighing about 20 g were used as background mice to induce a vitiligo mouse model.

[0099] On day 0, intradermal injection of well-cultured in vitro active mouse melanoma cell line B16F10 was inoculated into the shaved back of the mice, and 2×10 5 B16F10 cells were injected into each mouse.

[0100] On day 4, observe whether a tumor the size of a rice grain has formed at the inoculation site. And give the mice an intraperitoneal injection of CD4 neutralizing antibody, 10 μg of CD4 neutralizing antibody per gram of mouse.

[0101] On day 10, give the mice an intraperitoneal injection of the same dose of CD4 neutralizing antibody again.

[0102] On day 12, surgically remove the intradermal tumor tissue completely and suture the skin. Observe the state of the mice after tumor resection every day, and exclude the mice that develop recurrent melanoma after tumor resection from the group.

[0103] On the 30th day after induction of vitiligo in mice, whole-mount staining of the tail epidermis of vitiligo mice was performed to detect CD8 + T cell infiltration and judge the modeling situation of vitiligo mice.

[0104] 1.4 Medication: On the 35th day after induction of vitiligo in mice, mice with consistent CD8 + T cell infiltration in the tail epidermis of vitiligo mice were randomly divided into a negative control group and an experimental group, with 10 mice in each group.

[0105] Negative control group: Apply 50 mg of vaseline once a day; Experimental group: Apply 50 mg of STM2457 cream once a day. The mice in each group were smeared all over the body, and the medication was continued for 40 days. After stopping the drug for 15 days, observe the changes in the hair color and depigmentation area of the mouse tail.

[0106] 1.5 Evaluation criteria: Observe the hair depigmentation situation visually every day and record the scores. Divide the depigmented areas into the drug-treated areas and non-drug-treated areas, with a total score of 5 for each area and a total of 10 for each mouse.

[0107] Scoring criteria for hair depigmentation in mice:

[0108] 0 point indicates no hair depigmentation;

[0109] 1 point indicates a hair depigmentation area of 0 - 10%;

[0110] 2 points indicates a hair depigmentation area of 11% - 25%;

[0111] 3 points indicates a hair depigmentation area of 26% - 50%;

[0112] 4 points indicates a hair depigmentation area of 51% - 75%;

[0113] 5 points indicates a hair depigmentation area of 76% - 100%.

[0114] 1.6 Results and analysis:

[0115] The results obtained are shown in Table 1 and Figure 17 as follows:

[0116] Table 1

[0117] Group Incidence rate Time of decolorization appearance Average decolorization time Average integral Negative control group 100％ 40 days 44.1 days 4.5 Experimental group 62％ 65 days 68.3 days 3.1

[0118] Analysis:

[0119] Negative control group: Dot-like depigmentation started to appear on the 40th day of the experiment, and all mice showed depigmentation on the 44th day with the depigmented spots gradually expanding. When using petrolatum on the 40th day, the skin lesions expanded and partially merged; the average score of the depigmented area was 4.5 points.

[0120] Experimental group: Dot-like depigmentation started to appear on the 65th day of the experiment, and all mice showed depigmentation on the 67th day with the depigmented spots gradually expanding. When using STM2457 cream on the 40th day, the skin lesions slightly expanded and partially merged; the average score of the depigmented area was 3.1 points.

[0121] From the above results, it can be seen that STM2457 cream can delay the progression of vitiligo white patches and thus treat vitiligo.

[0122] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. Use of METTL3 enzyme inhibitor in the preparation of a medicament for treating vitiligo, characterized in that, The METTL3 enzyme inhibitor is STM2457.

2. The application according to claim 1, characterized in that, The METTL3 enzyme inhibitor treats vitiligo by inhibiting the combined expression of METTL3 enzyme and YTHDC1 and thereby reducing the expression of S100A9.

3. The application according to claim 1, characterized in that The STM2457 is made into a drug for use together with pharmaceutically acceptable excipients.

4. The application according to claim 3, wherein The drug is made into one of a cream, an ointment, a liniment, and an injection for use.

5. The application according to claim 4, wherein The excipients for making the cream include stearic acid, cetostearyl alcohol, liquid paraffin, triethanolamine, glycerol, disodium edetate, and petrolatum.

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