Use of mettl3 as a target in the preparation of a skin anti-aging drug or skin care product

Abstract

The application relates to the field of biological agents, and more specifically, to the application of METTL3 as a target point in the preparation of skin anti-aging drugs or skin care products, which promotes the expression of collagen in human skin fibroblasts by targeting and inhibiting the expression of METTL3, and the expression of METTL3 is also increased in the inflammation model, which also increases the expression of collagen in human skin fibroblasts; by m 6 A epigenetic modification increases the expression of human skin endogenous collagen, which provides a strategy for the development of anti-aging products.

Description

Technical Field

[0001] This application relates to the field of biological agents, and more specifically, to the application of METTL3 as a target in the preparation of drugs or skin care products for skin anti-aging. Background Technology

[0002] The skin is the largest organ in the human body. The collagen and elastin networks in the epidermis and dermis, along with subcutaneous adipose tissue, produce the skin's biomechanical and physiological properties. Factors such as hormones, ultraviolet radiation, and the external environment can affect the skin's appearance, structure, and integrity. During the aging process, the skin undergoes qualitative and quantitative changes, such as loss of elasticity, thinning of the epidermis, reduction in collagen content, and increased wrinkles.

[0003] Collagen plays a crucial role in skin anti-aging. It is a major structural protein of the skin, essential for its elasticity, firmness, and overall health. As we age, the amount of collagen in the skin gradually decreases, leading to signs of aging such as sagging and wrinkles. Therefore, supplementing collagen and promoting its synthesis are key anti-aging strategies. Currently, most skincare products on the market primarily supplement collagen from external sources, with fewer products promoting the skin's own synthesis. Therefore, this application aims to target and promote the expression of endogenous collagen in the skin.

[0004] Epigenetics is the science of influencing an organism's traits by regulating gene expression without altering the DNA sequence. Epigenetic regulatory mechanisms play a crucial role in the skin aging process. 6 A,N6-methyladenosine (AMA) modification is a post-transcriptional modification of mRNA and long non-coding RNA ubiquitous in eukaryotes, playing a crucial regulatory role in RNA splicing, maturation, degradation, and translation. In the field of skin, m... 6 A modification is closely related to skin regeneration, wound healing, scar formation, and the development of various skin diseases. In vivo, m 6 A modification is mainly regulated by three types of protein molecules, including the "writer" protein. 6 A methyltransferase, "eraser" m 6 A demethylase, "reader" m 6 A. A binding or recognition protein. METTL3, as an mRNA methyltransferase, primarily catalyzes the methylation of adenosine nucleotides on mRNA. 6METTL3 is modified and associated with the development of various diseases, including cancer, cardiovascular disease, and autoimmune diseases. Its role in skin health and disease has also attracted researchers' attention. For example, in inflammatory diseases, changes in METTL3 expression, target genes of modification, and pathogenesis are being studied. In research on skin photoaging, METTL3 is also considered to potentially influence the skin's response to photoaging by regulating certain key biological processes, such as autophagy. In general, the role of METTL3 in skin health and disease is a multifaceted research area involving various biological processes and disease mechanisms. With further research, METTL3 may become a new target for the treatment of skin-related diseases. Summary of the Invention

[0005] To promote the expression of endogenous collagen in the skin, this application provides the use of METTL3 as a target in the preparation of anti-aging drugs or skin care products.

[0006] The following technical solution is adopted: the application of METTL3 as a target in the preparation of anti-aging drugs or skin care products.

[0007] METTL3 as a target inhibitor can directly promote the increased mRNA and protein expression of various collagen proteins such as COL1A1, COL1A2, and COL3A1 in human skin fibroblasts. This reveals the role of METTL3 from a novel perspective of promoting endogenous collagen synthesis.

[0008] Preferably, the application includes the preparation of drugs or skin care products for skin anti-aging by METTL3 inhibitors that specifically inhibit or specifically interfere with METTL3 gene expression.

[0009] Preferably, the METTL3 inhibitor includes small molecule compounds that specifically inhibit METTL3 and interfering molecules that specifically interfere with METTL3 gene expression.

[0010] Preferably, the METTL3 inhibitor directly inhibits the activity of METTL3, reducing m 6 A level, or specifically inhibit METTL3 gene expression, or target and inhibit METTL3 protein activity.

[0011] Preferably, the drug or skincare product comprises a medically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient directly inhibits the activity of METTL3 and reduces m 6 A level, or specifically inhibit METTL3 gene expression, or target and inhibit METTL3 protein activity.

[0012] Preferably, the small molecule compound that specifically inhibits METTL3 is STM2457.

[0013] Preferably, the interfering molecule that specifically interferes with the expression of the METTL3 gene is shMETTL3, and the sequence of shMETTL3 is GCCAAGGAACAATCCATTGTT.

[0014] Preferably, the drug or skin care product includes one or more of the following: ointment, gel, cream, serum, and liquid.

[0015] In summary, this application has the following beneficial effects: This application provides the use of METTL3 inhibitors in the preparation of anti-aging drugs or skin care products. By targeting and inhibiting the expression of METTL3, it promotes the increase of collagen expression in human skin fibroblasts. Furthermore, in an inflammatory model, inhibiting the expression of METTL3 also increases the expression of collagen in human skin fibroblasts.

[0016] This application provides a novel target and application for skin anti-aging by promoting the synthesis of endogenous collagen in the skin.

[0017] This application was approved by m 6 Epigenetic modifications enhance the expression of endogenous collagen in human skin, providing a strategy for developing anti-aging products. Attached Figure Description

[0018] Figure 1 shMETTL3 induces increased collagen expression in human skin fibroblasts; a concentration of 1×10⁻⁶ was used. 8 Human skin fibroblasts were infected with viral fluid containing TU / ml and MOI=20. Fluorescence images (A), western blotting (B), and quantitative real-time PCR (C) showed that shMETTL3 successfully knocked down METTL3 expression. Quantitative real-time PCR results showed changes in mRNA levels of COL1A1 (D), COL1A2 (E), and COL3A1 (F) after METTL3 knockdown. A t-test was used for analysis. * indicates a comparison with SCR; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0019] Figure 2To investigate the effect of METTL3 inhibitors on increased collagen expression in human skin fibroblasts, different concentrations of inhibitors were used for treatment for 4 hours. Quantitative real-time PCR was used to detect changes in the gene levels of METTL3 (A), COL1A1 (C), COL1A2 (D), and COL3A1 (E). Western blotting was used to detect changes in the protein level of METTL3 (B). A t-test was used for analysis. * indicates a comparison with DMSO; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0020] Figure 3 To investigate the downregulation of collagen expression in an IL-1β-induced aging model, HSF was treated with 20 ng / ml IL-1β for different time periods, and the gene levels of IL-6 (A), COL1A1 (B), COL1A2 (C), and COL3A1 (D) were detected by quantitative real-time PCR. Two-way ANOVA analysis was performed. * indicates comparison with the control group (PBS), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0021] Figure 4 To investigate the effect of shMETTL3 on reversing collagen expression in a human skin fibroblast aging model, HSF cells were transfected with shMETTL3 for 18 hours, cultured for another 6 days, and then treated with IL-1β for 16 hours. Fluorescence images (A), western blotting (B), and quantitative PCR (C) showed that shMETTL3 successfully knocked down METTL3 expression. Western blotting detected changes in COL1A1 and COL3A1 protein levels, and ImageJ analysis of COL1A1 protein results (B) was performed. Quantitative PCR detected changes in the gene levels of IL-6 (D), COL1A1 (E), COL1A2 (F), and COL3A1 (G). Two-way ANOVA analysis was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0022] Figure 5To investigate collagen expression in a human skin fibroblast aging model induced by STM2457 reversal, HSF was treated with IL-1β for 16 h, followed by STM2457 treatment for 4 h. Quantitative real-time PCR was used to detect changes in the gene levels of IL-6 (A), METTL3 (B), COL1A1 (C), COL1A2 (D), and COL3A1 (E). Western blotting was used to detect changes in COL1A1 protein levels, and ImageJ analysis was performed (F). Two-way ANOVA was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0023] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0024] 1. Experimental Methods 1.1 Cell Culture 1.1.1 Cell passage HSF cells are adherent cells, spaced at 5 cm intervals. 2Cells were cultured in a culture flask with 1 ml of DMEM complete medium (containing 10% FBS). When the cells in the logarithmic growth phase reached 80%–90% confluence with the bottom of the flask, contact inhibition began to appear. At this point, the cells were digested with trypsin solution for passage. The original medium in the cell culture flask was aspirated, and 1 ml of 1×PBS buffer was added to the flask to cover the bottom. The flask was gently shaken to wash the bottom and remove apoptotic cells and residual medium. This washing was repeated twice. 1 ml of 0.25% trypsin (containing EDTA) was added to cover the bottom of the flask. The cell culture flask was then placed in a 37°C CO2 cell culture incubator for approximately 1 minute to digest the cells, disrupting cell junctions and cell-to-flask contact. The flask was then removed from the incubator and observed under a light microscope. At this point, most cells should have lost their original shape and detached from the bottom of the flask. When the cells were observed to move in a quicksand-like motion when gently tapped from the side of the flask, the cell digestion was successful. Then, quickly add 3 ml of complete cell culture medium to stop the digestion, and gently pipette the solution to completely detach the digested cells from the bottom of the cell culture flask. Transfer the cell suspension to a 15 ml sterile centrifuge tube and centrifuge at 1000×g for 5 min at room temperature. After centrifugation, carefully aspirate the supernatant and discard it into the waste container. Then, add an appropriate amount of fresh complete cell culture medium to the cell pellet at the bottom of the tube and gently pipette to mix the cell pellet, forming a homogeneous single-cell suspension. Seed the mixed cell suspension into cell culture flasks at a ratio of 1:3, mix gently, and then incubate at 37°C in a CO2 incubator.

[0025] 1.1.2 Cell cryopreservation Select cells in the logarithmic growth phase, i.e., with a cell confluence of approximately 70%–80%. Remove the culture medium from the culture flask using a pipette. Add 1 ml of 1×PBS solution to the culture flask until it just covers the bottom. Wash the culture flask, repeating twice. Add 0.25% trypsin containing EDTA and digest in a cell culture incubator for 1 min until most cells detach from the bottom of the flask. Then add 3 ml of complete DMEM culture medium to stop digestion. Gently pipette the cells to mix, then transfer them to centrifuge tubes and centrifuge at 1000×g for 5 min. After centrifugation, discard the supernatant, add an appropriate amount of serum-free cell cryopreservation medium, and gently pipette to mix and resuspend the cells. Aliquot the cell resuspended solution into sterile cryovials, adding 1 ml of cell suspension to each tube. Place the cryovials directly in a cryovial box and store at -80°C overnight. Afterward, transfer the frozen cells to a liquid nitrogen container for preservation.

[0026] 1.1.3 Cell resuscitation Take a vial of cryopreserved cells from the liquid ammonia tank and quickly place it in a 37°C water bath for about 1 minute to thaw. Once the cell suspension has thawed to a size of only a soybean, transfer the cryopreservation tube to room temperature. At this point, the cells have completely frozen and thawed. Transfer the thawed cells to a centrifuge tube using a pipette, add 3 ml of complete culture medium, and centrifuge at 1000×g for 5 minutes. Discard the supernatant, resuspend the cells in 4-5 ml of complete culture medium, and transfer them to a sterile culture flask of appropriate size. Gently shake the flask to mix using a cross-hatching method and observe under a microscope. Then, incubate the cells at 37°C in a 5% CO2 incubator. After overnight incubation, the thawed cells adhere to the culture medium. This application involves changing the culture medium after thaw to remove any non-adherent, dead cells.

[0027] 1.2 Lentiviral transfection experiment Prepare a density of 3×10 using a complete culture medium. 4 Inoculate 2 ml / well of cell suspension into 6-well plates using 1 / ml of cell suspension. Incubate at 37°C, 5% CO2 for 24 h until cell confluence reaches 20-30%. Calculate the required amount of virus based on cell MOI and viral titer, then add 40 μl of infection reagent, and finally add complete culture medium to a final volume of 1 ml to prepare the infection solution. Add 1 ml / well of infection solution to each 6-well plate and incubate at 37°C, 5% CO2 for 18 h. Then, replace with complete culture medium and continue incubation for 48 h. Add 5 μg / ml Puromycin to each 6-well plate and incubate at 37°C, 5% CO2 for 48 h. Change the medium and incubate for another 24 h, then harvest RNA or protein samples.

[0028] 1.3 RNA Extraction 1.3.1 Sample Processing Discard the cell culture medium and wash once with 1×PBS. Add 500 μl of RNA-easy to each well of a six-well plate, ensuring it fully covers the cell surface, and then pipette the cells off the plate. Transfer the lysis buffer to centrifuge tubes and pipette repeatedly until complete lysis is achieved.

[0029] 1.3.2 RNA Extraction Add 200 μl of RNase-free ddH2O to the above lysis buffer, mix by inverting, and incubate at room temperature for 5 min. Centrifuge at 12000×g for 15 min at room temperature. Remove the centrifuge tube; the solution will now separate into an upper aqueous phase (containing RNA) and a dark lower precipitate (containing proteins, DNA, polysaccharides, and other impurities). Carefully aspirate the upper aqueous phase into a new centrifuge tube. Add an equal volume of isopropanol, mix by inverting, and incubate at room temperature for 10 min. Centrifuge at 12000×g for 10 min at room temperature; a white precipitate will usually be visible. Carefully discard the supernatant. Add 500 μl of 75% ethanol (prepared with RNase-free ddH2O), gently tap the bottom of the tube to resuspend the precipitate, and invert several times. Centrifuge at 8000×g for 3 min at room temperature and discard the supernatant. Repeat the process once more, adding 500 μl of 75% ethanol, gently tapping the bottom of the tube to resuspend the precipitate, and inverting several times. Centrifuge at 8000×g at room temperature for 3 min, discarding the supernatant. Allow to air dry at room temperature, add an appropriate amount of RNase-free ddH2O to dissolve the precipitate, vortex at room temperature for 3 min to fully dissolve the RNA precipitate, and store at -80℃ for long-term storage.

[0030] 1.3.3 Purity and Concentration Detection Product purity was measured using an ELISA reader; an OD260 / OD280 ratio between 1.8 and 2.2 indicated high RNA purity. Product concentration was also measured using an ELISA reader.

[0031] 1.4 cDNA Synthesis 1.4.1 Genomic DNA Removal Reaction Prepare the reaction mixture on ice according to the ingredients in Table 1. To ensure the accuracy of the reaction mixture preparation, when performing each reaction, first prepare the Master Mix according to the reaction number + 1, then dispense it into each reaction tube, and finally add the RNA sample.

[0032] Table 1 Genomic DNA Removal System After thoroughly mixing the above solution, vortex and centrifuge, react at 42°C for 2 min in a PCR instrument, and then transfer to ice at 4°C.

[0033] 1.4.2 Reverse transcription reaction Prepare the reaction solutions on ice according to the ingredients in Table 2. To ensure the accuracy of the reaction solution preparation, prepare the Master Mix by the amount of reaction number + 1 before each reaction, and then dispense 10 μl into each reaction tube. Gently mix and immediately proceed with the reverse transcription reaction.

[0034] Table 2 Reverse Transcription Reaction System After the above reagents are mixed evenly, the reaction is carried out in a PCR instrument at 37°C for 15 min, 85°C for 5 sec, and finally maintained at 4°C until the end.

[0035] 1.5 Quantitative Real-Time PCR The reaction primers required for the experiment were ordered from the Hangzhou branch of Beijing Qingke Biotechnology Co., Ltd. The specific gene PCR primer sequences used in this experiment are shown in Table 3.

[0036] Table 3 Primer Sequence List for RT-qPCR Prepare the reaction system according to Table 4.

[0037] Table 4 RT-qPCR Reaction System The RT-qPCR reaction conditions are shown in Table 5.

[0038] Table 5 RT-qPCR Reaction Conditions 1.6 Western blot analysis of proteins 1.6.1 Protein Sample Extraction Wash the cells twice gently with 1×PBS, and thoroughly aspirate any remaining liquid from the flask using a pipette, ensuring no liquid remains. Then, add an appropriate amount of strong RIPA lysis buffer (containing a suitable concentration of protease inhibitors) according to the cell density and quantity, and place the flask on ice. Next, repeatedly scrape the cells with a cell scraper until visibly white cell clumps fall off. At this point, pipette the lysis buffer into a new 1.5 ml EP tube and incubate on ice for 10 min. Centrifuge at 4°C for 20 min (12000 rpm), then transfer the supernatant to a new 1.5 ml EP tube. This can be used for subsequent protein preparation or temporarily stored at -20°C.

[0039] 1.6.2 Protein concentration determination and denaturation Prepare a series of BSA standard solutions of known concentrations according to Table 6.

[0040] Table 6. Preparation of BSA Standard Solution Sample preparation: 3 μl of the protein sample to be tested + 27 μl of ddH2O + 600 μl of Bradford solution, i.e., diluting the protein sample to one-tenth of its original concentration. Add 200 μl of the prepared solution to each well of a 96-well plate. For each concentration of standard and protein sample, three replicates are set up for subsequent statistical analysis. Subsequently, the absorbance at 595 nm is measured using a microplate reader, and a standard curve is plotted based on the aforementioned protein standard. The concentration of the protein sample to be tested is calculated based on the obtained standard curve. Protein sample denaturation: Add an appropriate amount of 2 × SDS Loading Buffer and protein sample according to the protein concentration. Then, denature the protein sample in a 100℃ metal bath for approximately 10 min and store at -20℃ for later use.

[0041] 1.6.3 Preparation of adhesive (taking a 1.5mm mini adhesive as an example) Please invert and mix all components before use. Take equal volumes of the lower gel solution and lower gel buffer, 4.0 ml each, and mix well. Add 80 μl of coagulant and mix well. Pour the mixture into the casting plate, ensuring the liquid level is approximately 1.5 cm from the top edge of the short glass plate. Add 1 ml of isopropanol to cover the lower gel. After the lower gel solidifies (approximately 15-20 minutes), discard the top layer of isopropanol. Take equal volumes of the upper gel solution and colored upper gel buffer, 1 ml each, and mix well. Add 20 μl of coagulant to the mixture and mix well. Pour the mixture into the casting plate and insert the comb. After the upper gel solidifies (approximately 15-20 minutes), remove the comb; the plate is now ready for electrophoresis.

[0042] 1.6.4 Protein Electrophoresis Secure the gel plate in the electrophoresis tank as required, ensuring the clips are firmly in place to prevent leakage of the electrophoresis buffer. Fill the inner tank with 1× electrophoresis buffer, then gently pull the comb from the gel plate vertically upwards. Next, add an appropriate amount of 1× SDS electrophoresis buffer to the outer tank until it covers the bottom of the electrophoresis tank. Then, load the samples one side at a time, adding equal volumes of protein sample to each comb well in the experimental sequence. For the split protein samples, a control well should be included, with 1-3 μl of marker added to each well. Connect the power supply and select a constant voltage of 60V for approximately 30 minutes to concentrate the protein. After the sample has transferred from the stacking gel to the separating gel, increase the voltage to 100V and continue running the separating gel until the bromophenol blue reaches the bottom of the separating gel. The termination time of electrophoresis should be determined based on the experimental requirements.

[0043] 1.6.5 Transfer of film Cut a 0.45 μm PVDF membrane to the desired protein band size beforehand and activate it in methanol solution for about 5 minutes. Cut the required protein tape for electrophoresis using a plastic plate, discarding any unnecessary parts. Wet the transfer clamp in pre-cooled transfer buffer and place the following layers in sequence, starting with the black baffle: sponge, 3 layers of filter paper, protein tape, PVDF membrane (rinsed in TBST), 3 layers of filter paper, and sponge, ensuring air bubbles are removed. Finally, press the white baffle firmly. Place the transfer clamp in the transfer tank, paying attention to the polarity. Place a blue plastic ice cube to maintain a 4°C environment. Then, add the pre-cooled transfer buffer (pre-cooled to -20°C) to the top of the tank. Transfer conditions: constant voltage 100V for 100 minutes. After transfer, disconnect the power supply, remove the PVDF membrane, and mark it (usually a notch in the upper left corner of the PVDF membrane is used to indicate orientation).

[0044] 1.6.6 Antigen-antibody reaction and imaging Prepare 3% BSA using 1 × TBST. Remove the PVDF membrane and moisten it in 1 × TBST, then place it in an appropriate amount of blocking buffer for approximately 30 minutes (the exact time can be adjusted according to experimental requirements). Afterward, recover the blocking buffer and wash the PVDF membrane 2-3 times with 1 × TBST until any residual BSA is removed. Add an appropriate amount of primary antibody (prepared with primary antibody dilution buffer according to the specific concentration required by the antibody instructions), and incubate overnight at 4°C on a roller mixer with gentle shaking (approximately 12-16 hours, the exact time can be adjusted according to experimental requirements). Discard the primary antibody and wash the membrane 3 times × 10 minutes on a shaker with 1 × TBST (the shaker speed can be relatively fast in this step). Then, add an appropriate amount of secondary antibody (diluted at a ratio of 1:3000 with 1 × TBST) to cover the PVDF membrane at room temperature, and incubate slowly on a roller mixer for approximately 1 hour. Discard the secondary antibody and wash the membrane 3 times × 10 minutes with 1 × TBST. Development: Place the PVDF membrane in a pre-prepared luminescent dish, and evenly drop the luminescent solution (the color development solution A and reagent B are prepared in a 1:1 ratio) onto the membrane and incubate for about 10 seconds. Then, perform luminescence according to the instrument's instructions.

[0045] 2. Experimental Results 2.1 Inhibition of METTL3 promotes collagen expression in human skin fibroblasts 2.1.1 METTL3 shRNA increases collagen expression in human skin fibroblasts. Reference Figure 1 In order to study whether skin collagen is affected by m6 Modification regulation: This application uses shRNA to knock down METTL3. Firstly, green fluorescence can be observed under a microscope, indicating successful shRNA transfection. Figure 1 A). Next, to determine the optimal shRNA sequence for effectively knocking down METTL3, real-time quantitative PCR analysis of METTL3 mRNA levels showed that METTL3 shRNA-1 (shMETTL3-1) was the most effective shRNA for reducing METTL3 expression in HSF cells (Figure 1C). Simultaneously, Western blot results also confirmed that shMETTL3-1 can inhibit METTL3 protein expression (…). Figure 1 B). To investigate collagen expression in the skin after METTL3 inhibition, this application examined the mRNA levels of COL1A1, COL1A2, and COL3A1. qPCR results showed that in METTL3-silenced HSF cells, the mRNA levels of COL1A1, COL1A2, and COL3A1 were elevated (Figure 1D). Figure 1 (E and Figure 1F). These results indicate that inhibiting the expression of METTL3 in human skin fibroblasts promotes collagen expression. Specifically, the sequence of shMETTL3 is GCCAAGGAACAATCCATTGTT.

[0046] 2.1.2 STM2457 promotes collagen expression in human skin fibroblasts Reference Figure 2 To further determine the expression of collagen in the skin after METTL3 inhibition, this application pharmacologically reduced the activity of METTL3 using a METTL3 inhibitor (STM2457). After treating HSF with different concentrations of STM2457 for 4 hours, the mRNA levels of METTL3, COL1A1, COL1A2, and COL3A1 were measured. qPCR results showed that compared with the control group (DMSO), the mRNA levels of METTL3 were increased in the 0.1 μM, 1.0 μM, and 10.0 μM groups (Figure 2A), while the protein levels in each group remained unchanged (Figure 2B). This indicates that the small molecule compound STM2457 directly inhibits the activity of METTL3 and affects its expression. 6 Level A: The mRNA levels of COL1A1, COL1A2, and COL3A1 were increased in the 0.1 μM and 1.0 μM groups, while there was no change in the 10.0 μM group (Figures 2C, 2D, and 2E). From these results, it is concluded that 0.1 μM STM2457 was used for subsequent experiments.

[0047] 2.2 Aging Model The senescence-associated secretory phenotype (SASP) is one of the key markers of aging. It involves the secretion of a range of cytokines, including pro-inflammatory cytokines, growth factors, chemokines, and proteases. Therefore, this application selected the pro-inflammatory cytokine IL-1β to treat HSF cells to create an aging model.

[0048] 2.2.1 Pro-inflammatory factors reduce collagen expression in human skin fibroblasts. Reference Figure 3 In this study, HSF cells were first treated with IL-1β for different time periods. qPCR results showed an increase in IL-6 mRNA levels (Figure 3A), indicating successful model establishment. To detect collagen expression in the aging model, the mRNA levels of COL1A1, COL1A2, and COL3A1 were measured. qPCR results showed that the mRNA levels of COL1A1, COL1A2, and COL3A1 decreased in a time-dependent manner (Figures 3B, 3C, and 3D).

[0049] 2.2.2 METTL3 inhibitors promote collagen expression in a human skin fibroblast aging model Reference Figure 4 To determine whether METTL3 inhibition could reverse collagen expression in a human skin fibroblast aging model, this application first transfected HSF with shMETTL3, then treated HSF with IL-1β, and detected the expression of IL-6, METTL3, COL1A1, COL1A2, and COL3A1. First, green fluorescence was observed under a microscope (Figure 4A). Western blot results showed that the METTL3 protein level was decreased in the shMETTL3+PBS and shMETTL3+IL-1β groups (Figure 4B), and the METTL3 mRNA level was also decreased in the shMETTL3+PBS and shMETTL3+IL-1β groups. Figure 4C) indicates successful transfection of METTL3shRNA. Subsequent qPCR results showed increased IL-6 mRNA levels in the SCR+IL-1β and shMETTL3+IL-1β groups, while no change was observed in the shMETTL3+PBS group (Figure 4D), indicating successful model establishment. Compared to the control group (SCR+PBS), the shMETTL3+PBS group showed increased COL1A1, COL1A2, and COL3A1 mRNA levels, while the SCR+IL-1β group showed decreased COL1A1, COL1A2, and COL3A1 mRNA levels. However, compared to the SCR+IL-1β group, the shMETTL3+IL-1β group showed increased COL1A1, COL1A2, and COL3A1 mRNA levels (Figures 4E, 4F, and 4G). Furthermore, the Western blot results for COL1A1 and COL3A1 corresponded to the qPCR results. Figure 4 B) indicates that METTL3 inhibition can reverse collagen expression.

[0050] Reference Figure 5 Next, this application treated HSF with IL-1β and then applied STM2457 to HSF to detect the expression of IL-6, METTL3, COL1A1, COL1A2 and COL3A1. qPCR results showed that IL-6 mRNA levels were elevated in the DMSO+IL-1β and STM2457+IL-1β groups, while no change was observed in the STM2457+PBS group (Figure 5A), indicating successful model establishment. METTL3 mRNA levels remained unchanged in the STM2457+PBS, DMSO+IL-1β, and STM2457+IL-1β groups (Figure 5B). Compared to the control group (DMSO+PBS), the STM2457+PBS group showed elevated COL1A1, COL1A2, and COL3A1 mRNA levels, while the DMSO+IL-1β group showed decreased COL1A1, COL1A2, and COL3A1 levels. However, compared to the DMSO+IL-1β group, the STM2457+IL-1β group showed elevated COL1A1, COL1A2, and COL3A1 levels. mRNA levels were elevated (Figures 5C, 5D, and 5E), and the WB results of COL1A1 corresponded to the qPCR results. Figure 5 F) indicates that inhibiting METTL3 expression with drugs can also reverse collagen expression.

[0051] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Application of METTL3 as a target in the preparation of anti-aging drugs or skin care products. 2.The use of METTL3 as a target in the preparation of a skin anti-aging drug or skin care product according to claim 1, characterized in that: This includes the preparation of drugs or skin care products for skin anti-aging by METTL3 inhibitors that specifically inhibit or interfere with METTL3 gene expression. 3.The use of METTL3 as a target in the preparation of a skin anti-aging drug or skin care product according to claim 1, characterized in that: The METTL3 inhibitors include small molecule compounds that specifically inhibit METTL3 and interfering molecules that specifically interfere with METTL3 gene expression.

4. The application of METTL3 as a target in the preparation of anti-aging drugs or skin care products according to claim 2, characterized in that: The METTL3 inhibitor is to directly inhibit the activity of METTL3, to reduce m 6 A level, or target to inhibit the expression of METTL3 gene, or target to inhibit the activity of METTL3 protein.

5. The application of METTL3 as a target according to any one of claims 1-4 in the preparation of anti-aging drugs or skin care products, characterized in that: The pharmaceutical or skin care product comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, which is directly inhibiting the activity of METTL3, reducing m 6 A level, or specifically inhibiting METTL3 gene expression, or target inhibiting METTL3 protein activity.

6. The application of METTL3 as a target according to claim 3 in the preparation of anti-aging drugs or skin care products, characterized in that: The small molecule compound that specifically inhibits METTL3 is STM2457.

7. The application of METTL3 as a target in the preparation of anti-aging drugs or skin care products according to claim 3, characterized in that: The interfering molecule that specifically interferes with the expression of the METTL3 gene is shMETTL3, and the sequence of shMETTL3 is GCCAAGGAACAATCCATTGTT.

8. The application of METTL3 as a target in the preparation of anti-aging drugs or skin care products according to claim 1, characterized in that: The medicine or skin care product includes one or more of the following: ointment, gel, cream, serum, and liquid.

Images