Hepatoprotective Activity And Mechanism Of Cornus Officinalis Against Anti-Tuberculous Drug-Induced Liver Injury In Mice

Objective:Traditional Chinese medicine Cornus officinalis is widely used in clinic,which has biological effects such as anti-inflammatory,anti-oxidation,anti-aging,hypoglycemic,neuron protection,liver protection and so on.However,there are few reports about its possible influence on anti-tuberculosis drug-induced liver injury.Therefore,the method of network pharmacology is applied to explore the network relationship between the effective components of Cornus officinalis and the targets of anti-tuberculosis drug-induced liver injury,screen out the key targets and pathways that Cornus acting on anti-tuberculosis drug-induced liver injury,and explore the effects and mechanism of Cornus officinalis on ATB-DILI through experiments.Methods:The active components of Cornus officinalis were screened by TCMSP database and supplemented by literature reports.The molecular structure formula and Canonical SMILES number of the active components of Cornus officinalis were collected in PubChem database,then downloaded the SDF structure files.Targets of the active components were predicted by Swiss Target Prediction database and PharmMapper database.The protein targets supplemented by TCMSP platform and literature collection were integrated,and then the duplicate values were removed.With "anti-tuberculosis drug-induced liver injury","ATB-DILI" as keywords,the disease targets of anti-tuberculosis drug-induced liver injury were searched and collected in GeneCards database,OMIM database and TTD database,and the OMIM-GeneMap search function in OMIM database was used to search and supplement the disease targets,integrate the targets and then remove duplicate values.The active component targets and disease targets were intersected to obtain the targets of Cornus officinalis against ATB-DILI,and then,the intersection targets were input into String11.0 database to obtain protein-protein interaction data,and the PPI network diagram of protein-protein interaction was drawn through Cytoscape 3.7.2 software.The top 20 central targets and potential protein function modules in the network were obtained by calculation.Through Metascape platform,GO enrichment analysis and KEGG pathway analysis were performed on the targets of Cornus officinalis against anti-tuberculous drug-induced liver injury.The "component-target-pathway" network diagram was constructed through Cytoscape 3.7.2 software,and the main active components of Cornus officinalis against anti-tuberculous drug-induced liver injury were obtained by calculation.Objective to extract iridoid glycosides from Cornus officinalis(CIG)and then determine their concentrations.The liver injury model of mice was established by intragastric administration of isoniazid(INH)and rifampicin(RFP)for 14 days.Sixty male C57BL 16 mice were randomly divided into 6 groups:normal control group(NC),model group(IL),positive control group(PC),low-dose group(CIG50),medium-dose group(CIG 100)and high-dose group(CIG200).Isoniazid(150mg/kg)and rifampicin(150mg/kg)were used to establish the model by gavage for 14 days.Mice of normal control group were given 0.9%normal saline,the positive control group mice were given silymarin(100mg/kg),and mice of the traditional Chinese medicine group were given iridoid glycosides of Cornus officinalis(50mg/kg,100mg/kg and 200mg/kg).After the last administration,the mice were fasted for 16 hours,and then taken the samples.Blood and liver tissue were collected,liver coefficient was calculated,pathological changes of liver tissue were observed by HE staining,then detected the serum biochemical indexes including alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and total bile acid(TBA);serum and liver lipid metabolism indexes including total cholesterol(TC),triglyceride(TG),high density lipoprotein(HDL)and low density lipoprotein cholesterol(LDL-C);the levels of oxidative factors in liver tissue,including superoxide dismutase(SOD),reduced glutathione(GSH),catalase(CAT)and malondialdehyde(MDA);the levels of inflammatory factors in liver tissue,including tumor necrosis factor(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6).The mRNA expression levels of PPAR-α,FABP1 and STAT3 were detected by real-time quantitative PCR.Results:24 active components of Cornus officinalis were screened,and 482 drug targets were predicted;248 targets of anti tuberculosis drug-induced liver injury,and 41 targets of Cornus officinalis on anti-tuberculosis drug-induced liver injury were obtained.The main active components of Cornus officinalis are ursolic acid,tannins,lipids,sterols(β-sitosterol,sitosterol,stigmasterol),iridoid glycosides(loganin,morroniside);the core targets related to the main active components are IL-6,EGFR,TNF,NR1H3,TGFB1,BAX,VEGFA,CYP3A4,CYP2C19,IL-1β,KDR,MMP9,MTOR,SRC,STAT3,TP53,F2,FABP1,etc.KEGG pathway enrichment analysis revealed 61 pathways related to anti-tuberculosis drug-induced liver injury,mainly involving drug metabolism,retinol metabolism,nonalcoholic fatty liver disease,insulin resistance,etc;signal pathways such as MAPK,PI3K-Akt,HIF-1,tumor necrosis factor,p53,JAK-STAT,adipocytokines,PPAR,etc.A total of 40 representative clusters of enriched terms were obtained by GO enrichment analysis.Biological process(BP)mainly involves the positive regulation of lipid metabolism,steroid metabolism,JAK-STAT cascade,apoptotic signaling pathway,cell migration and cell stress response.Molecular function(MF)mainly involves cofactor and transcription factor binding,protease binding,lipid binding,enzyme activity(oxidoreductase,phosphotransferase,protein kinase),growth factor and cytokine activity.Isoniazid combined with rifampicin can significantly reduce the weight of mice and increase the liver index.The pathological observation of liver indicates that inflammatory cell infiltration,cell necrosis and moderate steatosis are observed in mice of the model group.However,the intervention of iridoid glycosides of Cornus officinalis can increase the weight of mice and reduce the liver index,the inflammatory necrosis and steatosis of liver tissue are also reduced in varying degrees.The levels of ALT,AST,ALP and TBA in serum of mice in model group are significantly increased.All dose of iridoid glycosides of Cornus groups could significantly inhibit the increase of ALT,AST and ALP levels caused by the combination.of isoniazid and rifampicin,and the high dose group of CIG can inhibit the increase of TBA level,but the effect of medium and low dose groups of CIG is not obvious.The levels of TC,TG and LDL-C in serum and liver of mice in model group are significantly increased,and the level of serum HDL-C is significantly decreased.The intervention of iridoid glycosides of Cornus officinalis can significantly improve the lipid metabolism disorder and improve the lipid metabolism index levels of mice induced by isoniazid and rifampicin.The levels of SOD,CAT and GSH in the liver of mice in model group are significantly decreased,while the level of MDA is significantly increased.The intervention of iridoid glycosides of Cornus officinalis can inhibit the decrease of SOD,CAT activity and GSH content induced by INH-RFP,and reduce the accumulation of MDA in the liver tissue.Isoniazid combined with rifampicin can significantly increase the levels of TNF-α,IL-1β,IL-6 and the expression level of STAT3 mRNA in liver tissue of mice,while iridoid glycosides from Cornus officinalis can significantly inhibit the levels of TNF-α,IL-1β and IL-6,and the high-dose group of CIG can down regulate the expression of STAT3 to a certain extent.In the model group,the expression levels of PPAR-αand FABP1 mRNA are significantly decreased,and the iridoid glycosides of Cornus officinalis may increase the expression of FABP1 by up regulating PPAR-α,thus improving the disorder of liver lipid metabolism caused by anti-tuberculosis drugs.Conclusion:1.Cornus officinalis acts on multiple targets through multiple components and pathways,which may involve cell growth,proliferation,differentiation,apoptosis,cell stress,migration and movement,immune regulation,inflammatory response,lipid metabolism,enzyme activity regulation,etc,so as to play a protective role in anti-tuberculosis drug-induced liver injury.2.Iridoid glycosides from Cornus officinalis have protective effects on liver injury induced by isoniazid combined with rifampicin in mice.3.The liver protective mechanism of iridoid glycosides from Cornus officinalis may be related to improving antioxidant capacity,reducing inflammatory reaction,regulating lipid metabolism and up regulating the expression levels of PPAR-α and FABP1.