Application of genistein to treatment of rifampicin-induced drug-induced liver injury

Abstract

The invention provides application of genistein to protection of rifampicin-induced drug-induced liver injury. GEN is an extract monomer of a leguminous plant, and intragastric administration is adopted in the invention. According to the technical scheme of the invention, a GEN protection rifampicin induced mouse liver injury model is carried out, and different liver cell lines are used for verifying that GEN of human liver cancer HepG2, human liver cancer Hep3B and normal mouse liver cells AML12 reduces damage of liver cells in vitro. GEN intragastric administration lasts for 16 days, the weight of the liver can be reduced, fatty degeneration can be remarkably improved, deposition of fat including triglyceride and cholesterol in the liver can be reduced, the blood bilirubin level can be reduced, and mice do not die in the whole process; the endogenous mechanism of the gene and GEN increases CH25H expression, the catalytic product 25HC of the latter is increased, and the latter combines and activates LXR alpha and enters the nucleus to promote ABCA transcription, so that cholesterol excretion of liver cells is promoted. The new pharmacological efficacy and liver protection mechanism of GEN are found for the first time, and a foundation is laid for future clinical application.

Description

Technical Field

[0001] The present invention relates to a new pharmaceutical use of genistein (GEN for short). It is found that the chemical composition (oral administration) of genistein has a strong protective effect against rifampicin-induced liver damage, which helps to expand its indications and provide a new option for liver protection treatment of anti-tuberculosis drugs. Background Art

[0002] Drug-induced liver injury (DILI) is one of the most common liver diseases in clinical practice. Rifampicin (RFP) is a first-line anti-tuberculosis drug recommended by the World Health Organization. In AT-DILI, rifampicin (RFP)-induced DILI is one of the common causes of AT-DILI. However, the treatment effect of AT-DILI is not ideal because the mechanism is not yet clear and precision medicine cannot be achieved.

[0003] Rifampicin-induced liver injury has significant characteristics, with early or short-term liver fat deposition, cholestasis, and no elevated transaminases. Long-term use can lead to liver cell rupture and elevated serum transaminases. However, there is currently no specific drug for the treatment of RFP-induced liver injury. Traditional liver protection drugs such as reduced glutathione, diammonium glycyrrhizinate, and glucuronolactone are only symptomatic treatments, with antioxidant and anti-inflammatory effects, and cannot act on the precise targets of rifampicin-induced liver injury. How to intervene early and protect against rifampicin-induced drug-induced liver injury is a top priority for basic research.

[0004] Genistein (GEN, also known as genistein, 5,7,4'-trihydroxyisoflavone, genistein) is a monomer of rhizome extracts of plants in the Leguminosae family (Sophora japonica, Sophora flavescens, and Broom). It is soluble in DMSO and ethanol, almost insoluble in water, and soluble in dilute alkali. It is used for anti-tumor, antibacterial, hypolipidemic, and estrogen-like effects. This invention proposes for the first time that GEN can protect RFP-induced drug-induced liver injury and its intrinsic mechanism both in vivo and in vitro, which will lay the foundation for future clinical applications. Summary of the invention

[0005] The present invention discloses that GEN can alleviate RFP-induced liver damage. This model uses a medium dose of RFP (150 mg / kg·d -1 , gavage, once a day) to create a liver injury model, inducing liver enlargement, liver fat deposition, significant increase in intrahepatic triglycerides and cholesterol, and increased serum bilirubin in mice. The present invention is based on the discovery in the process of GEN treating RFP-induced liver injury model that GEN has a very strong protective effect, reducing liver fat deposition, fatty degeneration, and bilirubin, and no death occurs. The specific contents are as follows: The technical solution of the present invention has the following beneficial effects: (1) RFP-induced liver injury is characterized by hepatic steatosis and cholestasis, but no increase in transaminase. -1 , once a day) was administered orally to C57BL / 6J mice (about 20 g, male, 8 weeks old) for a short period of 16 days. -1 According to the conversion ratio (9.1 times), 91 mg / kg is needed for oral administration. We consulted the literature and conducted preliminary experiments, and found that RFP 300 mg / kg·d -1 , with a mortality rate of about 1 / 3. 75 mg / kg is difficult to induce or requires a long time to induce liver damage. It was finally decided to use a medium dose and short-term induction of liver damage. The results showed that there was no death record in this model, with significant liver enlargement, liver fatty degeneration, and cholestasis. In addition, we found that there was no increase in transaminase levels in this model, which was an early non-inflammatory liver lesion. Our model belongs to RFP short-term mild liver injury, which is consistent with the clinical characteristics of mild symptoms (liver lipid deposition, cholestasis), and there was no death record; see the relevant steps of Example 4.

[0006] (2) GEN alleviates RFP-induced liver lipid droplet formation in a time-dependent and dose-dependent manner. Through in vitro experiments, RFP has a time-dependent and dose-dependent increase in lipid droplet formation in hepatocytes, especially cholesterol accumulation in the liver. Several liver cell lines have the same lipid droplet formation (human liver cancer HepG2, human liver cancer Hep3B, normal mouse AML12). 300mM RFP treatment of liver cell lines for 24 hours can successfully induce lipid accumulation in hepatocytes, and total cholesterol (TC) and total triglycerides (TG) have significant increases, but the increase in TC in the cell supernatant is not obvious; the inconsistent trend of TC changes inside and outside the cell suggests that there may be impaired cholesterol efflux function; at the same time, GEN can significantly reduce intracellular TC to the normal range. See the relevant steps of Examples 2 and 3.

[0007] (3) The intrinsic mechanism of GEN in alleviating RFP-induced cholesterol accumulation in the liver. This study used network pharmacology and transcriptomics to find that RFP, DILI, and GEN may have common targets (including CH25H, ABCA, and LXRα). Subsequent verification experiments found that RFP can significantly inhibit the CH25H-LXRα-ABCA1, 2, and 3 pathways, thereby inhibiting cholesterol efflux and leading to a large accumulation of cholesterol in cells; while GEN can activate the CH25H-LXRα-ABCA1, 2, and 3 pathways, thereby promoting cholesterol efflux, alleviating cholesterol accumulation and reducing liver lipid deposition. See the relevant steps of Example 5. Overexpression of the gene by transfecting the CH25H plasmid can alleviate RFP-induced lipid droplet deposition. This shows that CH25H plays a key role in RFP-induced lipid droplet deposition. See the relevant steps of Example 6.

[0008] (4) Molecular docking explains that RFP and GEN may competitively bind to CH25H. The Autodock molecular docking software found that three key amino acid residues (VAL-205, VAL-206, TRP-209) may be the sites of competitive binding between RFP and GEN. They may affect the affinity of RFP and CH25H by affecting the formation of mutual hydrogen bonds. See the relevant steps in Example 7.

[0009] (5) Method for preparing solvent for dissolving GEN. GEN is a light yellow powder crystal with a melting point of 297°C-298°C. It needs to be protected from light. It is soluble in DMSO and ethanol, almost insoluble in water, with a solubility of 8.7 μg / ml in water. It is soluble in common organic solvents, and changing the pH of the solution has no significant effect on genistein. This scheme uses a small volume of pure DMSO to dissolve GEN powder, and then dilutes it with a larger volume of PBS (the volume ratio of DMSO:PBS for animal experiments = 1:500; the volume ratio of DMSO:PBS for cell experiments = 1:17). On the one hand, it enhances the dissolution of GEN (avoids heating), and also reduces the possible impact of pure DMSO on cell and animal experiments. See the relevant steps in Example 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : Figure 1A shows that different concentrations of RFP induce lipid droplet formation in human hepatocellular carcinoma Hep3B cells (Oil Red O staining).

[0011] Figure 2 : Figure 2A shows BODIPY staining (green for lipid droplets, DAPI blue for cell nuclei) and Nile red staining (red for lipid droplets, Hoechst 33258 for cell nuclei).

[0012] Figure 3 : Figure 3A shows the enzymatic determination of intracellular cholesterol; Figure 3B shows the enzymatic determination of intracellular triglyceride content.

[0013] Figure 4 : Figure 4A shows the changes in intracellular cholesterol determined by enzymatic method; Figure 4B shows the increase in extracellular cholesterol determined by enzymatic method; Figure 4C determines the area under the curve (AUC) of the increase in supernatant cholesterol. Figure 5 : Network pharmacology analysis of the intersection target genes of RFP, GEN and drug-induced liver injury (DILI).

[0014] Figure 6 : Network pharmacology analysis of potential targets of GEN in treating DILI.

[0015] Figure 7 : Transcriptomic sequencing (RNA-seq) revealed potential targets in GEN compared with the control group (Vehicle, Veh).

[0016] Figure 8 :Transcriptomic sequencing (RNA-seq) found differential targets between RFP and RFP+GEN.

[0017] Fig. 9 : Changes of body weight, blood lipids and liver weight in RFP-induced liver injury model treated with GEN.

[0018] Fig.10 : Gross appearance, HE staining and Oil Red O staining of liver.

[0019] Fig.11 : Determination of total cholesterol (TC), triglyceride (TG) and serum transaminase in liver tissue.

[0020] Fig.12 : Serum total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) levels in mice.

[0021] Fig.13 : The protein expressions of LXRα and CH25H in liver tissues of RFP group and GEN group were detected by immunoblotting.

[0022] Fig.14 :In vitro experiments showed that GEN could reduce lipid deposition in hepatocytes induced by RFP (Oil Red O staining).

[0023] Fig.15 :In vitro experiments, RFP at different concentrations and time points improved the expression of CH25H protein in different hepatic cell lines (Western blotting).

[0024] Fig.16 :In vitro experiments showed the effect of genistein on the protein expression of LXRα and CH25H in RFP-treated hepatocytes (Western blotting).

[0025] Fig.17 : Figure 17A shows that GEN can reverse the nuclear export of LXRα induced by RFP. Figure 17B shows that GEN increases the mRNA expression of ABCA and CH25H in Hep3B (qRT-PCR method).

[0026] Fig.18 :18A Oil Red O staining showed that overexpression of mouse-derived CH25H plasmid could alleviate the increase of lipid droplets in Hep3B hepatocytes induced by RFP. 18B Enzymatic assay was used to determine the effect of overexpression of mouse-derived CH25H plasmid on the intracellular total cholesterol (TC) and triglyceride (TG) content induced by RFP.

[0027] Fig.19 : Autodock 4.2 molecular docking software predicted that RFP and GEN docked with human CH25H respectively. DETAILED DESCRIPTION

[0028] Example 1 1.1 Preparation of RFP solution (cell experiment) Take 100 mg of RFP (MCE, product number HY-B0272) + 1.215 mL of DMSO (dimethyl sulfoxide, cell culture grade, purity>99.5%, Sigma, product number D2650-100 mL, the same below), dissolve into 100 mM storage solution, store the storage solution at -20°C for 1 month, and at -80°C for 6 months; Take 2 μL of the above storage solution and add 1.998 mL of DMEM, mix well to get a working solution with a concentration of 100 μM, and use it immediately after preparation; Take 4 μL of the above storage solution and add 1.996 mL of DMEM, mix well to obtain a working solution with a concentration of 200 μM, and use it immediately after preparation; Take 6 μL of the above storage solution and add 1.994 mL of DMEM, mix well to obtain a working solution with a concentration of 300 μM, which can be used immediately after preparation.

[0029] 1.2GEN solution preparation (cell experiment) Take 5 mg of GEN (MCE, catalog number: HY-B0272) and 1.8502 mL of DMSO, dissolve into a 10 mM storage solution, store the storage solution at -20°C for 6 months and at -80°C for 1 year; Take 1 μL of the above storage solution and add 1.999 ml of DMEM, mix well to get a working solution with a concentration of 5 μM, and use it immediately after preparation; Take 2 μL of the above storage solution and add 1.998 ml of DMEM, mix well to get a working solution with a concentration of 10 μM, and use it immediately after preparation; Take 4 μL of the above storage solution and add 1.996 ml of DMEM, mix well to obtain a working solution with a concentration of 20 μM, and use it immediately after preparation.

[0030] 1.3 Preparation of RFP solution (animal experiment) Take 100 mg of RFP (same as above) + 1.215 mL of DMSO and dissolve into 100 mM stock solution. The stock solution can be stored at -20°C for 1 month and at -80°C for 6 months. Take 0.182 mL of the above storage solution and add 1.818 mL of PBS, mix well to obtain an intragastric solution with a concentration of 15 mg / mL, and store at 4°C for one week.

[0031] 1.4GEN solution preparation (animal experiment) Take 20 mg of GEN (same as above) + 0.370 mL of DMSO and dissolve into 200 mM stock solution. The stock solution can be stored at -20°C for 6 months and at -80°C for 1 year. Take 0.111 ml of the above storage solution + 1.889 ml of PBS, mix well to obtain an intragastric solution with a concentration of 3 mg / ml, and store at 4°C for one week.

[0032] Note: Phosphate Buffered Saline (PBS, Powder) is sterilized by steam sterilization at 121.3℃ (103.4KPa) under high pressure (30 minutes), and stored at room temperature or in a refrigerator at 4℃ (use up within 1 week).

[0033] PBS (pH 7.4, 10 mM) preparation: 1. Preparation of mother solution: 0.2M Na 2 HPO 4 :Weigh 71.6g Na 2 HPO 4 12H 2 O, dissolved in 1000mL water; 0.2M NaH 2 PO 4 :Weigh 2.0g NaH 2 PO 4 ·2H 2 O, dissolved in 1000mL water; Preparation of PBS (pH=7.4): First prepare 0.2M PBS (pH=7.4, 100mL): Take 19mL of 0.2mol / L Na 2 HPO 4 ,81ml 0.2mol / L Na 2 HPO 4 , that's it.

[0034] 2. Dilute the stock solution 20 times to obtain a 10mM working solution. 0.01M PBS (pH=7.4): Take 50mL of 0.2M PBS and dilute to 1000mL with water.

[0035] Example 2 In vitro experiments showed that RFP induced lipid droplet formation in hepatocyte cell lines.

[0036] Hep3B cells were treated with different concentrations of RFP (0, 100, 200, 300 μM, for 24 h) and then fixed with formaldehyde fixative. The cells were stained with lipid droplets using the Oil Red O kit. Figure 1 A Oil red O staining showed that RFP could induce the formation of a large number of lipid droplets in Hep3B cells with increasing concentration; Figure 1 B shows that RFP acts on human liver cancer cells HepG2, and Oil Red O staining indicates that different concentrations of RFP induce the increase of lipid droplets in HepG2. Figure 2 B is Nile red staining (red represents lipid droplets, and Hoechst33258 blue represents cell nuclei), indicating that different RFPs increase intracellular lipid droplets in HepG2 and Hep3B. Figure 2 A shows that the intracellular TG content of Hep3B cells treated with RFP (300 μm, 24 h) was measured using a triglyceride (TG) enzymatic kit, and it was found that RFP could induce an increase of about three times in intracellular TG. Figure 2 B shows the measurement of the intracellular total cholesterol (TC) content in Hep3B cells (300 μ m , 24 h) treated with RFP using an enzyme assay kit. It was found that RFP could induce an approximately three-fold increase in intracellular TC. Figure 4 A shows that treatment of Hep3B with different concentrations of RFP (0, 100, 200, 300 μM, 24 h) led to an increase in intracellular cholesterol (TC), and a statistically significant increase was found at 300 μM. Figure 4 B shows the changes in TC of cell supernatant over time. RFP can reduce the increase in TC of cell supernatant. Figure 4 C Display Figure 4 The area under the curve (AUC) of B showed that RFP could significantly inhibit the increase of TC in the supernatant compared with the control group. This indicates that RFP increases intracellular TC and reduces extracellular TC levels, which may be related to the reduction of cholesterol efflux.

[0037] Example 3 Network pharmacology analysis of the intersection genes of GEN and RFP: 3.1 Figure 5A obtained the molecular structure of chemical small molecules (PMOL format) through the chemical formula query website (https: / / pubchem.ncbi.nlm.nih.gov), and the code of rifampicin is CID: 135398735. Then, a collection of 349 potential target genes was found from 4 chemical small molecule target databases (https: / / www.bindingdb.org / bind / index.jsp) (https: / / ctdbase.org / ) (https: / / lilab-ecust.cn / pharmmapper / index.html) (http: / / swisstargetprediction.ch / ). 590 key genes were obtained by searching for drug-induced liver injury (DILI). The above two were intersected in a Venn diagram to obtain 120 genes.

[0038] 3.2 Figure 5 B obtained the code of genistein through the chemical formula query website (https: / / pubchem.ncbi.nlm.nih.gov ), which is CID: 5280961. Then, a collection of 386 potential target genes was found from 4 chemical small molecule target databases (https: / / www.bindingdb.org / bind / index.jsp) (https: / / ctdbase.org / ) (https: / / lilab-ecust.cn / pharmmapper / index.html) (http: / / swisstargetprediction.ch / ). A search for drug-induced liver injury (DILI) yielded 590 key genes. The above two were intersected in a Venn diagram to obtain 89 genes.

[0039] 3.3 Figure 5 C made the Venn diagram intersection of 5A and 5C and found that they had only 36 common genes.

[0040] 3.4 Figure 5 D shows the mutual connection of these 36 genes analyzed by the website STRING system (https: / / cn.string-db.org / ), that is, the possible interactions of albumin were obtained.

[0041] 3.5 Figure 5E shows that the software Cytoscape 3.80 software (using strict screening criteria ClosenessCentrality>0.3, Clustering Efficiency>0.2) was used to finally obtain 26 key genes that may interact with RFP and GEN from 36 genes.

[0042] 3.6 Figure 6 A adopts Figure 5 The same method as A was used to obtain 20 common target genes of GEN and DILI.

[0043] 3.7 Figure 6 B shows 20 genes that interact or associate.

[0044] 3.8 Figure 6 C adopts Figure 5 E method, and the interactions of 15 core genes were obtained.

[0045] Example 4 In vivo experiment of GEN intervening RFP-induced drug-induced liver injury C57Bl / 6J mice (8 weeks old, male) were divided into 3 groups: one group was the negative control group (Veh group): gavage with an equal volume of DMSO, about 0.2 mL of DMSO per mouse, once a day; one group was the RFP group (gavage with 150 mg / kg.d-1, for 16 consecutive days, once a day); one group was the RFP+GEN group, that is, gavage with rifampicin and GEN (30 mg / kg, once a day) at the same time, for 16 consecutive days, and then fresh liver tissues were obtained, quick-frozen in liquid nitrogen, and then transported to Shanghai Zhongke Purui Company for sequencing on dry ice.

[0046] 4.1 Figure 7 Figure A is the volcano plot result of mouse liver transcriptome sequencing RNA-seq. Figure 7 B is the bioinformatics analysis of RNA-seq - KEGG enrichment analysis. Figure 7 C is the bioinformatics analysis of RNA-seq - GO enrichment analysis. Figure 7 D is the bioinformatics analysis of RNA-seq - heat map analysis.

[0047] 4.2 Figure 8 Figure A shows the volcano plot results of mouse liver transcriptome sequencing RNA-seq. Figure 8 B is the bioinformatics analysis of RNA-seq - KEGG enrichment analysis. Figure 8 C is the bioinformatics analysis of RNA-seq - GO enrichment analysis. Figure 8 D is the bioinformatics analysis of RNA-seq - heat map analysis.

[0048] 4.3 Fig. 9 A shows the weight changes of mice (no difference among groups). Fig. 9 B. Serum cholesterol (TC) and triglyceride (TG) levels (no difference between the groups). Fig. 9 C is the change of liver index (liver weight / body weight). The liver index of the RFP group was higher than that of the Veh group, and it was significantly reduced to normal level after GEN treatment.

[0049] 4.4 Fig.10 The gross appearance, HE staining, and Oil Red O staining of the livers of the three groups were shown. The livers of the three groups were photographed and then stained with HE and Oil Red O. The results showed that the color of the livers of RFP was yellowish, and the color of the livers improved after GEN treatment. HE staining and Oil Red O staining showed that there were obvious small fat droplets formed in the livers of the RFP group, and the livers had fatty degeneration. The condition improved significantly after GEN treatment.

[0050] 4.5 Determination of total cholesterol (TC), triglycerides (TG) and serum transaminase in liver tissue. Fig.11 A and Fig.11 B is in accordance with Fig. 9 The mice were divided into groups and the TC and TG contents of fresh liver tissues were determined using an enzymatic kit built in Nanjing. It was found that the triglyceride and total cholesterol contents in the liver tissues of the RFP group were significantly higher than those in the control group, but GEN treatment could significantly improve the TC and TG contents. Fig.11 C The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice were determined using the Lysine method or microplate kit (C009-2-1, C010-2-1) produced by Nanjing Jiancheng Company (no difference between the groups), indicating that this model is an early injury without severe hepatocyte inflammation and hepatocyte necrosis.

[0051] 4.6 Levels of total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) in mouse serum. Fig.12 A and Fig.12 B is in accordance with Fig. 9 The three types of bilirubin in serum were measured according to the grouping method using the chemical oxidation kit (C019-1-1, C019-2-1) produced by Nanjing Jiancheng. Fig.12 B is the ratio of DBIL / IBIL, which indicates that RFP has obvious cholestasis or obstruction on the liver, but GEN can significantly reduce serum bilirubin levels.

[0052] 4.7 Immunoblotting was used to detect the protein expression of CH25H and LXRα in liver tissue. Fig.13 The protein expressions of LXRα and CH25H in liver tissues of RFP group and GEN group are shown. Fig. 9The mice were divided into groups and liver tissues were collected for immunoblotting to detect the protein expression of LXRα and CH25H. RFP could significantly reduce the expression of the above two genes, but GEN could significantly restore the expression of the above two genes.

[0053] Example 5 In vitro study on the intervention of GEN on RFP-induced drug-induced liver injury Effect of 5.1GEN on RFP-induced lipid droplet formation in Hep3B. Fig.14 A is the negative control group (Veh), RFP (300μM, 24h), and RFP (300μM, 24h) + GEN group (20μM, 24h) treated Hep3B, and then Oil Red O was used to stain lipid droplets. Fig.14 B is the intracellular total cholesterol (TC) content of the liver cell line Hep3B cells. C shows that different concentrations of GEN reduce the TC content of the supernatant of Hep3B cells.

[0054] 5.2 Effect of RFP on CH25H protein induction in Hep3B Effect of RFP at different concentrations and time on CH25H protein expression in different liver cell lines (Western blotting). Fig.15 A is the effect of different concentrations of RFP on the expression of C25H protein in liver cell lines (mouse normal liver cell AML12, human liver cancer cell line HepG2, human liver cancer cell line Hep3B). It was found that RFP could significantly reduce the expression of CH25H as the concentration increased. Fig.15 B is the effect of RFP (concentration is 300μM) treatment for different time periods on C25H protein expression in different liver cell lines (AML12, HepG2, human immortalized liver cell C3A). It was found that RFP could significantly reduce the expression of CH25H as time went on.

[0055] 5.3 In vitro experiments showed that genistein affects the protein expression of LXRα and CH25H in RFP-treated hepatocytes (Western blotting). Fig.16 A shows the effect of different concentrations of RFP + different concentrations of GEN on the expression of LXRα protein in Hep3B cells. Fig.16 B shows the effect of different concentrations of RFP + different concentrations of GEN on the expression of CH25H protein in Hep3B cells. Both showed that in in vitro experiments, RFP reduced the expression of CH25H and LXRα, but GEN increased the expression of CH25H and LXRα.

[0056] 5.4 Immunofluorescence showed that GEN could reverse the nuclear export of LXRα induced by RFP. Fig.17A shows that LXRα is normally expressed in the nucleus of hepatocytes. RFP can induce LXRα to move from the nucleus to the cytoplasm. GEN can promote LXRα in RFP-treated hepatocytes to re-enter the nucleus from the cytoplasm. Since LXRα is a nuclear transcription factor, GEN can promote LXRα to enter the nucleus and promote the transcription of downstream genes (such as the ABCA family). Fig.17 B shows the mRNA expression of cholesterol efflux genes ABCA1, ABCA2, and ABCA3 downstream of LXRα. RFP can reduce the mRNA expression of ABCA1-3, but GEN can increase the mRNA expression of ABCA1-3. Fig.17 B shows that RFP can reduce the mRNA expression of ABCA1, 2, 3 and CH25H in Hep3B; while GEN increases the mRNA expression of ABCA1, 2, 3 and CH25H in Hep3B.

[0057] Example 6 In vitro study on the effect of overexpression of CH25H gene on RFP-induced drug-induced liver injury In vitro experiments suggest that overexpression of mouse-derived CH25H plasmid (for details, see the published literature of this experiment, iScience, 2024, 27(3):109204) can alleviate the increase in lipid droplets induced by RFP. Fig.18 A shows the effect of Oil Red staining on lipid droplets of Hep3B cells overexpressing CH25H plasmid (mouse-derived wild-type CH25H transiently transfected for 48 h, during which time rifampicin treatment was added for 24 h). Fig.18 B and Fig.18 C is the total cholesterol level (TC) and triglyceride level (TG) in Hep3B cells transfected with wild-type CH25H and compared with the control blank plasmid group using an enzymatic kit.

[0058] Example 7 Autodock 4.2 molecular docking software predicted that RFP and GEN docked with human CH25H respectively.

[0059] The specific steps are divided into 4 steps: 1) Ligand treatment: Use Chemidraw 14.0 to process the structure of small molecules, save it in mo12 format, and then save it as pdbqt file using Mgtools1.5.6 2) Receptor processing The protein three-dimensional structure was obtained through the alphafold database, and the protein structure was processed with Mgtools1.5.6. After adding hydrogen, calculating charges, and merging non-polar hydrogen, it was saved as a pdbqt file. 3) Autogrid operation Open the pdbqt file of the large and small molecules, then build a 60*60*60 box centered on the active site of the protein (center x=-3.265, center y=-11.735, center z=8.217), save it as a gpf file, and generate a glg file through autogrid4.exe operation 4) Autodock operation Open the pdbqt file of large and small molecules, use the Lamarckian genetics algorithm, and use the flexible method for small molecule structures. Calculate 100 times and save it as a dpf file. Generate a dlg file through autodock4.exe calculation The results show: Fig.19 A is the three-dimensional structure prediction diagram of CH25H protein (CH25H has not yet been successfully crystallized, so its crystal structure cannot be found in the PDB data. Therefore, the predicted structure is used instead and queried in the AlphaFold structure prediction database, see in website: https: / / www.uniprot.org / uniprotkb / O95992 / entry, the code is O95992). Fig.19 B is the molecular structure diagram of rifampicin. Fig.19 D is the molecular structure diagram of genistein. Fig.19 C shows that RFP can bind to multiple amino acid sites of CH25H (LEU121, LEU124, LEU125, ILE128, THR202, VAL205, VAL206, TRP209); Figure E shows that GEN can bind to multiple amino acid sites of CH25H (PHE51, VAL205, VAL206, TRP209, LEU210). Two of the three amino acid sites may have competitive binding (VAL-205, VAL-206, TRP-209). Table 1 shows the affinity, hydrogen bond formation, and docking amino acid residues of the small molecule and CH25H protein molecule after software settlement.

Claims

1. Use of genistein in preparing a drug for treating or improving rifampicin-induced drug-induced liver injury.

2. The use according to claim 1, characterized in that The genistein is used in preparing a drug for lowering total cholesterol index, lowering triglyceride index and reducing blood bilirubin level in treating or improving rifampicin-induced drug-induced liver injury.

3. The use according to claim 1, characterized in that The genistein is used in preparing a drug for increasing the expression of CH25H and LXRα in treating or improving rifampicin-induced drug-induced liver injury.

4. The use according to claim 1, characterized in that The liver damage includes human liver cancer HepG2 or human liver cancer Hep3B.

5. The use according to claim 1, characterized in that: When the active ingredient is genistein, the dosage is 25-50 mg / Kg.

6. The use according to any one of claims 1 to 5, characterized in that: Genistein was dissolved in DMSO, diluted in PBS and then directly gavaged for induction.

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