Use of mettl3 inhibitor in preparation of a medicament for treating drug-induced liver injury

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a METTL3 inhibitor in preparation of a medicine for treating drug-induced liver injury; the METTL3 inhibitor comprises STM2457 or JNJ64619178; the drug-induced liver injury is acetaminophen-induced acute liver injury. It is found for the first time that the METTL3 inhibitor STM2457 or JNJ64619178 has a significant curative effect on treatment of acetaminophen-induced drug-induced liver injury, STM2457 or JNJ64619178 can effectively alleviate acute liver tissue injury caused by acetaminophen, inhibit further apoptosis and necrosis of hepatocytes, help promote recovery of liver injury, and the treatment effect is better than that of a traditional medicine N-acetylcysteine, thus providing a new treatment means for acetaminophen-induced drug-induced liver injury and expanding the clinical application range of the inhibitor.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a METTL3 inhibitor in preparation of a drug for treating drug-induced liver injury. BACKGROUND

[0002] Drug-induced liver injury is a major global health problem. In China, drug-induced liver injury is one of the main causes of unexplained liver injury encountered in clinical practice and is one of the most common causes of acute liver injury, which can lead to acute liver failure and even death in severe cases. Due to the lack of specific clinical manifestations and diagnostic markers of drug-induced liver injury, especially the insidious onset of chronic liver injury caused by drugs, it is often not discovered or diagnosed in clinical practice. In China, the main drugs causing liver injury are various health products and traditional Chinese medicines, antituberculosis drugs, antitumor drugs or immunomodulators. In China, the incidence of drug-induced liver injury is increasing year by year. Acetaminophen (APAP) is one of the most widely used drugs in the world, and although its recommended therapeutic dose is safe and effective, the therapeutic window is narrow. The use of APAP has become the main cause of drug-induced liver injury in Western countries. In the United States, the United Kingdom and Australia, APAP is the main cause of acute liver failure (in the United States, more than 50% of acute liver failure is caused by APAP).

[0003] Currently, there is only one antidote for APAP, namely N-acetyl-L-cysteine (NAC), which is approved for clinical use. The mechanism of action of NAC depends on restoring the level of glutathione in liver cells, but only within 8 hours of intoxication can it exert the maximum effect. At the same time, NAC has a greater gastrointestinal irritation, and high-dose use can have other toxic side effects, such as allergies. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art and provide a drug with fewer side effects, low toxicity, expanded treatment time window, and greatly improved treatment effect for acetaminophen-induced liver injury in clinical practice.

[0005] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is as follows:

[0006] In a first aspect, the present application provides application of a METTL3 inhibitor in preparation of a drug for treating drug-induced liver injury, wherein the METTL3 inhibitor comprises STM2457 or JNJ64619178.

[0007] The inventors first discovered through experimental exploration that N6-methyladenosine (m 6A) The expression level of METTL3 (Methyltransferase-like 3) in the RNA methylation enzyme complex is significantly related to drug-induced liver injury induced by APAP. When acute liver injury is induced by APAP treatment, the expression level of METTL3 in the liver of the body increases significantly, and when the expression level of METTL3 gene decreases, the acute liver injury induced by APAP can be significantly alleviated. The inventors have found through experiments that STM2457 can effectively inhibit the methylation of m 6 A methyltransferase METTL3, so that STM2457 is used for treating acute liver injury caused by acetaminophen. When STM2457 is used for treating acute liver injury caused by acetaminophen, it can effectively alleviate acute liver tissue injury, inhibit further apoptosis and necrosis of hepatocytes, and promote repair of liver injury. The small molecule inhibitor JNJ64619178 reduces the expression of METTL3 to treat drug-induced liver injury induced by APAP. The therapeutic effect of STM2457 or JNJ64619178 inhibitor is significant, and the therapeutic effect is significantly better than that of traditional drugs NAC, thereby providing a new treatment for drug-induced liver injury induced by acetaminophen.

[0008] Preferably, the drug-induced liver injury is acute liver injury induced by acetaminophen.

[0009] Preferably, the dose of STM2457 applied to animals is 45-55 mg / kg / d, preferably 50 mg / kg / d, and the dose of STM2457 applied to humans is 4.95-6.06 mg / kg / d.

[0010] Preferably, the dose of JNJ64619178 applied to animals is 5-15 mg / kg / d, preferably 10 mg / kg / d, and the dose of JNJ64619178 applied to humans is 0.55-1.65 mg / kg / d.

[0011] Preferably, STM2457 inhibits the methylation of mRNA by inhibiting the enzyme activity of METTL3. 6 A methylation modification to treat drug-induced liver injury.

[0012] Preferably, JNJ64619178 inhibits the expression level of METTL3 to treat drug-induced liver injury.

[0013] RNAm 6 A methylation is the most abundant mRNA modification form in eukaryotes, and is dynamic and reversible, which is involved in almost all important biological processes. 6 A modification determines the fate of modified RNA molecules at the transcription and post-transcriptional levels, and affects almost all important biological processes. Through experiments, it has been found that in the process of drug-induced liver injury, the methylation of m6 Among the enzyme complexes related to methylation modification, METTL3 has a significant influence, and when the enzyme activity of METTL3 or the expression level of METTL3 is inhibited by a METTL3 inhibitor, the m 6 A methylation modification can significantly affect the m 6 A mediated post-transcriptional regulation, thereby exerting a good drug effect on the treatment of drug-induced liver injury.

[0014] In a second aspect, the present application provides a drug for treating drug-induced liver injury, the drug comprising a METTL3 inhibitor.

[0015] Preferably, the METTL3 inhibitor comprises STM2457 or JNJ64619178.

[0016] Preferably, the dose of STM2457 applied to animals is 45-55 mg / kg / d, preferably 50 mg / kg / d, and the dose of STM2457 applied to humans is 4.95-6.06 mg / kg / d.

[0017] Preferably, the dose of JNJ64619178 applied to animals is 5-15 mg / kg / d, preferably 10 mg / kg / d, and the dose of JNJ64619178 applied to humans is 0.55-1.65 mg / kg / d.

[0018] Preferably, the drug-induced liver injury is acetaminophen-induced acute liver injury.

[0019] Preferably, the STM2457 inhibits the m 6 A methylation modification to exert the effect of treating drug-induced liver injury.

[0020] Preferably, the JNJ64619178 exerts the effect of treating drug-induced liver injury by inhibiting the expression level of METTL3.

[0021] In a third aspect, the present application also provides a method for constructing a mouse model of drug-induced liver injury, comprising the following steps:

[0022] APAP is added to physiological saline, dissolved at 50-60°C water bath, and the mouse is fasted for 16 hours, then injected intraperitoneally with the APAP solution, thereby obtaining a mouse model of drug-induced liver injury.

[0023] Preferably, the concentration of the APAP solution is 15 mg / mL, and the injection dose is 300 or 500 mg / kg.

[0024] In a fourth aspect, the present application provides application of the above-mentioned drug-induced liver injury mouse model in drug screening for treating drug-induced liver injury.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application first discovers through cell experiments and animal experiments that METTL3 inhibitors STM2457 and JNJ64619178 have a significant therapeutic effect on treating acetaminophen-induced drug-induced liver injury, STM2457 can effectively alleviate the acute liver tissue injury caused by acetaminophen, inhibit further apoptosis and necrosis of hepatocytes, and help promote recovery of liver damage, and JNJ64619178 also reduces the expression of METTL3, thereby playing a role in treating drug-induced liver injury caused by APAP, thereby improving the quality of life of patients and prolonging the survival expectation, and the therapeutic effect of the two is better than that of the traditional NAC drug, thereby providing a new treatment for acetaminophen-induced drug-induced liver injury, and expanding the clinical application range of the inhibitor, and providing a new direction for the study of the action mechanism of the inhibitor. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The flowchart for constructing METTL3 flox / flox / Albumin-Cre ERT2 (ALB-CRE ERT2 ) mice in Example 1 is shown in the figure.

[0028] Figure 2 The mRNA expression level of m 6 A modification in Example 1 (Figure A) and the change in METTL3 expression level at the protein expression level and the statistical graph (Figure B) are shown in the figure.

[0029] Figure 3 The representative liver gross morphology (Figure A), hematoxylin-eosin (H&E) staining and necrosis area score (Figure B), representative TUNEL immunohistochemical staining and positive area statistics (Figure C), serum aspartate aminotransferase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) levels (Figure D) of control mice and liver-specific METTL3 knockout mice in Example 2 at different time points after APAP-induced drug-induced liver injury, and the survival rate of mice after high-dose APAP administration (Figure E) are shown in the figure.

[0030] Figure 4Figure for the liver injury of mice after intervention at 1.5h and 8h after APAP modeling in Example 3 using normal saline (NS), STM2457, JNJ64619178 and NAC respectively; A-D are the results of intervention at 1.5h after APAP modeling, in which A is the schematic diagram of modeling process, B is the level of AST, ALT and LDH in serum, C is the liver tissue sample after intervention of different drugs, D is the liver tissue H&E staining and liver tissue cell necrosis area statistics results after intervention of different drugs; E-H are the results of intervention at 8h after APAP modeling, in which E is the schematic diagram of modeling process, F is the level of AST, ALT and LDH in serum, G is the liver tissue sample after intervention of different drugs, H is the liver tissue H&E staining and liver tissue cell necrosis area statistics results after intervention of different drugs.

[0031] Figure 5 Figure for the mechanism of JNJ64619178 in treating APAP drug-induced liver injury in Example 4, in which A is the mRNA expression of main regulatory factors in HepG2 cells treated with different concentrations of JNJ64619178 for 24h, B is the proliferation activity of HepG2 cells treated with different concentrations of JNJ64619178 for 24h, C is the level of LDH in the supernatant of HepG2 cells treated with different concentrations of JNJ64619178 for 24h, D is the light microscope of HepG2 cells treated with 20mM APAP and different concentrations of JNJ64619178 for 24h, E is the proliferation activity detection of HepG2 cells treated with 20mM APAP and different concentrations of JNJ64619178 for 24h, F is the liver function level detection of the supernatant of HepG2 cells treated with 20mM APAP and different concentrations of JNJ64619178 for 24h. 6 A is the mRNA expression of main regulatory factors; B is the proliferation activity of HepG2 cells treated with different concentrations of JNJ64619178 for 24h; C is the level of LDH in the supernatant of HepG2 cells treated with different concentrations of JNJ64619178 for 24h; D is the light microscope of HepG2 cells treated with 20mM APAP and different concentrations of JNJ64619178 for 24h; E is the proliferation activity detection of HepG2 cells treated with 20mM APAP and different concentrations of JNJ64619178 for 24h; F is the liver function level detection of the supernatant of HepG2 cells treated with 20mM APAP and different concentrations of JNJ64619178 for 24h. DETAILED DESCRIPTION

[0032] The above content of the present application will be further explained in detail by the following specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present application being limited to the following examples.

[0033] STM2457 in the present application is purchased from Selleck Company in the United States, and the drug item number is S9870; JNJ64619178 is purchased from Selleck Company in the United States, and the drug item number is S8624.

[0034] METTL3 flox / floxThe mice were gifted by Professor Zhou Qi of the Institute of Animal Sciences of the Chinese Academy of Sciences, and the Albumin-Cre ERT2 (ALB-Cre ERT2 ) mice were purchased from Beijing Boao Situ Gene Biotechnology Co., Ltd. The C57BL / 6J mice were purchased from Guangdong Yaoke Biological Technology Co., Ltd.

[0035] The reagents used in the examples are conventional reagents in the art unless otherwise specified, and can be purchased through commercial channels. The experimental operations not specifically mentioned in the examples are conventional operations in the art or can be understood or known by those skilled in the art according to the existing technology or common knowledge mastered by them.

[0036] Example 1. Liver tissue conditions of mice with acute liver injury induced by acetaminophen (APAP)

[0037] 1. Preparation of experimental mice

[0038] METTL3 flox / flox mice were crossed with ALB-Cre ERT2 mice to obtain METTL3 flox / flox / ALB-Cre ERT2 mice Figure 1 ), and the obtained METTL3 flox / flox / ALB-Cre ERT2 mice were intraperitoneally injected with olive oil (Oil) at 4-5 weeks of age, and then normally fed for 1 week after 5 consecutive injections, to obtain METTL3 flox / flox / Alb-Cre ERT 2 + Oil treated mice, i.e. Control mice;

[0039] (1) Control group: no injection of any drug in Control mice, time point is 0h;

[0040] (2) APAP experimental group: the Control mice were fasted for 16 hours, then intraperitoneally injected with APAP solution, the final concentration of APAP solution was 15mg / mL, the injection dose was 300mg / kg, and the treatment time points were 3h, 6h and 24h, respectively. After treatment, they were returned to a specific pathogen-free facility with a temperature of 24±2℃, a humidity of 30-70%, and sufficient food and water.

[0041] 2. Experimental method

[0042] (1) qPCR quantitative experiment:

[0043] After 0h, 3h, 6h and 24h of APAP treatment, 0.2g of fresh mouse liver tissue after blood removal by cold PBS perfusion was collected for total RNA extraction, genomic DNA removal and reverse transcription cDNA.

[0044] Quantitative qPCR reaction system: 2*PCR premix 5μL, cDNA 1μL, upstream primer 0.5μL, downstream primer 0.5μL, DEPC water 3μL, total volume 10μL; a total of three duplicate wells were set, and the program was set as 95℃ 10min, 95℃ 15s, 60℃ 30s, 72℃ 30s, a total of 40 cycles; melting curve analysis: 55℃-95℃, read once per minute.

[0045] (2) Mouse liver tissue total protein extraction and Western blot experiment:

[0046] 0.2g of fresh mouse liver tissue after blood removal by cold PBS perfusion was collected, frozen in liquid nitrogen, and then 400uL of RIPA lysis buffer (phosphatase inhibitor and protease inhibitor were added before use) and two 3mm steel balls were added for rapid grinding and lysis of the liver tissue. The mixture was centrifuged at 4℃, 14000rpm for 10 minutes, and the supernatant was collected to obtain the total protein of the liver tissue.

[0047] The total protein of the liver tissue obtained above was subjected to Western blot experiment, METTL3 specific primary antibody (Servicebio, GB124688-100) was used to detect the level of METTL3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primary antibody (Cell Signaling Technology, Cat#2118) was used to detect the level of GAPDH as an internal reference protein. After incubation with horseradish peroxidase (HRP) conjugated anti-mouse secondary antibody, the Bio-Rad gel imaging system was used for development.

[0048] 3. Experimental results

[0049] The results are shown in Figure 2 Compared with the control group without APAP injection, the expression levels of METTL3 in the liver of the experimental group mice increased significantly with time after APAP treatment, and the related mRNA and protein expression levels also increased significantly; and the expression of m 6 Among the enzymes related to methylation modification, Mettl3 mRNA expression changed most significantly, indicating that the expression level of METTL3 was significantly related to drug-induced liver injury induced by APAP. When acute liver injury was induced by APAP treatment, the expression level of METTL3 in the liver of the organism increased significantly.

[0050] Example 2. Mice with liver cell METTL3 specifically knocked out were induced by APAP to acute liver injury

[0051] 1. Preparation of experimental mice

[0052] (1) Control mice: the obtained METTL3 flox / flox / ALB-Cre ERT2 mice were intraperitoneally injected with olive oil (Oil) at the age of 4-5 weeks, and after continuous injection for 5 days, they were normally fed for 1 week to obtain METTL3 flox / flox / ALB-Cre ERT2 + Oil treated mice, i.e. Control mice;

[0053] (2) METTL3 cKO mice: the obtained METTL3 flox / flox / ALB-Cre ERT2 mice were intraperitoneally injected with tamoxifen (10 mg / mL stock solution; when the weight of the mice was less than 15 g, 100 μL per mouse per day was administered; when the weight of the mice was > 15 g, 7.5 μL / g per day was administered) at the age of 4-5 weeks, and after continuous injection for 5 days, they were normally fed for 1 week to obtain liver cell-specific METTL3 knockout mice, i.e. METTL3 cKO mice.

[0054] 2. Experimental method

[0055] (1) APAP modeling: APAP powder was added to the corresponding volume of normal saline, and dissolved completely in a 55°C water bath, with a final concentration of 15 mg / mL; the Control mice and METTL3 cKO mice were fasted for 16 h, and then injected intraperitoneally with APAP at a dose of 300 mg / kg (regular modeling) or 500 mg / kg (survival rate observation), to obtain the Control+APAP group and the METTL3 cKO+APAP group (i.e. a drug-induced liver injury mouse model with liver-specific METTL3 gene knockout), respectively, and the same volume of normal saline was injected as a blank group.

[0056] (2) Hematoxylin-eosin staining experiment: the liver tissues of the mice sampled at 3 h, 6 h and 24 h after APAP modeling were immersed in 4% paraformaldehyde, and fixed at room temperature for 8-24 h, then sent to the Sivier Company for further paraffin embedding, sectioning and H&E staining treatment, and the necrotic cell area was counted.

[0057] (3) TUNEL immunohistochemical staining: After the paraffin white film of each experimental group was dewaxed and hydrated, the liver tissue section area was circled using a histological pen, 20 ug / mL of proteinase K (Beyotime, ST533, Shanghai, China) without DNAse was added for incubation for 15 minutes, 3% H2O2 was added for incubation for 15 minutes; the corresponding reagent was prepared according to the manufacturer's instructions, biotin-labeled liquid was added for reaction at 37°C for 1 hour in the dark; after the reaction was completed, the termination liquid was incubated for 10 min; streptavidin-HRP reaction liquid was added for incubation for 30 min, then diaminobenzidine (DAB) was used for color development, and finally hematoxylin was used to stain the nucleus; the TUNEL stained film was scanned by TissueFAXS SL high-throughput cell instrument (Tissue Gnostics, Austria), and the TUNEL positive cells of each individual were quantitatively analyzed using TissueFAXS software.

[0058] (4) Serum liver function detection: After the mice at different sampling time points were deeply anesthetized with ketamine mixed anesthetics, 500 uL of whole blood was collected from the orbital venous plexus capillary pipette, the blood sample was centrifuged at 5000 rpm for 10 min at 4°C, a small amount of serum was diluted 10 times with PBS, and then the automatic biochemical analyzer (Hitachi, Tokyo, Japan) was used to detect the serum AST, ALT and LDH levels.

[0059] 3. Experimental results

[0060] The results are shown in Figure 3 By constructing a liver cell-specific METTL3 knockout mouse (METTL3 cKO mouse) and performing APAP modeling at two doses, liver tissue samples were collected from mice at 3h, 6h and 24h after APAP regular dose modeling (300mg / kg) ( Figure 3 A), it was found that compared with the Control group, the liver tissue necrosis area of the METTL3 cKO group mice was significantly reduced ( Figure 3 B), and the liver function levels AST, ALT and LDH were also significantly decreased ( Figure 3 D); and after APAP high dose (500mg / kg) modeling, it was found that compared with the Control group, the survival rate of the METTL3 cKO group mice was significantly increased ( Figure 3 E); the above results also prove that the expression level of METTL3 is significantly related to APAP-induced drug-induced liver injury, when the METTL3 gene is deleted, the expression level is decreased, which can significantly alleviate APAP-induced acute liver injury.

[0061] Example 3. STM2457 and JNJ64619178 intervene in the liver damage of acute liver injury mice induced by APAP

[0062] 1. Preparation of experimental mice

[0063] C57BL / 6J male mice aged 6-8 weeks were purchased from Guangdong Jisui Yekang Biotechnology Co., Ltd. and were subjected to 16 h fasting treatment in advance.

[0064] 2. Experimental grouping

[0065] (1) Control group: C57BL / 6J mice were not added with any drug;

[0066] (2) APAP+NS group: C57BL / 6J mice were intraperitoneally injected with APAP 300 mg / kg for 1.5 h, and then injected with an equal volume of solvent normal saline (NS), and the detection time point was 24 h after APAP injection;

[0067] (3) APAP+JNJ64619178 experimental group: C57BL / 6J mice were intraperitoneally injected with APAP 300 mg / kg for 1.5 h, and then intraperitoneally injected with JNJ64619178 solution, with a final concentration of 27 mg / mL and a dose of 10 mg / kg, and the detection time point was 24 h after APAP injection;

[0068] (4) APAP+STM2457 experimental group: C57BL / 6 mice were intraperitoneally injected with APAP 300 mg / kg for 1.5 h, and then intraperitoneally injected with STM2457, with a final concentration of 6.75 mg / mL and a dose of 50 mg / kg, and the detection time point was 24 h after APAP injection;

[0069] (5) APAP+NAC experimental group: C57BL / 6J mice were intraperitoneally injected with APAP 300 mg / kg for 1.5 h, and then gavaged with NAC drug, with a final concentration of 30 mg / mL and a dose of 300 mg / kg, and the detection time point was 24 h after APAP injection;

[0070] (6) APAP+NS group: C57BL / 6J mice were intraperitoneally injected with APAP 300 mg / kg for 8 h, and then injected with an equal volume of solvent normal saline (NS), and the detection time point was 24 h after APAP injection;

[0071] (7) APAP+JNJ64619178 experimental group: C57BL / 6J mice were intraperitoneally injected with APAP 300 mg / kg for 8 h, and then intraperitoneally injected with JNJ64619178 solution, with a final concentration of 27 mg / mL and a dose of 10 mg / kg, and the detection time point was 24 h after APAP injection;

[0072] (8) APAP+STM2457 experimental group: C57BL / 6 mice were treated with APAP 300mg / kg intraperitoneally for 8h, and then STM2457 was injected intraperitoneally. The final concentration was 6.75mg / mL and the dose was 50mg / kg. The detection time point was 24h after APAP injection.

[0073] (9) APAP+NAC experimental group: C57BL / 6J mice were treated with APAP 300mg / kg intraperitoneally for 8h, and then NAC was administered by gavage to a final concentration of 30mg / mL and a dose of 300mg / kg. The detection time point was 24h after APAP injection.

[0074] 3. Test methods

[0075] (1) H&E staining experiment:

[0076] Mouse liver tissues collected from each experimental group were immersed in 4% paraformaldehyde and fixed at room temperature for 8-24 hours. They were then sent to Cellular for further paraffin embedding, sectioning, and H&E staining, and the area of ​​cell necrosis was counted.

[0077] (2) Liver function test results:

[0078] Mice in each experimental group were deeply anesthetized with a mixture of ketamine and anesthetic. 500 μL of whole blood was collected from each group via capillary aspiration of the retro-orbital venous plexus. The blood samples were incubated overnight at 4°C and then centrifuged at 5000 rpm for 10 min. A small amount of serum was taken and diluted 10 times with PBS. The serum AST, ALT and LDH levels were then detected using a fully automated biochemical analyzer (Hitachi, Tokyo, Japan).

[0079] 4. Experimental Results

[0080] The results are as follows Figure 4 As shown, when intervention was performed 1.5 h after APAP injury in mice ( Figure 4 A) The Control group served as the negative control, and the APAP+NS group served as the positive control. Compared with the APAP+NS group, the use of the METTL3 specific inhibitor STM2457, the small molecule inhibitor JNJ64619178, and the only antidote for APAP, NAC, significantly reduced liver function indicators AST, ALT, and LDH. Figure 4 B) Reduce the overall swelling of the liver ( Figure 4 C) Reduce the area of ​​liver tissue necrosis ( Figure 4 D), promotes the recovery of liver damage in mice ( Figure 4(BD). Notably, when comparing liver function levels, the serum AST and ALT levels in the APAP+STM group were lower than those in the APAP+NAC group (Figure B); and when comparing the area of ​​liver necrosis, the area of ​​liver necrosis in the APAP+STM group was smaller than that in the APAP+NAC group. Figure 4 D) suggests that the STM treatment group had a more significant therapeutic effect. Intervention was performed 8 hours after APAP injury in mice (…). Figure 4 E), the Control group served as the negative control, and the APAP+NS group served as the positive control; compared with the APAP+NS group, among the METTL3-specific inhibitor STM2457, the small molecule inhibitor JNJ64619178, and the NAC treatment intervention groups, only JNJ64619178 showed a role in promoting the recovery of liver injury in mice. Figure 4 FH): When comparing serum liver function indicators, it was found that the serum AST and ALT levels in the APAP+JNJ group were significantly lower than those in the APAP+NS group. Figure 4 F); When comparing the gross liver, it was found that the hepatic redness and swelling were reduced in the APAP+JNJ group compared with the APAP+NS group. Figure 4 G), when comparing the area of ​​liver necrosis, it was found that the area of ​​liver necrosis in the APAP+JNJ group was smaller compared with that in the APAP+NS group. Figure 4 H), the degree of liver damage is reduced, and the treatment effect is significant. The above experimental results show that STM2457 and JNJ64619178 have significant efficacy in treating acetaminophen-induced drug-induced liver injury. They can effectively alleviate acute liver tissue damage caused by acetaminophen, inhibit further apoptosis and necrosis of hepatocytes, help promote liver damage recovery, thereby improving patients' quality of life and prolonging survival. Moreover, their therapeutic effect is significantly better than traditional NAC drugs, and they can still exert their effects beyond the 8-hour treatment window, providing a new treatment method for acetaminophen-induced drug-induced liver injury.

[0081] Example 4. Investigation into the mechanism of action of JNJ64619178 in treating APAP-induced liver injury

[0082] This embodiment investigates the mechanism of action of the small molecule inhibitor JNJ64619178 in a hepatocellular carcinoma model. Different concentrations of JNJ64619178 (0, 0.5, 1, 2, 2.5, 5, and 10 μM) were added to the HepG2 hepatocellular carcinoma cell line to detect the effects of various concentrations. 6 A regulates the level of change in molecules, and the result is as follows: Figure 5(A) shows that the inhibitor JNJ64619178 can specifically reduce the expression of METTL3 in hepatoma cell lines; further, this embodiment selects different JNJ64619178 drug concentrations (1, 2 μM) to affect the hepatoma cell lines, compared with the control group only adding DMSO, the JNJ64619178 inhibitor can affect the proliferation activity of hepatoma cell lines Figure 5 B), but does not affect the supernatant liver function level Figure 5 C); however, in the APAP hepatoma cell damage model, different concentrations of JNJ64619178 small molecule inhibitors were added for treatment, and then through the cell light microscope graph Figure 5 D) and cell proliferation activity detection Figure 5 E) found that JNJ64619178 can enhance the cell activity after APAP treatment, the cell number increases significantly, and the supernatant liver function level detection Figure 5 F) shows that compared with the DMSO group, the AST level of the culture supernatant in the experimental group intervened by JNJ64619178 is significantly reduced, that is, the damage degree is reduced, and the above conclusions all show that the JNJ64619178 small molecule inhibitor can reduce the acute cell damage caused by APAP by inhibiting the expression level of METTL3, and the treatment effect is significant.

[0083] Finally, it should be explained that the above embodiments are only used to illustrate the technical solutions of the present application, not to limit the protection scope of the present application, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Use of a METTL3 inhibitor in the preparation of a medicament for treating drug-induced liver injury, characterized in that, The METTL3 inhibitor is JNJ64619178; The drug-induced liver injury is acetaminophen-induced acute liver injury.

2. Use according to claim 1, characterized in that, The JNJ64619178 exerts the effect of treating drug-induced liver injury by inhibiting the expression level of METTL3.

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