Use of inhibitors of mettl14 in the preparation of a medicament for the prevention and treatment of skin damage

Abstract

This invention discloses the application of METTL14 inhibitors in the preparation of drugs for the prevention and treatment of skin injuries. This invention also reveals for the first time the downregulation of METTL14 in human keratinocytes (HaCaT cells), human skin fibroblasts (WS1 cells), and animal tissues by ultraviolet B (UVB) radiation. 6 The downregulation of METTL14 protein levels, mediated by the reduction of METTL14 expression, can treat skin damage, providing a new approach for developing interventional targets for drugs treating UV-induced skin radiation damage.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the use of METTL14 inhibitors in the preparation of drugs for the prevention and treatment of skin lesions. Background Technology

[0002] Ultraviolet radiation is a type of non-ionizing radiation that is widely present in nature. Long-term or excessive exposure to ultraviolet radiation can lead to various skin diseases, including skin cancer, photoaging, and photosensitive dermatitis. Among them, ultraviolet radiation B (UVB) in sunlight has a wavelength of 280nm to 320nm and can penetrate the skin surface, making it the main type of skin damage. For the treatment of UVB radiation-induced skin damage, steroids and non-steroidal anti-inflammatory drugs are generally used to treat the erythema, pain, and itching caused by ultraviolet radiation. However, to date, there is no specific targeted therapy that can completely cure skin damage (Am J Clin Dermatol, 2004, 5(1)).

[0003] 9-47.). Therefore, the search for new drugs to prevent and treat ultraviolet (UV) skin damage is of practical significance.

[0004] N6-methyladenine (m 6 A) is the most abundant source of dynamic and reversible chemical modifications within RNA in eukaryotic cells, m 6 A-methylation plays a crucial role in regulating cell growth, differentiation, and response to environmental stimuli.

[0005] (FrontBioeng Biotechnol,2023,11:1143866.). m 6 A-methylation modification is composed of multiple components m 6 A-methyltransferase complex (MTC) catalyzes RNAm. 6 Methyltransferase 3 / methyltransferase 14 (METTL3 / METTL14) have been identified as two core components of MTC. They form a stable heterodimer and coordinately induce m6A methylation modification. Furthermore, METTL14 is an RNAmase. 6 A key factor in homeostasis. Methyltransferases such as METTL14 are formed by transferring methyl groups to the sixth nitrogen atom of adenine using S-adenosylmethionine (SAM) as a methyl donor. S-adenosylhomocysteine ​​(SAH) is a byproduct of the SAM reaction, which can regulate the methylation process by inhibiting the activity of methyltransferases (J BiomolScreen, 2016, 21(3):290-7.). RNAm 6There are no reports on precise intervention strategies targeting key molecules involved in UVB-induced skin damage mediated by METTL14 methylation. Therefore, the application of METTL14 inhibitors in UVB-induced skin radiation damage requires further research and exploration. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention investigated the correlation between METTL14 expression and UVB-induced skin damage, finding that during UVB irradiation, RNA m in human keratinocyte line HaCaT cells and skin fibroblast WS1 cells... 6 Downregulation of A levels, and subsequent studies on methylation-related enzymes, revealed that skin cells underwent UVB radiation-induced m... 6 The decrease in A levels is caused by a decrease in the expression of the methyltransferase METTL14.

[0007] The first objective of this invention is to provide the use of an inhibitor of METTL14 in the preparation of a medicament for the prevention and treatment of skin lesions.

[0008] Furthermore, the skin damage is UVB-induced skin damage.

[0009] Furthermore, the skin-damaging drug is used to reduce the expression of METTL14.

[0010] Furthermore, the inhibitor of METTL14 is SAH.

[0011] Furthermore, the dosage of SAH is 1-20 mg / kg; preferably 5 mg / kg.

[0012] Furthermore, SAH powder is added to a co-solvent to dissolve it, forming a homogeneous solution, which is then diluted with physiological saline to form a mixed solution for injection.

[0013] Furthermore, the cosolvent is dimethyl sulfoxide (DMSO).

[0014] Furthermore, the drug is used to prevent and treat skin tissue damage and / or inflammatory infiltration caused by UVB irradiation.

[0015] Furthermore, the drug is administered via subcutaneous injection.

[0016] A second objective of this invention is to provide a medicament for the prevention or treatment of skin lesions, which targets METTL14 and reduces the expression of METTL14.

[0017] Furthermore, the drug includes SAH.

[0018] Furthermore, the skin damage is UVB-induced skin damage.

[0019] Furthermore, the drug also includes pharmaceutical excipients, such as pharmaceutically acceptable salts, excipients, or carriers.

[0020] Furthermore, the dosage form of the drug is selected from water extract, powder, lotion, tincture, oil or aerosol.

[0021] The beneficial effects of this invention are:

[0022] This invention regulates m by targeting the METTL14 protease. 6 This invention uses the level of METTL14 protease to prevent and treat UVB-induced skin damage, revealing that decreased expression of METTL14 leads to reduced methylation levels in cells and animals, thereby treating skin damage. This invention has significant practical implications and promising applications for drugs and applications used to prevent and treat UV radiation-induced skin damage. Attached Figure Description

[0023] Figure 1 The m after 24 hours of UVB irradiation of human keratinocytes (HaCaT) and human skin fibroblasts (WS1) in Example 1 of this invention. 6 A. Methylation level;

[0024] Figure 2 In Example 2 of this invention, HaCaT and WS1 cells were irradiated with UVB for different durations (m). 6 Expression of A-related proteins;

[0025] Figure 3 This is the m of total RNA from mouse skin tissue in Example 3 of the present invention. 6 A methylation level ( Figure 3 A) and immunohistochemical detection of METTL14 protein expression in mouse skin tissue on day 4 after UVB irradiation ( Figure 3 B);

[0026] Figure 4 In Example 3 of this invention, SAH inhibits the expression of METTL14 protease, thereby regulating m 6 A schematic diagram of level A;

[0027] Figure 5 This is a diagram showing the skin damage of mice in different groups of mice on day 9 after UVB irradiation in Example 4 of the present invention;

[0028] Figure 6 This is a graph showing the number of days of UVB irradiation and skin damage scores for different mouse groups in Example 4 of this invention;

[0029] Figure 7 This refers to the changes in the pathological morphology of mouse skin tissue in each group on the 9th day after UVB irradiation in Example 3 of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] Example 1: HaCaT and WS1 cells after UVB irradiation m 6 Level A testing

[0032] In this embodiment, m 6 An RNA methylation quantitative detection kit was purchased from Abcam (catalog number ab185912), and an RNA rapid extraction kit was purchased from Shanghai Yishan Biotechnology Co., Ltd. (RN001).

[0033] The instruments used in this embodiment are as follows: the cell UVB irradiation uses a Japanese SANKYO DENKI UVB lamp with an emission wavelength of 280-320nm and an irradiation distance of approximately 20cm. The irradiation time is adjusted using a UV test chamber from Shenzhen Xinhengsen Instrument Equipment Co., Ltd., and the dose is calibrated using a Korean Genuv ultraviolet radiation meter.

[0034] Human keratinocytes (HaCaT) and human skin fibroblasts (WS1) in the exponential growth phase were seeded into culture dishes. When the cell confluence reached 70%-80%, they were irradiated with UVB and cultured for another 24 hours at 37°C in a 5% CO2 incubator. Cells were then collected and analyzed using a rapid RNA extraction kit and m... 6 A quantitative RNA methylation detection kit for m 6 A. Methylation level detection.

[0035] like Figure 1 As shown, the m of total RNA in HaCaT and WS1 cells 6 A levels were significantly reduced (t = 4.75, 4.36, P < 0.05).

[0036] Example 2: HaCaT and WS1 cells after UVB irradiation m 6 A-methylation-related enzyme detection

[0037] The cell irradiation instrument used in this embodiment is the same as that in Embodiment 1.

[0038] HaCaT and WS1 cells in logarithmic growth phase were seeded into culture dishes. When the cell confluence reached 70%-80%, they were irradiated with UVB and cultured at 37°C in a 5% CO2 incubator for 6, 12, and 24 hours. Cells were then lysed using RIPA and Western blot analysis was performed. 6 A-related enzymes were identified, and the expression level of the target protein was analyzed using ImageJ software.

[0039] like Figure 2 As shown, in HaCaT and WS1 cells, METTL14 protein expression decreased over time after irradiation, exhibiting a time-dependent effect; while in other m 6 A-related proteases showed no time-dependent changes (t = 6.39, 2.93, 4.76, P < 0.05), suggesting that skin cells m after UVB radiation... 6 The decrease in A levels is caused by a decrease in the expression of the methyltransferase METTL14.

[0040] Example 3: Mouse skin tissue after UVB irradiation (m) 6 Level A testing

[0041] In this embodiment, m 6 The ARNA methylation quantitative detection kit and the RNA rapid extraction kit are the same as in Example 1. The immunohistochemical staining kit was purchased from Beijing Solarbio Co., Ltd.

[0042] In this example, SPF-grade C57BL / 6J mice, female, 6-8 months old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and raised in the SPF-grade animal room of the Animal Experiment Center of Soochow University under constant temperature of 37℃, relative humidity of 60%, circadian rhythm alternation of 12h each, and free access to food and water.

[0043] The instrument used in this embodiment is a French Bio-Sun ultraviolet irradiator, with a UVB lamp emitting a wavelength of 312nm and a dose of 150mJ / cm². 2 The images were observed and analyzed using an optical microscope (Olympus IX73, Japan). Pairwise independent t-tests were performed using GraphPadprism 6 statistical software, with p < 0.05 considered statistically significant.

[0044] Experimental conditions: Eight SPF-grade C57BL / 6J mice were randomly divided into two groups: an irradiated group and a non-irradiated group. All mice were shaved on their backs for 48 hours and then given a single dose of 150 mJ / cm². 2 Skin tissue was collected on day 4 after UVB irradiation and analyzed using a rapid RNA extraction kit and m 6 ARNA methylation quantitative detection kit for m 6 Methylation levels were detected, and the expression of METTL14 protein was detected using an immunohistochemical kit.

[0045] like Figure 3 As shown in Figure A, the m-value of total RNA in mouse skin tissue after irradiation... 6 A levels were significantly reduced (t = 3.07, P < 0.05).

[0046] like Figure 3 As shown in Figure B, the expression of METTL14 protein in mouse skin tissue decreased after 4 days of UVB irradiation.

[0047] like Figure 4 The diagram shown illustrates the experimental principle and mechanism of this invention, which regulates m by inhibiting the expression of METTL14 protein through SAH. 6 The level of A can then be used to treat skin lesions.

[0048] Example 4: Subcutaneous injection of SAH solution to treat UVB-induced skin damage in mice

[0049] The animal source and breeding conditions in this implementation case are the same as in Example 3.

[0050] The reagents used in this example were sourced as follows: S-adenosyl homocysteine ​​(SAH) was purchased from MedChemexpress, USA. The HE staining kit was purchased from Solarbio, Beijing.

[0051] The instruments used in this embodiment are the same as those in Embodiment 3. GraphPadprism 6 statistical software was used to perform pairwise independent t-tests, with p < 0.05 considered statistically significant.

[0052] Experimental conditions: Twelve SPF-grade C57BL / 6J mice were randomly divided into three groups: a control group (saline group), a SAH 1 mg / kg group, and a SAH 5 mg / kg group. All mice were shaved on their backs for 48 hours and then given a single dose of 150 mJ / cm². 2 Mice were subjected to UVB irradiation, and twice consecutively injected subcutaneously with physiological saline or SAH solutions of different concentrations before and after irradiation. Observation continued until skin damage in mice healed. The preparation process of the SAH solution in the treatment group was as follows: SAH powder was dissolved in dimethyl sulfoxide (DMSO) to form a 10 mM SAH solution. The SAH solution was diluted with physiological saline to a certain concentration of mixed solution. The SAH mixed solution was injected into the mice, and the dosage was related to the mouse's body weight. The mouse model skin UVB radiation damage was scored based on erythema, skin swelling and hardening, and skin integrity. A four-level objective score from 0 to 3 was established for the color of the erythema on the mouse's back, the degree of skin swelling and hardening, and the depth of skin damage, where 0 represents none, 1 represents mild, 2 represents moderate, and 3 represents severe. The scores were combined to comprehensively assess the degree of UVB radiation damage to the mouse skin. The specific scoring criteria are shown in Table 1.

[0053] Table 1

[0054]

[0055] Experimental results: such as Figure 5 As shown in Figure 6, 150 mJ / cm 2On day 7 after UVB irradiation, the skin lesion score of the SAH 5mg / kg group was significantly different from that of the control group; by day 9 after irradiation, most of the skin lesions in the SAH 5mg / kg group mice had healed. These results indicate that SAH can alleviate 150mJ / cm² irradiation. 2 The severity of skin damage caused by UVB irradiation and the promotion of skin recovery.

[0056] After fixing, dehydrating, and paraffin-embedding mouse dorsal tissue, sections were stained with hematoxylin and eosin (HE). The HE staining results showed that... Figure 7 In the control group (saline group), the tissue structure of mice was disordered, and a large number of inflammatory cells were infiltrated. The SAH 1mg / kg group was similar to the control group, but less severe. The skin of mice in the SAH 5mg / kg group had orderly arranged structure in each layer, with a few inflammatory cells infiltrated.

[0057] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The use of METTL14 inhibitors in the preparation of drugs for the prevention and treatment of skin lesions, characterized in that, The skin damage is UVB-induced skin damage, and the inhibitor of METTL14 is S-adenosylhomocysteine.

2. The application as described in claim 1, characterized in that, The dosage of the METTL14 inhibitor is 1-20 mg / kg.

3. The application as described in claim 1, characterized in that, The inhibitor of METTL14 is administered via subcutaneous injection.

4. The application as described in claim 1, characterized in that, The drug is used to prevent and treat skin tissue damage and / or inflammatory infiltration caused by UVB irradiation.

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