Use of prmt4 inhibitors in the preparation of medicaments for the treatment of metabolic-associated steatohepatitis
By using PRMT4 inhibitors TP-064 or EZM2302 to regulate the PRMT4-YAP signaling axis, the problem that existing MASH treatments cannot meet clinical needs has been solved, achieving targeted therapy for MASH and significantly improving liver pathology and metabolic disorders.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
- Filing Date
- 2026-05-07
- Publication Date
- 2026-07-10
AI Technical Summary
Existing treatments for metabolic-associated steatohepatitis (MASH) primarily focus on single targets, which is insufficient to meet clinical treatment needs. Furthermore, the pathogenesis is complex, involving multiple factors such as genetics, epigenetics, metabolism, diet, and environment.
By using PRMT4 inhibitors TP-064 or EZM2302, the liver fat deposition, fibrosis and inflammation induced by a high-fat diet in MASH mice were significantly reduced by inhibiting protein arginine methyltransferase 4 (PRMT4), and the PRMT4-YAP signaling axis was regulated, thus intervening in the core pathological mechanism of MASH.
It significantly reduces the degree of hepatic steatosis, inflammatory infiltration and fibrosis in MASH mice, improves lipid metabolism disorders in serum and liver tissue, reduces body weight, lowers serum triglyceride and alanine aminotransferase and aspartate aminotransferase levels, improves insulin resistance, and provides a targeted and precise treatment option with a clear mechanism.
Smart Images
Figure CN122351237A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fatty liver disease treatment technology, and in particular to the application of PRMT4 inhibitors in the preparation of therapeutic drugs for metabolic-associated steatohepatitis. Background Technology
[0002] Metabolic fatty liver disease (MAFLD) is currently the most common metabolic dysfunction-related fatty liver disease caused by the global obesity epidemic. The latest epidemiological data published in 2025 indicates that 30%–40% of adults worldwide have MAFLD. This disease is often asymptomatic before progressing to metabolic-associated steatohepatitis (MASH). MASH, the advanced stage of MAFLD, is characterized by hepatic steatosis, inflammation, fibrosis, and liver damage, and may eventually progress to life-threatening stages, including cirrhosis and hepatocellular carcinoma, imposing a heavy health and economic burden on society.
[0003] Despite years of basic and clinical research and the fact that several drugs have entered preclinical trials, current guidelines in various countries still only recommend metabolic weight-loss surgery, lifestyle interventions, and dietary modifications for the treatment of MASH. In 2024 and 2025, the US FDA and the European Union approved the first drug for MASH, Rezdiffra, which acts as an agonist by activating thyroid hormone receptor β in the liver to reduce liver fat accumulation. In addition, in August 2025, the US FDA approved another GLP-1 receptor agonist (semaglutide) for the treatment of MASH.
[0004] However, existing research has found that the pathogenesis of MASH is complex, involving multiple factors such as genetics, epigenetics, metabolism, diet and environment. Currently approved drugs for clinical use all focus on single targets, which is difficult to meet clinical treatment needs. Therefore, it is urgent to explore new target drugs and use them in combination to alleviate the progression of MASH. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of PRMT4 inhibitors in the preparation of therapeutic drugs for metabolism-related steatohepatitis.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of PRMT4 inhibitors in the preparation of medicaments for treating metabolic-associated steatohepatitis.
[0007] This invention, through HE, Oil Red, Sirius, and F4 / 80 immunohistochemical staining of liver tissue, revealed that, compared with the control group, treatment with PRMT4 inhibitors significantly reduced hepatic steatosis, fibrosis, and inflammation in MASH mice induced by a high-fat diet. This confirms that PRMT4 inhibitors can significantly reduce the degree of hepatic steatosis, inflammatory infiltration, and fibrosis in MASH model mice. Therefore, PRMT4 inhibitors can be used to treat metabolic-associated steatohepatitis.
[0008] Protein arginine methyltransferase 4 (PRMT4), also known as coactivator-associated arginine methyltransferase 1 (CARM1), participates in biological processes such as signal transduction, transcriptional regulation, RNA splicing, cell cycle regulation, and autophagy by catalyzing monomethylation or asymmetric dimethylation of histone or non-histone arginine residues.
[0009] As a preferred embodiment of the first aspect, the PRMT4 inhibitor is TP-064 or EZM2302.
[0010] TP-064 is a chemical probe and a potent selective protein arginine methyltransferase 4 (PRMT4; CARM1) inhibitor, CAS No. 2080306-20-1.
[0011] EZM2302 is an effective oral active PRMT4 inhibitor, CAS No. 1628830-21-6.
[0012] The PRMT4 inhibitors used in this invention mainly include TP-064 and EZM2302. During the research process, TP-064 has undergone more extensive in vivo animal studies compared to EZM2302, resulting in more substantial research evidence and verified safety. Furthermore, regarding its mechanism of action, TP-064 has a broader inhibitory range on intracellular PRMT4-related methylation events compared to EZM2302. Therefore, this invention selected TP-064 as a potential drug target for further validation. It should be noted that although TP-064 is used in the embodiments of this invention, this does not negate the fact that EZM2302 also possesses the same efficacy. TP-064 was chosen simply because its effect is superior to EZM2302.
[0013] Secondly, the present invention provides the use of PRMT4 inhibitors in any of the following: (1) Application in the preparation of products that reduce the weight of patients with metabolic-associated steatohepatitis; (2) Application in the preparation of products that reduce liver weight in patients with metabolic-associated steatohepatitis; (3) Application in the preparation of products that reduce serum triglyceride and / or liver triglyceride levels in patients with metabolic-associated steatohepatitis; (4) Application in the preparation of products that reduce serum alanine aminotransferase and / or aspartate aminotransferase levels in patients with metabolic-associated steatohepatitis; (5) Application in the preparation of products that reduce fat deposition in the liver tissue of patients with metabolic-associated steatohepatitis; (6) Application in the preparation of products that improve liver tissue fibrosis in patients with metabolic-associated steatohepatitis; (7) Application in the preparation of products that reduce liver tissue inflammation in patients with metabolic-associated steatohepatitis; (8) Application in the preparation of products that improve insulin resistance in patients with metabolic-associated steatohepatitis.
[0014] This invention, through animal experiments, detected relevant indicators in MASH mice, including body weight, liver weight, serum triglycerides, liver triglycerides, serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), liver fat deposition, liver fibrosis, liver inflammation, and insulin resistance. The results showed that PRMT4 inhibitors significantly reduced body weight and liver wet weight in MASH mice, improved serum and liver tissue lipid metabolism disorders, effectively alleviated hepatocyte steatosis and inflammatory infiltration, and significantly inhibited the progression of liver fibrosis. These ameliorative effects are closely related to the specific inhibition of PRMT4-mediated methylation modification by TP-064. Therefore, PRMT4 inhibitors can be used for drug development and clinical translation for the aforementioned indications.
[0015] Thirdly, the present invention provides the use of PRMT4 inhibitors in any of the following: (1) Application in the preparation of products that inhibit YAP activity; (2) Application in the preparation of products that inhibit the expression levels of CYR61, CTGF and / or H3R17me2 proteins; (3) Application in the preparation of products that control the PRMT4-YAP signal axis.
[0016] This invention validates the effects of PRMT4 inhibitors on the levels of YAP, a key transcriptional activator of lipid and inflammatory metabolism, and its downstream target gene proteins. It was found that PRMT4 inhibitors significantly inhibit YAP nuclear translocation and transcriptional activity, downregulate CYR61 and CTGF expression, and simultaneously reduce H3R17me2 levels. These results collectively reveal that PRMT4 activates the YAP signaling pathway, further confirming the crucial regulatory role of the PRMT4-YAP signaling axis in the development and progression of MASH. Therefore, PRMT4 inhibitors can be used to target the core pathological mechanism of MASH—namely, the abnormal activation of the PRMT4-YAP signaling axis—providing a solid theoretical foundation and translational pathway for developing innovative drugs with precise targeting, clear mechanisms, and promising therapeutic effects.
[0017] Fourthly, the present invention provides a medicament for treating fatty liver, the medicament comprising a PRMT4 inhibitor and pharmaceutically acceptable excipients.
[0018] As a preferred embodiment of the third aspect, the PRMT4 inhibitor is a pharmaceutically acceptable salt of TP-064 or EZM2302.
[0019] As a preferred embodiment of the third aspect, the excipients include fillers, disintegrants, lubricants, adhesives, coating materials, and stabilizers.
[0020] As a preferred embodiment of the third aspect, the filler includes microcrystalline cellulose, lactose, mannitol, or pregelatinized starch.
[0021] As a preferred embodiment of the third aspect, the disintegrant includes crospovidone, sodium carboxymethyl starch, or low-substituted hydroxypropyl cellulose; the lubricant includes magnesium stearate, talc, or silica.
[0022] As a preferred embodiment of the third aspect, the drug is in the form of tablets, capsules, injections, or sustained-release microspheres.
[0023] Compared with the prior art, the present invention has the following advantages: This invention is the first to discover that PRMT4 inhibitors can serve as novel drug targets for the treatment of MASH. In vitro cell experiments revealed that PRMT4 inhibitors can inhibit PA-induced hepatocyte steatosis. In vivo animal experiments confirmed that PRMT4 inhibitors can improve hepatic steatosis, inflammatory response, hepatocyte damage, fibrosis, and insulin resistance in high-fat-fed (HFD) mice, and reduce liver tissue and serum triglyceride and transaminase levels. Further mechanistic investigation revealed that PRMT4 inhibitors primarily inhibit the activation of YAP, a key transcriptional activator involved in lipid and inflammatory metabolism, and the expression of its downstream target genes. Therefore, PRMT4 inhibitors can be used to prepare drugs for the treatment of MASH, with definite and significant efficacy, demonstrating promising medical application prospects. Attached Figure Description
[0024] Figure 1 A schematic diagram showing the experimental results of PRMT4 and H3R17me2 protein levels in mouse liver tissue; Figure 2 A schematic diagram showing the experimental results of mouse liver weight; Figure 3 A schematic diagram showing the experimental results of mouse body weight; Figure 4 This is a schematic diagram showing the experimental results of liver weight to body weight ratio in mice; Figure 5This is a schematic diagram showing the experimental results of triglyceride (TG) content in mouse serum; Figure 6 A schematic diagram showing the experimental results of triglyceride (TG) content in mouse liver; Figure 7 A schematic diagram showing the experimental results of alanine aminotransferase (ALT) levels in mouse serum; Figure 8 A schematic diagram showing the experimental results of aspartate aminotransferase (AST) levels in mouse serum; Figure 9 A schematic diagram showing the experimental results of H&E and Oil Red O staining for detecting fat deposition in mouse liver tissue and a comparison of the proportion of positive areas. Figure 10 A schematic diagram showing the experimental results of detecting collagen in mouse liver tissue using Sirius staining and a comparison of the proportion of positive areas; Figure 11 A schematic diagram showing the experimental results of F4 / 80 immunohistochemical detection of macrophages in mouse liver tissue and the comparison of the proportion of positive areas; Figure 12 This is a schematic diagram showing the experimental results of mRNA levels of inflammatory factors in mouse liver tissue. Figure 13 A schematic diagram of the experimental results for mRNA markers of liver fibrosis in mice; Figure 14 This is a schematic diagram of the results of the mouse insulin tolerance test; Figure 15 This is a schematic diagram of the results of a mouse glucose tolerance test. Figure 16 This is a schematic diagram showing the experimental results of the levels of p-YAP, YAP and their downstream target gene proteins in mouse liver tissue.
[0025] Figure 17 A schematic diagram illustrating the experimental results of assessing PA-induced steatosis in primary liver cells using Nile Red staining. Detailed Implementation
[0026] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0027] This invention is the first to discover that PRMT4 inhibitors can serve as novel drug targets for the treatment of MASH. The PRMT4 inhibitors used in this study primarily include TP-064 and EZM2302. During the research process, TP-064 had more extensive in vivo animal studies and more robust evidence compared to EZM2302, and its safety has been verified. Furthermore, regarding its mechanism of action, TP-064 has a broader inhibitory range on intracellular PRMT4-mediated methylation events compared to EZM2302. Therefore, TP-064 was selected for subsequent experimental verification in this invention. It should be noted that although TP-064 was used in this invention, this does not negate the fact that EZM2302 also possesses the same efficacy; TP-064 was chosen simply because its effects are superior to EZM2302.
[0028] TP-064 is a chemical probe and a potent selective protein arginine methyltransferase 4 (PRMT4; CARM1) inhibitor, CAS No. 2080306-20-1.
[0029] EZM2302 is an effective oral active PRMT4 inhibitor, CAS No. 1628830-21-6.
[0030] Example 1: Investigating the difference in PRMT4 expression levels between MASH mice and normal mice 1. Constructing the MASH mouse model After a week of environmental adaptation, male C57BL / 6J mice were fed a high-fat diet (CDAHFD) with choline deficiency and L-amino acid definition starting from week 8 to establish the MASH model. The control group (normal mice) were fed a normal diet (ND) at the same time.
[0031] 2. Investigate the effect of PRMT4 inhibitors on H3R17me2 protein levels: H3R17me2 protein is a key indicator of PRMT4 methyltransferase activity; higher levels indicate higher PRMT4 methyltransferase activity, while lower levels indicate lower PRMT4 methyltransferase activity.
[0032] (1) Experimental method: Protein sample preparation: Weigh 20 mg of liver tissue, homogenize it, and add it to 0.5 ml of 1×RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors. Centrifuge at 3000 r / min for 15 minutes at 4℃, and determine the protein content of the supernatant (BCA method). Take 100 μg of protein, add 5×loading buffer, heat at 99℃ for 10 minutes to denature, and perform a Western blot experiment to detect the expression level of the target protein in mouse liver tissue.
[0033] (2) Experimental results: like Figure 1 As shown, compared with the control group (normal mice), the expression level of PRMT4 was significantly increased in the MASH model. At the same time, the expression level of H3R17me2 protein, which reflects the PRMT4 methyltransferase activity, was also significantly increased in the MASH model, indicating that the PRMT4 protein level and its methyltransferase activity were significantly increased in the MASH model.
[0034] Example 2 Animal Experiment 1. Construction of the MASH mouse model and experimental grouping treatment: Seven-week-old male C57BL / 6J mice were acclimatized to their environment for one week. Starting from week 8, they were fed a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) to establish a MASH model. At week 4 of modeling, mice were randomly assigned to a control group (Vehicle treatment group) and an experimental group (TP-064 treatment group). The experimental group received intraperitoneal injections of 5 mg / kg PRMT4 inhibitor (TP-064) three times a week for two weeks, while the control group received an equal dose of PBS. The body weight of mice in each group was recorded for later data analysis. The mice were fed the CDAHFD diet for four weeks, followed by overnight fasting. Subsequently, the mice were anesthetized, blood was collected, and the mice were euthanized and their liver tissue was harvested.
[0035] 2. Investigating the effects of PRMT4 inhibitors on liver and body weight in MASH mice: (1) Experimental method: After constructing the MASH model, the body weight and liver weight of the mice were recorded at certain time intervals. After the experiment, all plotting and statistical analysis were performed using GraphPad Prism 8.1. All data are expressed as mean ± standard error (SE). Differences with p < 0.05 were considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance).
[0036] (2) Experimental results: like Figure 2-4 As shown, compared to the control group, the liver weight of mice treated with TP-064 ( Figure 2 ),weight( Figure 3 ) and liver-to-body weight ratio ( Figure 4 () decreased significantly.
[0037] 3. Investigate the effects of PRMT4 inhibitors on serum and liver triglycerides (TG) in MASH mice: (1) Experimental method: Serum collection: Blood was collected from the orbital cavity of anesthetized mice, incubated at low temperature for 4 hours, centrifuged, and the supernatant serum was transferred to a new centrifuge tube to obtain serum. The TG content was measured according to the standard kit instructions.
[0038] Lipid extraction from liver tissue: 30 mg of liver tissue was homogenized in 0.5 ml of PBS and allowed to stand for 30 minutes. After centrifugation, 60 μl of the supernatant was collected for BCA quantification. The remaining sample was mixed with 1 ml of organic extraction reagent (chloroform:methanol = 2:1 volume fraction), vortexed, and centrifuged for 3 hours. Then, 0.5 ml of 0.9% physiological saline was added, vortexed again, and centrifuged. The lower organic phase was transferred to a new centrifuge tube and dried overnight in a fume hood. 80 μl of 0.1% Triton X-100 (prepared with anhydrous ethanol) was added to dissolve the lipids at the bottom of the tube, and the tube was stored at 4°C. The TG content was detected according to the standard kit instructions.
[0039] (2) Experimental results: like Figure 5-6 As shown, compared with the control group, the serum of the TP-064-treated group ( Figure 5 ) and liver ( Figure 6 The TG levels in the MASH model showed a significant decreasing trend. This indicates that PRMT4 inhibitors can reduce serum and liver TG levels in the MASH model.
[0040] 4. To investigate the effects of PRMT4 inhibitors on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in MASH mice: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are markers of liver damage in serum. Elevated levels indicate worsening liver damage, while decreased levels indicate lessening liver damage.
[0041] (1) Experimental method: Serum was obtained following the same steps described above. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were measured according to the instructions of a standard kit.
[0042] (2) Experimental results: The results are as follows Figure 7-8 As shown, compared with the control group, the levels of ALT and AST in the TP-064 group were significantly reduced. This indicates that TP-064 can reduce serum AST and ALT levels, suggesting that TP-064 can alleviate liver damage caused by a high-fat diet.
[0043] 5. Investigate the effects of PRMT4 inhibitors on fat deposition, fibrosis, and inflammation in the liver tissue of MASH mice: 5.1 Detection of lipid deposition, fibrosis, and inflammation in liver tissue: (1) Experimental method: The effects of PRMT4 inhibitors on lipid (triglyceride) deposition in the liver tissue of MASH mice were observed using HE and Oil Red staining; the effects of PRMT4 inhibitors on fibrosis (collagen) in the liver tissue of MASH mice were observed using Sirius staining; and the effects of PRMT4 inhibitors on inflammation (macrophages) in the liver tissue of MASH mice were observed using F4 / 80 immunohistochemical staining. The specific methods are as follows: HE and Oil Red staining: Liver tissue was fixed in 4% paraformaldehyde solution for more than 24 hours, then embedded in paraffin. The paraffin-embedded tissue was stained with hematoxylin and eosin (H&E) to observe lipid deposition. Frozen sections of liver embedded in OCT embedding agent were stained with Oil Red O.
[0044] Sirius staining: Tissue was fixed with 4% paraformaldehyde for more than 24 hours, routinely dehydrated, embedded in paraffin, sectioned, dewaxed and then immersed in Sirius red staining solution for 1 hour. After staining, rinsed with running water to remove excess dye, stained with hematoxylin for 8-10 minutes, rinsed with running water for 10 minutes, then dehydrated with graded alcohols, and finally cleared with xylene and mounted. Observed and photographed under a microscope.
[0045] F4 / 80 immunohistochemical staining: The sections were first treated with 3% H2O2 at room temperature for 30 minutes to quench endogenous peroxidase activity, and then blocked with 3% BSA for 1 hour. Subsequently, the tissue sections were incubated overnight at 4°C with anti-F4 / 80 antibody (#GB11027, Xavier Biotech), and then incubated with HRP-conjugated secondary antibody (#GB1213, Xavier Biotech).
[0046] Finally, the images were analyzed and quantified using ImageJ software, and graphs and statistics were performed using GraphPad Prism 8.1.
[0047] (2) Experimental results: The results are as follows Figure 9-11 As shown, immunohistochemical staining of liver tissue sections with HE, Oil Red, Sirius, and F4 / 80 revealed that, compared with the control group, TP-064 treatment significantly reduced liver fat deposition induced by a high-fat diet. Figure 9 ), fibrosis ( Figure 10 ) and inflammation ( Figure 11 ).
[0048] 5.2 Detection of liver inflammatory factors and fibrosis markers: (1) Experimental method: The mRNA levels of marker inflammatory factors (IL-1β, IL-6, TNF-α) and fibrotic factors (Tgfβ1, col1a1, col3a1, ctgf, cyr61) were detected. Elevated levels indicated increased inflammation and fibrosis, while decreased levels indicated decreased inflammation and fibrosis. The specific method is as follows: RNA sample preparation and mRNA detection: Take 20 mg of liver tissue, add 1000 μl of TRIzol to dissolve the tissue, then add 200 μl of chloroform, mix well and let stand for 5 min, then centrifuge at 12000 g for 15 min at 4 °C, and transfer 200 μl of the supernatant to another EP tube, add 200 μl of isopropanol, mix thoroughly and let stand for 5 min, then centrifuge at 12000 g for 15 min at 4 °C, discard the supernatant, wash the precipitate with 75% alcohol, then centrifuge at 7500 g for 5 min at 4 °C, dry the precipitate at room temperature for 5-10 min, add 10 μl of RNase-free water to dissolve the precipitate to obtain RNA, reverse transcribe it into cDNA using a reverse transcription kit (Takara), and detect the mRNA level of the target gene by qRT-PCR.
[0049] (2) Experimental results: The results are as follows Figure 12-13 As shown, compared with the control group, TP-064 treatment significantly reduced the levels of inflammatory factors IL-1β, IL-6, and TNF-α in liver tissue. Figure 12 ), and fibrotic factors Tgfβ1, col1a1, col3a1, ctgf, cyr61 ( Figure 13 The mRNA levels of TP-064 indicate that TP-064 can inhibit liver inflammation and fibrosis.
[0050] 6. Investigate the effects of PRMT4 inhibitors on insulin tolerance and glucose tolerance in MASH mice. (1) Experimental method: Insulin tolerance test: After fasting for 6 hours, mice were intraperitoneally injected with 0.75 IU / kg human insulin (Eli Lilly) dissolved in physiological saline.
[0051] Glucose tolerance test: Mice were fasted for 16 hours and then injected intraperitoneally with 20% D-glucose solution (2.0 mg / g body weight).
[0052] The blood glucose concentration in the rat tail was measured at 0, 30, 60, 90 and 120 minutes after insulin or glucose injection using an automated blood glucose monitor (LifeScan).
[0053] (2) Experimental results: Insulin tolerance ( Figure 14 ) and glucose tolerance ( Figure 15The experimental results showed that, compared with the control group, the blood glucose levels of mice treated with TP-064 were significantly reduced at different time points, indicating that TP-064 can improve insulin resistance in MASH mice.
[0054] 7. Investigate the effects of PRMT4 inhibitors on the levels of YAP, a key transcriptional activator of lipid and inflammatory metabolism, and its downstream target gene proteins: (1) Experimental method: Protein sample preparation: Weigh 20 mg of liver tissue and homogenize it in 0.5 ml of 1×RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors. Centrifuge at 3000 r / min for 15 minutes at 4℃, and collect the supernatant for protein content determination (BCA method). Add 100 μg of protein to 5×loading buffer and heat at 99℃ for denaturation for 10 minutes to obtain the protein sample. Perform Western blot experiments on the protein sample to detect the expression levels of target proteins (p-YAP1, CYR61, CTGF, H3R17me2) in mouse liver tissue.
[0055] (2) Experimental results: The results are as follows Figure 16 As shown, compared with the control group, the expression level of p-YAP1 protein was significantly increased in the experimental group after TP-064 treatment, while the expression levels of CYR61, CTGF, and H3R17me2 proteins were significantly decreased. This indicates that TP-064 significantly increased the phosphorylation level of YAP, thereby reducing YAP activation and inhibiting the expression of its downstream target proteins such as inflammation and fibrosis (CYR61, CTGF), thus suppressing the inflammatory response and fibrosis process.
[0056] Example 3 Cell Experiment Investigating the effects of PRMT4 inhibitors on PA-induced hepatocellular steatosis: (1) Experimental method: Isolation of primary mouse liver cells: Mice were anesthetized with CO2, their abdomens were disinfected with alcohol spray, and they were fixed to the operating table. The abdominal cavity was cut open to expose the inferior vena cava and portal vein, and the superior vena cava was clamped with hemostatic forceps. An indwelling needle was inserted into the inferior vena cava, connected to a peristaltic pump, and calcium- and magnesium-free PBS buffer was perfused at a rate of 5-10 ml / min. At the same time, the portal vein was cut open for drainage until all blood in the liver was drained. The solution was then replaced with digestion solution containing 0.5 mg / ml collagenase IV, and perfused at a rate of 5 ml / min. The digested liver was transferred to a culture dish containing cell washing solution. The gallbladder and capsule were removed, and the tissue was dispersed with forceps to allow the cells to flow out. The cell suspension was filtered through a sieve to remove undigested tissue fragments. The cells were centrifuged at 4°C and 50-100 g for 5-10 minutes, the supernatant was discarded, the cells were resuspended, centrifuged again, and the cell density was adjusted to 1×10⁶ cells / mL. 6 / ml, inoculated into collagen-coated culture dishes, and incubated in a 37℃, 5% CO2 incubator.
[0057] Nile Red staining of hepatocytes: Primary hepatocytes were stimulated with 0.5 mM palmitic acid (PA) or bovine serum albumin (BSA), then treated with TP-064 or Vehicle, respectively. The culture medium was discarded, and the cells were gently washed with PBS, digested with trypsin, centrifuged to collect the hepatocytes, and resuspended in PBS to 1×10⁻⁶ cells. 6 / mL. Add 500 µL of Nile Red working solution to each tube and incubate at 37°C in the dark for 10 minutes. Centrifuge to remove staining solution, wash twice with PBS, and resuspend in buffer. Detect fluorescence signal using a fluorescence microscope, and photograph after counterstaining with DAPI.
[0058] (2) Experimental results: The results are as follows Figure 17 As shown, compared with the control group, PA stimulation induced hepatocyte steatosis, while treatment with TP-064 significantly alleviated PA-induced hepatocyte steatosis. This indicates that PRMT4 inhibitors effectively alleviate hepatocyte steatosis.
[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. Application of PRMT4 inhibitors in the preparation of drugs for the treatment of metabolic-associated steatohepatitis.
2. The application as described in claim 1, characterized in that, The PRMT4 inhibitor is TP-064 or EZM2302.
3. Application of PRMT4 inhibitors in any of the following: (1) Application in the preparation of products that reduce the weight of patients with metabolic-associated steatohepatitis; (2) Application in the preparation of products that reduce liver weight in patients with metabolic-associated steatohepatitis; (3) Application in the preparation of products that reduce serum triglyceride and / or liver triglyceride levels in patients with metabolic-associated steatohepatitis; (4) Application in the preparation of products that reduce serum alanine aminotransferase and / or aspartate aminotransferase levels in patients with metabolic-associated steatohepatitis; (5) Application in the preparation of products that reduce fat deposition in the liver tissue of patients with metabolic-associated steatohepatitis; (6) Application in the preparation of products that improve liver tissue fibrosis in patients with metabolic-associated steatohepatitis; (7) Application in the preparation of products that reduce liver tissue inflammation in patients with metabolic-associated steatohepatitis; (8) Application in the preparation of products that improve insulin resistance in patients with metabolic-associated steatohepatitis.
4. Application of PRMT4 inhibitors in any of the following: (1) Application in the preparation of products that inhibit YAP activity; (2) Application in the preparation of products that inhibit the expression levels of CYR61, CTGF and / or H3R17me2 proteins; (3) Application in the preparation of products that control the PRMT4-YAP signal axis.
5. A drug for treating fatty liver, characterized in that, The drug includes a PRMT4 inhibitor and pharmaceutically acceptable excipients.
6. The drug as described in claim 5, characterized in that, The PRMT4 inhibitor is a pharmaceutically acceptable salt of TP-064 or EZM2302.
7. The drug as described in claim 5, characterized in that, The excipients include fillers, disintegrants, lubricants, adhesives, coating materials, and stabilizers.
8. The drug as described in claim 7, characterized in that, The filler includes microcrystalline cellulose, lactose, mannitol, or pregelatinized starch.
9. The drug as described in claim 7, characterized in that, The disintegrant includes crospovidone, sodium carboxymethyl starch, or low-substituted hydroxypropyl cellulose; the lubricant includes magnesium stearate, talc, or silica.
10. The drug as described in claim 5, characterized in that, The drug is in the form of tablets, capsules, injections, or sustained-release microspheres.