Application of substance for promoting expression of METTL3 in treatment of obesity adipose tissue inflammation and metabolic diseases

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a substance for promoting METTL3 expression in treatment of obesity adipose tissue inflammation and metabolic diseases. A monomeric compound licoflavone A capable of promoting expression of METTL3 is analyzed from a traditional Chinese medicine prescription, METTL3 is low in expression in obese adipose tissue, and overexpression METTL3 can enable a mouse to be slower in weight increase, higher in insulin sensitivity and improved in glucose tolerance when the mouse is fed with high-fat diet feed, and meanwhile, the average food intake of the mouse every day is not affected; the licoflavone A can remarkably promote protein expression of METTL3, can improve adipocyte inflammation and remarkably improve adipose tissue inflammation of obese mice, and the effect of the licoflavone A in improving the adipose tissue inflammation of the obese mice is verified by detecting inflammation-related gene expression or inflammatory cell infiltration conditions in the adipose tissue of the obese mice.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, specifically relating to the application of a substance that promotes METTL3 expression in the treatment of inflammation and metabolic diseases in obese adipose tissue. Background Technology

[0002] The increasing prevalence of obesity globally has become a major public health problem threatening human health. Obesity increases the risk of various metabolic complications and comorbidities, including type 2 diabetes, cardiovascular metabolic diseases, and fatty liver disease. However, the mechanisms by which obesity participates in the development of metabolic diseases are complex. Chronic inflammation of adipose tissue plays a crucial role in this process. Under normal physiological conditions, the primary function of adipose tissue is to store excess metabolic energy. However, in an obese state, adipose tissue is in a state of persistent hypoxia, resulting in impaired angiogenesis, increased adipocyte size, the emergence of insulin resistance phenotypes, increased expression and secretion of pro-inflammatory adipokines, and increased immune cell infiltration and related inflammatory phenotype changes. These phenomena lead to adipocyte dysfunction, affecting the limited healthy expansion of adipose tissue, exacerbating various adverse metabolic processes, and causing adipose tissue to enter a chronic, low-grade inflammatory state. This inflammatory state damages metabolic organs such as the liver and pancreas through multiple pathways, further aggravating the progression of metabolic diseases. Although current research confirms that targeted therapy using drugs such as dasatinib and quercetin can effectively slow the development and progression of obesity-related adipose tissue dysfunction, the inflammatory regulation and triggering mechanisms of obese adipose tissue remain unclear and require further investigation.

[0003] Epigenetics refers to the process of influencing gene expression by chemically modifying DNA and proteins on chromosomes. These changes do not alter the DNA sequence. Epigenetic modifications play multiple roles in transcriptional regulation. Common epigenetic modifications include DNA methylation, histone modification, non-coding RNA, RNA modification, and chromatin remodeling. RNA epigenetics is an emerging field of scientific research, with over 100 different types of RNA chemical modifications identified to date. Among these, m6A (N6-adenosylmethyl) is the most common dynamic and reversible RNA methylation modification. It is mainly regulated by m6A "writing enzymes" (methyltransferases METTL3 / METTL14 / WTAP), "erasing enzymes" (demethylases FTO and ALKBH5), and "reading proteins" (RNA-binding proteins YTHDF and IGF2BPs). These factors can affect the stability and translation of specific transcripts under different conditions, thereby altering cell phenotypes. Methyltransferase-like protein 3 (METTL3) is widely distributed in the human body. Abnormal expression of METTL3 is closely related to tissue development delay, malignant tumors, and the development of cardiovascular and cerebrovascular diseases. Recent studies have found that METTL3-mediated m6A modification plays an important role in the development and differentiation of adipose tissue, but its specific mechanism of action in adipose tissue inflammation remains unclear.

[0004] Traditional Chinese medicine (TCM) has thousands of years of clinical experience in treating obesity, offering benefits such as safety, gentleness, and long-lasting effects. Currently, traditional Chinese medicine formulas reported to have regulatory effects on obesity and adipose tissue inflammation include Fangji Huangqin Decoction, Sanhuang Decoction, Peilian Mahuang Formula, Compound Pingfei Tea, Qigong Pill, Berberine, Fenugreek, and Dendrobium officinale polysaccharide. Among them, Fangji Huangqi Decoction, originating from the ancient medical masterpiece *Jinkui Yaolue* by Zhang Zhongjing, is a classic TCM formula. This formula is carefully composed of four herbs: Fangji, Huangqi, Baizhu, and Gancao, with ginger and jujubes added during decoction to enhance its efficacy. It has the effects of dispelling wind and dampness, tonifying qi and strengthening the exterior, and is used to treat symptoms caused by wind-dampness invasion and exterior deficiency. Fangji Huangqi Decoction is a classic TCM formula and one of the earliest formulas for treating obesity accepted by foreign scholars. Clinical studies have revealed its potential in regulating glucose and lipid metabolism and promoting significant weight loss. However, despite the preliminary verification of its efficacy, research on the specific active ingredients and mechanisms of action of Fangji Huangqi Decoction remains relatively limited, which provides ample room for future scientific research. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an application of a substance that promotes METTL3 expression in the treatment of inflammation and metabolic diseases in obese adipose tissue.

[0006] The technical content of this invention is as follows: This invention provides an application of METTL3 as a target in screening products for the prevention and / or treatment of obesity and glucose-lipid-related metabolic disorders; The products include pharmaceuticals or laboratory reagents, hereinafter the same; The obesity and glucose-lipid-related metabolic disorders include obesity, insulin resistance, impaired glucose tolerance, or adipose tissue inflammation; The product works by improving the inflammatory state of adipose tissue and / or increasing the body's insulin sensitivity.

[0007] The present invention also provides the use of a substance that promotes METTL3 expression in the preparation of products for the prevention and / or treatment of obesity and glucose-lipid-related metabolic disorders; The substance is an agonist, an overexpression plasmid vector, or nanoparticles carrying METTL3 gene mRNA. Furthermore, the agonist includes glycyrrhizin A; The present invention also provides a monomeric compound that promotes METTL3 expression, wherein the monomeric compound is glycyrrhizin A.

[0008] The present invention also provides a pharmaceutical composition comprising an effective dose of a substance that promotes METTL3 expression, and a pharmaceutically acceptable carrier. The substance is an agonist, an overexpression plasmid vector, or nanoparticles carrying METTL3 gene mRNA. Furthermore, the agonist includes glycyrrhizin A; The pharmaceutically acceptable carriers include at least one of tablets, capsules, oral liquids, granules, suspensions, injections, powder for injection, pellets, sustained-release agents, controlled-release agents, and targeted formulations.

[0009] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of products for the prevention and / or treatment of obesity-related metabolic disorders.

[0010] The beneficial effects of this invention are as follows: This invention relates to glycyrrhizin A, a monomeric compound that promotes METTL3 expression. Glycyrrhizin A was derived from traditional Chinese medicine formulas and is known to promote METTL3 expression. METTL3 is expressed at low levels in obese adipose tissue, and overexpression of METTL3 in mice fed a 45% or 60% high-fat diet resulted in slower weight gain, improved insulin sensitivity, and improved glucose tolerance, without affecting the average daily food intake. Glycyrrhizin A significantly promotes METTL3 protein expression and improves adipocyte inflammation and adipose tissue inflammation in obese mice. The efficacy of glycyrrhizin A in improving adipose tissue inflammation in obese mice was verified by detecting the expression of inflammation-related genes or inflammatory cell infiltration in the adipose tissue of obese mice. This invention reveals that overexpression of METTL3 can significantly improve adipose tissue inflammation and glucose-lipid metabolism disorders, and that the monomeric compound glycyrrhizin A can effectively improve inflammation in obese adipose tissue and correct metabolic disorders. This provides a potential drug target for the treatment of obesity-related metabolic diseases and offers a new treatment strategy for adipose tissue dysfunction and obesity-related metabolic diseases. Attached Figure Description

[0011] Figure 1 METTL3 expression in adipose tissue of obese and non-obese individuals; Figure 2 The changes of various indicators in a mouse model of obesity induced by a 45% high-fat diet. Figure 3 The changes of various indicators in a mouse model induced by a 60% high-fat diet; Figure 4 To investigate the changes in various indicators in a cell adipogenesis model induced by the cocktail induction method for 3T3-L1 cells; Figure 5 The changes in various parameters in adipocytes overexpressing adeno-associated virus (AAV) after bilateral injection of METTL3-overexpressing adipocytes into mouse eWAT cells were observed. Figure 6 The changes in various indicators in eWAT adipocyte-specific overexpression of METTL3; Figure 7 The 16 drug monomers with the highest affinity and binding free energy fraction for METTL3 protein were selected. Figure 8 To use the MTT assay to detect the toxicity of 16 drug monomeric compounds to 3T3-L1 cells; Figure 9 Western blot analysis was conducted to detect the effects of 16 drug monomer compounds on METTL3 expression. Figure 10To investigate the effect of glycyrrhizin isoflavone A on LPS-induced inflammation in adipogenic 3T3-L1 cells; Figure 11 To investigate the effects of glycyrrhizin isoflavone A on macrophage infiltration and inflammatory factor expression in adipose tissue of obese mice. Detailed Implementation

[0012] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0013] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0014] Example 1 METTL3 is expressed at low levels in obese adipose tissue. ① The expression of METTL3 in adipose tissue of obese and non-obese individuals was analyzed using the GEO databases (GSE156906, GSE162653). The results are as follows: Figure 1 As shown, in the GSE156906 and GSE162653 databases, the expression of METTL3 mRNA in adipose tissue of obese individuals was lower than that of normal-weight individuals.

[0015] ② An obese mouse model was induced by feeding the mouse with a 45% high-fat diet (HFD) for 16 weeks, and the results were confirmed by the increase in body weight and blood glucose, as well as changes in the morphology and weight of iWAT and eWAT. Figure 2 As shown in the AD diagram, the body weight and blood lipids of mice fed a high-fat diet were significantly increased, and the fat volume was also significantly larger. Notably, compared with CD mice, HFD mice had significantly higher levels of iWAT and eWAT. 6 The level of modification A decreased significantly, such as Figure 2 As shown in E, METTL3 mRNA and protein levels also showed a significant decreasing trend in obese iWAT and eWAT, as indicated by [reference needed]. Figure 2 As shown in FH.

[0016] The 45% high-fat feed used (24% protein, 41% carbohydrates, 24% fat) is formulated as follows: casein, 200 gm, L-cysteine, 3 gm, corn starch, 72.8 gm, maltodextrin 10, 100 gm, sucrose, 172.8 gm, cellulose BW200, 50 gm, soybean oil, 25 gm, lard, 177.5 gm, mineral mixture, S10026 10 gm, dicalcium phosphate, 13 gm, calcium carbonate, 5.5 gm, potassium citrate, 16.5 gm, vitamin mixture V10001, 10 gm, choline tartrate, 2 gm, FD&C red dye #40, 0.05 gm, Total: 858.15 gm.

[0017] ③ An obese mouse model was induced by feeding the mouse with a 60% high-fat diet (HFD) for 12 weeks, and the results were confirmed by increases in body weight, blood lipids, blood glucose, and changes in the morphology and weight of iWAT and eWAT. Figure 3 As shown in the AE diagram, the body weight and blood lipids of infants fed a high-fat diet increased significantly, and the fat volume also increased significantly; it is worth noting that, as Figure 3 As shown in F, the protein level of METTL3 showed a significant decreasing trend in obese iWAT and eWAT.

[0018] The 60% high-fat feed used (protein 26.2%, carbohydrates 26.3%, fat 34.9%) is formulated as follows: casein, 200 gm, L-cysteine, 3 gm, corn starch, 0 gm, maltodextrin 10, 125 gm, sucrose, 68.8 gm, cellulose BW200, 50 gm, soybean oil, 25 gm, lard*, 245 gm, mineral mixture, S10026 10 gm, dicalcium phosphate, 13 gm, calcium carbonate, 5.5 gm, potassium citrate, 16.5 gm, vitamin mixture V10001, 10 gm, choline tartrate, 2 gm, FD&C red dye #40, 0.05 gm, Total: 773.85 gm.

[0019] ④ A cocktail induction method was used to induce adipogenesis in 3T3-L1 cells, constructing a cell adipogenesis model: 3T3-L1 cells were cultured in high-glucose DMEM + 10% fetal bovine serum (FBS) and grown to confluence in T25 cell flasks or six-well cell plates. The original ordinary culture medium was then replaced with adipogenic differentiation medium containing 0.5 mM isobutylmethylxanthine (IBMX), 1 µM dexamethasone, and 1 µg / ml insulin. After culturing under these conditions for 3 days, the medium was replaced with DMEM containing only 10% FBS and 1 µg / ml insulin, and this replacement was repeated every two days until day 8. Oil Red O confirmed the successful induction of the model. Figure 4 (A) Consistent with animal results, m in adipogenic 3T3-L1 cells 6 A levels decreased significantly ( Figure 4 (B in the text), and RT-qPCR, Western blot, immunofluorescence, etc., showed that METTL3 expression was significantly reduced in the adipogenic 3T3-L1 cell model ( Figure 4 (CF in the middle).

[0020] As can be seen from the above, METTL3 is expressed at low levels in obese adipose tissue, indicating that METTL3 can be used as a target for screening to prevent and / or treat obesity and glucose-lipid-related metabolic disorders, including obesity, insulin resistance, glucose intolerance, or adipose tissue inflammation.

[0021] Example 2 Overexpression of METTL3 significantly improves inflammation in obese adipose tissue. ① To further investigate whether METTL3 overexpression in adipocytes can improve adipose tissue inflammation, adipocyte METTL3-overexpressing adeno-associated virus (OE-METTL3-AAV) or control CON-AAV was injected into bilateral eWATs of 6-week-old mice, respectively. After 2 weeks of normal diet feeding, the mice were fed an HFD diet (60%) for 8 weeks to establish an obese mouse model. Figure 5 A in the text). Western blot and PCR were used to detect METTL3 expression in eWAT, and the results showed that the METTL3 overexpression model was successfully constructed. Figure 5 (BC in the middle). We observed that, compared with HFD mice injected with control CON-AAV, METTL3 overexpressing mice gained body weight more slowly after HFD, and there was no significant difference in average daily food intake between the two groups (BC in the middle). Figure 5 METTL3 overexpression mice showed better insulin sensitivity and glucose tolerance (DF in the D), and METTL3 overexpression mice had better insulin sensitivity and glucose tolerance (DF in the D), Figure 5GH in the middle). The level of FFA in the serum of METTL3-overexpressing mice was significantly reduced, and the levels of TG, TC, and LDL-C were significantly decreased, while the level of HDL-C was significantly increased ( Figure 5 (IJ in the text). These changes in metabolic indicators suggest that METTL3 overexpression in eWAT adipocytes improves obesity-induced glucose and lipid metabolism disorders.

[0022] ②For example Figure 6 As shown, overexpression of METTL3 in eWAT significantly reduced the weight and volume of mouse adipose tissue. Immunofluorescence analysis of the macrophage marker F4 / 80 indicated that increased METTL3 expression in adipose tissue reduced macrophage infiltration. Compared with the control group, overexpression of METTL3 in adipose tissue significantly decreased leptin levels and significantly increased adiponectin levels. Simultaneously, overexpression of METTL3 significantly reduced the mRNA levels of inflammatory cytokines such as IL-1β, IL-18, IL-6, and IL-8, and improved the degree of eWAT fibrosis. In conclusion, these results indicate that adipocyte-specific overexpression of METTL3 in eWAT can inhibit HFD diet-induced adipose tissue inflammation.

[0023] As can be seen from the above, overexpression of METTL3 can significantly improve inflammation in obese adipose tissue, indicating that the use of substances that promote METTL3 expression can help improve inflammation in obese adipose tissue.

[0024] Example 3 Searching for monomeric compounds in Fangji Huangqi Decoction that promote METTL3 expression ① Using the TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform) website, we analyzed the four main components (Atractylodes macrocephala, Stephania tetrandra, Glycyrrhiza uralensis, and Scutellaria baicalensis) in the traditional Chinese medicine formula Fangji Huangqin Decoction, and a total of 528 drug monomer compounds were identified.

[0025] ② The above 528 monomeric compounds were molecularly docked with the METTL3 protein (5TEY) in Discovery Studio, and the interaction between the drug monomers and the METTL3 protein and the molecular force field were measured to evaluate the affinity and biological activity of the molecules. The docking binding score was ranked according to the affinity and binding free energy of the molecules by LibDock.

[0026] ③ For example Figure 7As shown, 16 drug monomers were selected from the top-ranked compounds. The MTT assay was used to detect the cytotoxicity of these 16 drug monomer concentrations on 3T3-L1 cells, and the optimal drug treatment concentration with the least cytotoxicity was screened. Figure 8 As shown. Then according to Figure 8 3T3-L1 cells were treated with the optimal concentration for 48 h, and the effects of the above-mentioned drug monomers (with DMSO as the control) on METTL3 expression were detected by Western blot. Results are as follows: Figure 9 As shown, compared with other drug monomer compounds, licorice isoflavone A (LIC-A) can significantly promote the protein expression of METTL3.

[0027] As can be seen from the above, a monomeric compound that can promote METTL3 expression, glycyrrhizin A, was found in Fangji Huangqi Decoction, revealing that glycyrrhizin A can be used to prepare products that improve inflammation of obese adipose tissue, such as drugs or experimental products.

[0028] Example 4 In vitro and in vivo verification of the effects of lithoside A (LIC-A) on adipose tissue inflammation. ① To further investigate the regulatory effect of glycyrrhizin on adipose tissue inflammation, TNF-α-induced adipogenic 3T3-L1 cells were treated with glycyrrhizin A. After 48 hours, the expression of inflammatory cytokines was detected. It was found that glycyrrhizin A treatment reduced the expression of TNF-α-induced inflammatory cytokines such as IL-6, IL-18, and IL-1β, suggesting that glycyrrhizin A can improve TNF-α-induced inflammation in adipogenic 3T3-L1 cells (e.g., IL-6, IL-18, and IL-1β). Figure 10 (AE in the middle). Simultaneously, lipopolysaccharide (LPS)-induced adipogenic 3T3-L1 cells were treated with glycyrrhizin isoflavone A. After 48 hours, the expression of inflammatory factors was detected, and it was found that glycyrrhizin isoflavone A treatment could reduce the expression of LPS-induced inflammatory cytokines such as IL-6, IL-18, IL-1β, and MCP-1 (e.g., AE in the middle). Figure 10 The results (FJ) suggest that glycyrrhizin isoflavone A can improve LPS-induced inflammation in adipogenic 3T3-L1 cells.

[0029] ②For example Figure 11 As shown in Figure A, obese mice were treated with glycyrrhizin isoflavone A, and the results were as follows: Figure 11 As shown in the BE study, glycyrrhizin isoflavone A significantly improved glucose and lipid metabolism disorders in obese mice and further improved macrophage infiltration and inflammatory factor expression in adipose tissue of obese mice, suggesting that glycyrrhizin isoflavone A plays an important role in improving inflammation in obese adipose tissue.

[0030] As can be seen from the above, the monomeric compound glycyrrhizin A can effectively improve inflammation of obese adipose tissue and correct metabolic disorders, providing a potential drug target for the treatment of obesity-related metabolic diseases and offering a new treatment strategy for adipose tissue dysfunction and obesity-related metabolic diseases.

Claims

1. Use of METTL3 as a target in screening products for preventing and / or treating obesity and glucose-lipid related metabolic disorders.

2. Use according to claim 1, characterized in that, The products include drugs or experimental reagents.

3. Use according to claim 1, characterized in that, The obesity and glucose-lipid related metabolic disorders include obesity, insulin resistance, glucose intolerance, or adipose tissue inflammation.

4. Use of a substance for promoting METTL3 expression in the preparation of a product for preventing and / or treating obesity and glucose-lipid related metabolic disorders.

5. Use according to claim 4, characterized in that, The substance includes an agonist, an overexpression plasmid vector, or a nanoparticle loaded with METTL3 gene mRNA.

6. Use according to claim 4, characterized in that, The agonist includes glycyrrhizin isoflavone A.

7. A monomer compound for promoting METTL3 expression, characterized in that, The monomer compound is glycyrrhizin isoflavone A.

8. A pharmaceutical composition, characterized by, It includes an effective dose of a substance for promoting METTL3 expression, and a pharmaceutically acceptable carrier; The substance for promoting METTL3 expression is as shown in claim 5 or 6 or 7.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutically acceptable carrier includes at least one of tablets, capsules, oral liquids, granules, suspensions, injections, powder injections, dripping pills, sustained-release preparations, controlled-release preparations, and targeted preparations.

10. Use of the pharmaceutical composition of claim 8 or 9 in the preparation of a product for preventing and / or treating obesity related metabolic disorders.

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