Application of a polypeptide LLTRAGL in the preparation of drugs for resisting isoniazid-induced liver damage

Abstract

The present invention provides an application of a polypeptide LLTRAGL in preparing a drug for resisting isoniazid-induced liver damage, belonging to the field of biomedicine technology, and specifically provides an application of a polypeptide in preparing a drug for protecting the liver, wherein the amino acid sequence of the polypeptide is LLTRAGL; and an application of a polypeptide in preparing a drug for resisting isoniazid-induced liver damage, wherein the amino acid sequence of the polypeptide is LLTRAGL. The present invention discovers for the first time that the polypeptide LLTRAGL has a liver-protecting effect, and that the polypeptide LLTRAGL has an anti-INH-induced liver damage activity, providing a new idea for the prevention and treatment of INH-induced liver damage.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of a polypeptide LLTRAGL in the preparation of a drug for resisting isoniazid-induced liver damage. Background Art

[0002] Isoniazid (INH) is a first-line drug for the treatment of tuberculosis (TB). Liver injury is its primary adverse reaction, accounting for 2.51% of drug-induced liver injury (DILI). The occurrence of INH-induced liver injury (INH-ILI) forces patients who cannot tolerate INH-ILI to repeatedly discontinue medication. Repeated interruptions in treatment lead to the emergence of multidrug-resistant Mycobacterium tuberculosis, complicating clinical treatment and posing a significant threat to health and safety. Currently, modern medicine lacks specific medications for the treatment of INH-ILI; most patients rely on rest, enhanced nutrition, vitamin supplements, and symptomatic treatment. Therefore, identifying new therapeutic targets and effective liver-protective agents is an urgent need for addressing TB treatment.

[0003] Marine bioactive substances have become an important resource for new drugs. Due to their novel chemical structure, unique physiological effects and relatively high safety, they are worthy of in-depth research.

[0004] Zebrafish genes share up to 87% similarity with human genes. Their liver, kidneys, cardiovascular system, and immune system share remarkably similar developmental mechanisms and characteristics with their human counterparts, demonstrating highly conserved structure and function. The zebrafish liver begins to morphologically form 48 hours after fertilization (48 hpf), rapidly growing between 60 and 72 hpf until it reaches its proper size. Because zebrafish are transparent and development can be visualized, in vivo imaging techniques can be widely used in the screening of hepatoprotective drugs.

[0005] Currently, there are no reports on the effect of peptides derived from Rhizoctonia ulmoides against isoniazid-induced liver damage. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides an application of a polypeptide LLTRAGL in the preparation of a drug for resisting isoniazid-induced liver damage.

[0007] The present invention uses an INH-induced zebrafish liver injury model to study the hepatoprotective activity and mechanism of action of the polypeptide LLTRAGL, providing a reference for the treatment of INH-ILI.

[0008] The technical solutions of the present invention are as follows:

[0009] A use of a polypeptide in preparing a liver protection drug, wherein the amino acid sequence of the polypeptide is LLTRAGL.

[0010] A polypeptide is used in the preparation of a drug for resisting isoniazid-induced liver damage, wherein the amino acid sequence of the polypeptide is LLTRAGL.

[0011] According to the present invention, preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0012] More preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0013] According to the present invention, preferably, the dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

[0014] A drug for resisting isoniazid-induced liver damage, wherein the active ingredient comprises a polypeptide with an amino acid sequence of LLTRAGL.

[0015] According to the present invention, preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0016] More preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0017] According to the present invention, preferably, the dosage form of the drug is capsule, pill, tablet, oral liquid, granule, tincture or injection.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention discovers for the first time that the polypeptide LLTEAGL derived from Rhizoctonia venezuelae has a hepatoprotective effect and can resist the liver damage caused by isoniazid, and has the effect of preventing or treating isoniazid-induced liver damage, providing a new idea for the prevention and treatment of INH-induced liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the effect of the peptide LLTRAGL on liver morphology after 24-48 hours of drug treatment on isoniazid-induced liver damage.

[0021] Figure 2 This is a statistical diagram of the effect of the peptide LLTRAGL on isoniazid-induced liver damage;

[0022] In the figure: Figure A shows the statistical graph of liver area and fluorescence intensity of zebrafish in each group after 24 hours of drug treatment; Figure B shows the statistical graph of liver area and fluorescence intensity of zebrafish in each group after 48 hours of drug treatment;

[0023] Compared with the blank group, ##p<0.01, ####p<0.0001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 3 The effect of peptide LLTRAGL on zebrafish liver cell apoptosis;

[0025] In the figure: the yellow line area is the zebrafish liver, and the white arrows indicate apoptotic hepatocytes.

[0026] Figure 4 This is a statistical graph showing the effect of peptide LLTRAGL on the number of apoptotic cells in zebrafish liver;

[0027] Compared with the blank group, ####p<0.0001; compared with the model group, ***p<0.001, ****p<0.0001.

[0028] Figure 5 Statistical diagram of differentially expressed genes among groups;

[0029] In the figure: Figure A is the experimental process; Figure B is the statistical bar chart of differentially expressed genes in each group.

[0030] Figure 6 This is a GO enrichment analysis diagram.

[0031] Figure 7 KEGG enrichment analysis diagram of differentially expressed genes;

[0032] In the figure: Figure A shows the KEGG enrichment analysis of differentially expressed genes (DEGs) between the model group and the control group; Figure B shows the KEGG enrichment analysis of DEGs between the drug-treated group and the model group.

[0033] Figure 8 This is a diagram showing the effect of the peptide LLTRAGL on the expression levels of zebrafish MAPK pathway and apoptosis-related genes;

[0034] In the figure: compared with the blank group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001.

[0035] Figure 9 This is a graph showing the effect of the peptide FGINLIQ on liver morphology in isoniazid-induced liver injury after 24-48 hours of administration.

[0036] Figure 10 This is a statistical chart showing the effect of peptide FGINLIQ on isoniazid-induced liver injury;

[0037] In the figure: Figure A shows the statistical graph of liver area and fluorescence intensity of zebrafish in each group after 24 hours of drug treatment; Figure B shows the statistical graph of liver area and fluorescence intensity of zebrafish in each group after 48 hours of drug treatment;

[0038] Compared with the blank group, ####p<0.0001; compared with the model group, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto.

[0040] The contents in the examples without specific conditions were carried out under conventional conditions; the reagents or instruments used without indicating the manufacturer were all common commercial products.

[0041] (1) Study on the effect of peptide LLTRAGL on isoniazid-induced liver damage

[0042] The polypeptide LLTRAGL is a polypeptide extracted and identified from Rhizoctonia venosa.

[0043] SEQ ID NO. 1: Leu-Leu-Thr-Arg-Ala-Gly-Leu LLTRAGL.

[0044] hpe is hours after drug administration.

[0045] 1 Materials and Instruments

[0046] 1.1 Reagents

[0047] The peptide LLTRAGL (Leu-Leu-Thr-Arg-Ala-Gly-Leu, 95.29% purity) was synthesized by Hefei Cermanno Biotechnology Co., Ltd. Isoniazid (INH) was purchased from Sigma-Aldrich; reduced glutathione (GSH) was purchased from Merck Chemical Technology (Shanghai) Co., Ltd.; and an RNA extraction kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd. Experimental water was zebrafish culture water (5.0 mmol / L NaCl, 0.17 mmol / L KCl, 0.4 mmol / L CaCl2, 0.16 mmol / L MgSO4).

[0048] 1.2 Instruments

[0049] Zebrafish breeding equipment (Beijing Aisheng Technology Co., Ltd.), illumination incubator (SPX-280B-G, Shanghai Jixing Biotechnology Co., Ltd., China), stereo fluorescence microscope (SZX16, Olympus, Japan), Zeiss fluorescence microscope (AXIO-V16, Carl Zeiss Optical Co., Ltd., Germany), gradient PCR instrument (c1000 Touch, Bio-Rad, USA), real-time fluorescence quantitative PCR instrument (Light Cycler @ 96, Roche Ltd., Switzerland).

[0050] 2 Methods

[0051] 2.1 Obtaining zebrafish

[0052] The transgenic zebrafish Tg (fabp10a:EGFP) with liver-specific fluorescent expression used in the present invention can be purchased from the National Zebrafish Resource Center. Other transgenic zebrafish with liver-specific fluorescent expression can also be used. Male and female zebrafish are housed separately under standard conditions of 28°C, 14 hours of light / 10 hours of darkness. One day before the experiment, healthy, sexually mature zebrafish are selected and placed in a mating tank with the partitions separated, in a ratio of 1:1 or 1:2 female to male. The next day, the mating tank is removed, and fertilized eggs are collected between 11:00 and 12:00, cleaned and disinfected, and then transferred to zebrafish culture water containing 0.02% methylene blue and cultured at 28°C under controlled light.

[0053] 2.2 Zebrafish processing

[0054] Normally developed zebrafish at 72 hpf (72 hours post-fertilization, hpf) were randomly selected under a microscope and transferred to 24-well plates, with 10 per well. The plates were divided into a blank control group (zebrafish culture water), an INH-induced liver injury model group (INH 6 mmol / L), a positive drug group (INH 6 mmol / L + GSH 10 μmol / L), and a drug-treated group (INH 6 mmol / L + LLTRAGL 25, 50, and 100 μg / mL). Each experimental group was set up in triplicate, and the drug solution was changed every 24 hours. At 24 and 48 hours post-exposure (24 / 48 hpe), zebrafish liver morphology was observed under a fluorescence microscope, and liver area and liver fluorescence intensity were calculated.

[0055] 2.3 TUNEL staining of zebrafish liver

[0056] Zebrafish from each experimental group were selected at 48 h p.i., fixed with 4% paraformaldehyde, and placed in a 4°C refrigerator overnight. The next day, after washing three times with 1X PBS, the cells were treated with 30% H2O2 and methanol at a 1:10 dilution for 15 min. 0.3% Triton X-100 and sodium citrate antigen retrieval solution (1X) were mixed at a 1:300 ratio and incubated in a 4°C refrigerator for 10 min. Tdt enzyme and fluorescent labeling solution were mixed at a 1:10 ratio. Each zebrafish group was placed in a 200 μL centrifuge tube, and 50 μL of the fluorescent labeling mixture was added to each group. The cells were incubated in a 37°C incubator in the dark for 1.5 h. Cell apoptosis in the zebrafish liver region was observed under a fluorescence microscope.

[0057] 2.4 Transcriptomic Analysis (RNA-seq)

[0058] Zebrafish were crushed at 48 hpe in the blank control group (zebrafish culture water), the INH-induced liver injury model group (INH 6 mmol / L), and the drug-treated group (INH 6 mmol / L + LLTRAGL 50 μg / mL), and mRNA was extracted. The mRNA samples that passed the test in each group were used to construct a library, and transcriptome sequencing was performed by Qingdao Ouyi Biotechnology Co., Ltd. Genes with an absolute value of an expression change of more than 1.5 times (|Fold Change|>1.5) and a significant difference of less than 0.05 (P < 0.05) were considered significantly differentially expressed genes (DEGs). Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the DEGs.

[0059] 2.5 Protein interaction analysis and molecular docking

[0060] Protein-protein interaction (PPI) analysis was performed on DEGs in core pathways identified by transcriptomics using the STRING database (https: / / cn.string-db.org / ), selecting the source "Danio Rerio" with a confidence interval of 0.700. Protein interaction networks were visualized using Cytoscape (v.3.10.0) software, and core targets were identified by performing the MCC algorithm using the CytoHubba plugin.

[0061] Molecular docking was performed with the core target as the receptor and the peptide LLTRAGL as the ligand. The structure of the core target was obtained from the PDB protein structure database (RCSB PDB, https: / / www.rcsb.org / ). Discovery Studio 2019 was used to define the docking pocket of the original ligand in the protein crystal structure as the center of the active site for semi-flexible docking. The radius was set to 20, the water molecules and the original ligand in the crystal structure were deleted and hydrogenated, and the CDOCKER module was used to perform the docking task. The docking algorithm CDOCER based on CHARMm can accurately predict the protein-ligand interaction relationship, and the CDOCKER energy scoring function is used to evaluate the binding ability of the ligand to the receptor. The root mean square deviation (RMSD) between the initial conformation of the original ligand and the docked conformation was calculated. This indicates that the conformation obtained by docking can reproduce the binding mode of ligand and receptor, reflecting the reliability of the docking model.

[0062] 2.6 Real-time fluorescence quantitative PCR

[0063] Zebrafish from each experimental group were homogenized at 48 hpi and RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2. cDNA was reverse transcribed from each sample, and the relative expression levels of apoptosis-related genes were determined using a Light Cycler@96 real-time quantitative PCR instrument. Real-time quantitative PCR amplification conditions were as follows: pre-denaturation at 95°C for 30 s for one cycle; denaturation at 95°C for 10 s, annealing at 60°C for 10 s for a total of 40 cycles; and then 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s for one cycle. β-actin was used as an internal reference for relative quantitative analysis. PCR primers for the internal reference gene β-actin and the target gene were synthesized, purified, and quality-tested by Boshang Biotechnology (Shanghai) Co., Ltd. Primer sequences are shown in Table 1.

[0064] Table 1 Quantitative PCR primer sequences

[0065] Tab.1 Primers for quantitative PCR

[0066]

[0067]

[0068] 2.7 Statistics

[0069] The experimental results were analyzed by one-way ANOVA between groups using GraphPad Prism 9. All data are expressed as mean ± standard error (mean ± SE). P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference.

[0070] 3 Results

[0071] 3.1 Effect of LLTRAGL on isoniazid-induced liver injury

[0072] like Figure 1 、 Figure 2 As shown in the figure, at 24hpe and 48hpe, the liver area and fluorescence intensity of zebrafish in the INH-induced liver injury model group were significantly reduced compared with the blank control group. INH causes liver damage in zebrafish. Compared with the model group (INH 6mmol / L), the liver area and fluorescence intensity of zebrafish in the positive drug group (INH 6mmol / L + GSH 10μmol / L) and the drug-treated groups (INH 6mmol / L + LLTRAGL 25, 50, 100μg / mL) were significantly restored.

[0073] 3.2 Effect of peptide LLTRAGL on apoptosis of zebrafish liver cells

[0074] like Figure 3 、 Figure 4 As shown in the figure, at 48hpe, the number of apoptotic cells in the zebrafish liver in the model group increased significantly compared with the blank control group. Compared with the model group, the apoptotic cells in the 25, 50, and 100μg / mL treatment groups all decreased significantly.

[0075] 3.3 RNA-seq analysis of the anti-INH-induced liver injury activity of the peptide LLTRAGL

[0076] like Figure 5 As shown in the figure, there were 5889 DEGs in the model group and the control group (INH vs. CON), of which 3485 were upregulated genes and 2404 were downregulated genes; there were 197 DEGs in the drug-treated group and the model group (LLTRAGL vs. INH), of which 78 were upregulated genes and 119 were downregulated genes.

[0077] like Figure 6As shown in the figure, GO enrichment analysis of DEGs in the model and drug-treated groups yielded a total of 64 GO terms. GO enrichment analysis yielded 23 biological processes (BPs), 20 cellular components (CCs), and 21 molecular functions (MFs). GO analysis revealed that BPs primarily involved cell killing, response to stimulus, regulation of biological processes, and signaling; CCs primarily involved cell junctions, macromolecular complexes, and membrane-encloselumen; and MFs primarily involved enzyme regulator activity, receptor regulator activity, and translation regulator activity. These GO functional annotations, such as cell death, stress, macromolecular complexes, membrane-encloselumen, and enzyme regulator activity, are associated with apoptosis.

[0078] like Figure 7 As shown in the figure, KEGG enrichment analysis was performed on the DEGs of the model group and the control group, and the DEGs of the drug-treated group and the model group, and important signaling pathways were obtained, including MAPK signaling pathway, FoxO signaling pathway, and apoptosis.

[0079] Based on the RNA-seq results, it is speculated that the mechanism by which the peptide LLTRAGL protects against INH-induced liver injury is related to the regulation of MAPK signaling pathway and apoptosis.

[0080] 3.4 PPI network analysis and molecular docking of the anti-INH-induced liver injury activity of the peptide LLTRAGL

[0081] The MAPK signaling pathway DEGs obtained by KEGG enrichment analysis were selected to construct the PPI network, and the core targets were screened out using the MCC algorithm: EGFRa, FGFR1, PDGFRa, PDGFRb, and INSR.

[0082] Before molecular docking, the ligand conformation in the original crystal of the core target protein overlaps with the ligand conformation after docking, and the RMSD values ​​are less than This demonstrates that the proposed molecular docking method and parameters are reasonable. Molecular docking of the core target with the peptide LLTRAGL revealed that higher CDOCKER docking energy scores indicate better binding of the docking system. Polar and nonpolar bonds form between the receptor and ligand, contributing to the stability of the bound conformation. As shown in Table 2, the peptide LLTRAGL exhibits good ligand-based binding to the core targets EGFRa, FGFR1, PDGFRa, PDGFRb, and INSR. Among these, the core target with the highest ligand-based binding energy was EGFRa (8A27). The amino acid residues forming hydrogen bonds are THR790, MET793, LYS745, CYS775, ASP855, and LEU788, while the amino acid residues forming hydrophobic bonds are LEU777, LEU788, MET766, TYR869, and LEU718. The second-highest core target for ligand docking energy was FGFR1 (3DPK). The amino acid residues forming hydrogen bonds were LEU588, ASP670, CYS666, ASP796, ARG801, and ASP802, while the amino acid residues forming hydrophobic bonds were LEU588, MET637, and ARG782. Subsequent experiments selected EGFRa and FGFR1 as core targets for further validation.

[0083] Table 2 Molecular docking results

[0084] Tab.2 The results of molecular docking

[0085]

[0086] 3.5 RT-qPCR validation

[0087] To further investigate the mechanism of action of peptide LLTRAG against INH-induced liver injury, RT-qPCR was used to detect the mRNA expression levels of MAPK signaling pathway and apoptosis-related genes egfra, fgfr1, map2k2, map3k1, map3k7, mapk12a, caspase1, caspase3, caspase8, and caspase9 in zebrafish in each experimental group (n=30). Compared with the blank control group, the expression of egfra, fgfr1, map2k2, map3k1, map3k7, mapk12a, caspase1, caspase3, caspase8, and caspase9 was significantly upregulated in the INH-induced liver injury model group. Compared with the INH-induced liver injury model group, the expression of egfra, fgfr1, map2k2, map3k1, map3k7, mapk12a, caspase1, caspase3, caspase8, and caspase9 in the peptide LLTRAGL administration group was significantly downregulated with statistical significance (see Table 1). Figure 8 .

[0088] The results showed that after treatment with the peptide LLTRAGL, the mRNA expression levels of MAPK signaling pathway and apoptosis-related genes in zebrafish were significantly changed, indicating that the peptide LLTRAGL can alleviate the abnormal gene expression caused by INH.

[0089] The present invention uses INH to construct a zebrafish liver injury model. At 48hpe, the fluorescence intensity and area of ​​the zebrafish liver were significantly reduced, and the apoptosis of cells in the liver region was aggravated, indicating that the liver injury model was established. Compared with the model group, the apoptosis of cells in the liver region, the fluorescence intensity and area of ​​the liver in the drug-treated group were restored. In order to further explore the hepatoprotective mechanism of the polypeptide, the DEGs of the blank control group, model group and drug-treated group were detected by transcriptomics, and it was found that it was involved in regulating apoptosis and MAPK signaling pathways. Further molecular docking and RT-qPCR technology were used to verify that the polypeptide LLTRAGL has good docking binding energy with the core targets EGFRa and FGFR1, and can improve the abnormal expression of MAPK signaling pathway genes (egfra, fgfr1, map2k2, map3k1, map3k7, mapk12a) and apoptosis genes (caspase1, caspase3, caspase8, caspase9) caused by INH.

[0090] The present invention's analysis of transcriptomics, PPI networks, and molecular docking revealed that the peptide's anti-INH-induced liver injury activity is involved in regulating the MAPK signaling pathway, and the peptide has good docking binding energy with the core targets EGFRa and FGFR1. Therefore, it is speculated that the peptide LLTRAGL acts on EGFRa and FGFR1, thereby participating in the regulation of downstream MAPK signaling pathways and improving INH-induced liver injury.

[0091] In the analysis of the expression levels of MAPK signaling pathway and apoptosis-related genes in this study, it was found that compared with the blank control group, the INH-induced liver injury model group caused increased expression of map2k2, map3k1, map3k7, and mapk12a in zebrafish. At the same time, INH led to increased expression of caspase3, caspase8, and caspase9, indicating that INH activates the MAPK signaling pathway to induce cell apoptosis. After treatment with the peptide LLTRAGL, the expression levels of map2k2, map3k1, map3k7, mapk12a, caspase3, caspase8, and caspase9 were significantly downregulated. Therefore, this study speculates that the peptide LLTRAGL exerts its anti-INH-induced liver injury activity by inhibiting the activation of the MAPK signaling pathway and alleviating apoptosis in zebrafish liver cells.

[0092] In summary, the experimental results showed that compared with the INH-induced liver injury model group, the zebrafish liver area and liver fluorescence intensity in the positive control group and the peptide-treated group were significantly restored, and liver cell apoptosis was significantly reduced. Transcriptomic analysis showed that GO enrichment analysis included 64 terms, including those related to cell apoptosis, and KEGG enrichment analysis of differentially expressed genes all involved the MAPK signaling pathway. Molecular docking results showed that the peptide LLTRAGL had good docking ability with the core targets of the MAPK signaling pathway, EGFRa, FGFR1, PDGFRa, PDGFRb, and INSR. RT-qPCR results showed that compared with the INH-induced liver injury model group, the peptide LLTRAGL significantly downregulated the mRNA expression levels of egfra, fgfr1, map2k2, map3k1, map3k7, and mapk12a, as well as the mRNA expression levels of the apoptosis-related factors caspase1, caspase3, caspase8, and caspase9.

[0093] The polypeptide LLTRAGL has the activity of resisting INH-induced liver injury and can prevent or treat isoniazid-induced liver injury, providing a new idea for the prevention and treatment of INH-induced liver injury.

[0094] (II) Study on the effect of peptide FGINLIQ on isoniazid-induced liver injury

[0095] The polypeptide FGINLIQ is a polypeptide extracted and identified from Rhizoctonia venosa.

[0096] SEQ ID NO. 2: FGINLIQ.

[0097] 1 Materials and Instruments

[0098] 1.1 Reagents

[0099] The peptide FGINLIQ (Phe-Gly-Ile-Asn-Leu-Ile-Gln, purity 95.29%) was synthesized by Hefei Saimanuo Biotechnology Co., Ltd. Isoniazid (INH) was purchased from Sigma-Aldrich, USA.

[0100] 1.2 Instruments

[0101] Zebrafish breeding and rearing equipment (Beijing Aisheng Technology Co., Ltd.), illumination incubator (SPX-280B-G, Shanghai Jixing Biotechnology Co., Ltd., China), stereo fluorescence microscope (SZX16, Olympus Corporation, Japan).

[0102] 2 Methods

[0103] 2.1 Obtaining zebrafish

[0104] The present invention utilizes transgenic zebrafish (fabp10a:EGFP) expressing specific fluorescent protein in the liver. Male and female zebrafish were housed separately at 28°C under standard conditions of 14 hours of light / 10 hours of darkness. One day prior to the experiment, healthy, sexually mature zebrafish were selected and placed in a mating tank with the partitions separated, in a ratio of 1:1 or 1:2 female to male ratio. The next day, mating was performed by removing the partitions. Fertilized eggs were collected between 11:00 and 12:00, cleaned, and disinfected, then transferred to zebrafish culture water containing 0.02% methylene blue and cultured at 28°C under controlled light.

[0105] 2.2 Zebrafish processing

[0106] Under a microscope, normally developed zebrafish (72 hpf) were randomly selected and transferred to 24-well plates, with 10 per well. The plates were divided into a blank control group (zebrafish culture water), an INH-induced liver injury model group (INH 6 mmol / L), a positive drug group (INH 6 mmol / L + GSH 10 μmol / L), and a drug-treated group (INH 6 mmol / L + FGINLIQ 25, 50, and 100 μg / mL). Each experimental group was set up in triplicate, and the drug solution was changed every 24 hours. At 24 and 48 hours after drug treatment, zebrafish liver morphology was observed under a fluorescence microscope, and liver area and liver fluorescence intensity were calculated.

[0107] 3 Results

[0108] 3.1 Effect of peptide FGINLIQ on isoniazid-induced liver injury

[0109] like Figure 9 、 Figure 10 As shown, at 24 and 48 hpe, the liver area and fluorescence intensity of zebrafish in the INH-induced liver injury model group were significantly reduced compared with the blank control group. INH causes liver damage in zebrafish. Compared with the model group (INH 6 mmol / L), the liver area of ​​zebrafish in the positive drug group (INH 6 mmol / L + GSH 10 μmol / L) was significantly increased at 24 hpe, and the liver area and fluorescence intensity of zebrafish in the positive drug group (INH 6 mmol / L + FGINLIQ 25, 50, 100 μg / mL) were significantly increased at 48 hpe. Compared with the model group (INH 6 mmol / L), there were no significant differences in the liver area and fluorescence intensity of zebrafish in the drug groups (INH 6 mmol / L + FGINLIQ 25, 50, 100 μg / mL).

[0110] The results showed that the peptide FGINLIQ had no effect on the reduction in liver area and weakening of fluorescence intensity in zebrafish caused by isoniazid, and had no activity against isoniazid-induced liver damage.

[0111] The present invention discovered for the first time that the polypeptide LLTRAGL has a liver-protecting effect and has an anti-INH-induced liver injury activity, providing a new approach for the prevention and treatment of INH-induced liver injury.

Claims

1. Use of a polypeptide in the preparation of a drug for resisting isoniazid-induced liver damage, wherein the amino acid sequence of the polypeptide is LLTRAGL.

2. The use according to claim 1, characterized in that The medicine contains one or more pharmaceutically acceptable carriers or adjuvants.

3. The use according to claim 2, characterized in that The auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant or a lubricant.

4. The use according to claim 1, wherein The dosage form of the medicine is capsule, pill, tablet, oral liquid, granule, tincture or injection.

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