Study On The Mechanism Of Breviscapine On Non-alcoholic Steatohepatitis And Liver Fibrosis

Objective: Non-alcoholic steatohepatitis(NASH),developed from nonalcoholic fatty liver disease(NAFLD),is a leading cause of cirrhosis and liver-related death worldwide.NASH is typically characterized by fatty inflammation of the liver and fibrosis driven by metabolic disorders such as diabetes,obesity and dyslipidemia,which increases the risk of cirrhosis,hepatocellular carcinoma and liver failure.Because the pathophysiology of NASH is complex and unclear,there are currently no specific drugs approved to treat NASH.Breviscapine is a flavonoid compound extracted from traditional Chinese medicine breviscapus breviscapus,which is widely used in the treatment of coronary heart disease,hypertension,hyperlipidemia and other cardiovascular and cerebrovascular diseases in China.In recent years,breviscapine has been reported to have protective and anti-inflammatory effects on liver,but whether it has preventive and therapeutic effects on NASH remains unclear.Therefore,this study aims to elucidate the therapeutic effect of breviscapine on NASH and reveal its potential molecular mechanism.Method:1.Effect of breviscapine on PO-induced L02 hepatocyte model: POinduced L02 liver cells are used as NASH model,and then the cells are treated with 50μM and 100μM breviscapine,respectively.The effect of breviscapine on lipid deposition in vitro induced metabolic environment was investigated by oil-red O staining and TG content in cells.The expression levels of genes related to lipid metabolism and inflammation were detected by qPCR to investigate the improvement of lipid metabolism and inflammation under metabolic stress at the molecular level by breviscapine.2.Effects of breviscapine on HFHC diet-induced NASH mouse model:C57BL/6J mice induced by HFHC diet were treated with breviscapine of 15mg/kg and 30mg/kg,respectively.The weights of body and liver of the mice were weighed,and liver body ratio was calculated.Serum ALT and AST levels were measured to evaluate liver function.The contents of TG and TC in liver and serum were detected,liver HE staining and oil red O staining,lipid metabolism related indicators were detected by qPCR assay,and lipid synthesis related proteins were detected by Western blotting assay to investigate the improvement of liver lipid deposition induced by HFHC diet in vivo by breviscapine.By CD11b immunofluorescence staining,qPCR assay was used to detect inflammatory indicators and Western blotting assay was used to detect NF-κB signaling pathway related proteins to investigate the effect of breviscapine on liver inflammation caused by metabolic stress.PSR staining and qPCR were used to detect fibrosis related indexes in liver slices,and the effect of breviscapine on fibrosis was investigated.3.Effects of breviscapine on MCD diet-induced NASH mouse model:MCD induced C57BL/6J mice were treated with breviscapine of15mg/kg and 30mg/kg,respectively.Liver TG,serum ALT and AST were detected by H&E staining,oil red O staining,CD11b immunofluorescence staining and PSR staining.And qPCR detection of inflammation and fibrosis related indicators further verified the effects of breviscapine on steatosis,inflammation and fibrosis of NASH in MCD model.4.Multi-omics combined analysis: Breviscapine treated PO-induced L02 hepatocytes and HFHC-induced mice livers were selected for transcriptome sequencing.Transcriptome data were analyzed to obtain differential gene enrichment pathways in cells and liver tissues,and the intersection of the two enrichment pathways was found,and MAPK signaling pathway with the most differential genes was selected.Then proteomics and phosphorylomics analysis were performed on mouse liver to identify the phosphorylated proteins with the highest correlation with differential genes and proteins in MAPK signaling pathway.Finally,Western blotting experiments were performed to verify the proteins,so as to determine the possible therapeutic targets of berviscapine for NASH.Result:1.In vitro,oil-red O staining results showed that lipid deposition was significantly reduced after breviscapine administration,and cell TG detection also showed that lipid accumulation was significantly reduced,as well as m RNA content of lipid metabolism and inflammatory genes.2.In the in vivo model induced by HFHC diet,after breviscapine administration,liver weight and hepatobody ratio decreased significantly,the contents of TG and TC in liver serum decreased,and the staining results of pathological sections showed that lipid in liver decreased,and steatosis was significantly improved.In addition,serum ALT and AST levels decreased,and liver function gradually recovered.The down-regulation of lipid transport and synthesis related indexes and up-regulation of fatty acid β oxidation related indexes suggested that breviscapine could reduce lipid accumulation in liver.Immunofluorescence staining of breviscapine CD11b showed a decrease in the number of positive cells and inflammatory factors were down-regulated,and the expression of NF-κB signaling pathway related proteins IKBα was up-regulated,p-IKKβ and p-p65 were down-regulated.PSR staining showed decreased collagen deposition and fibrosis related indicators was down-regulated.These results suggest that breviscapine administration improves lipid metabolism disorder and alleviates liver inflammation and liver fibrosis.3.In the vivo model induced by MCD diet,similar to the HFHC in vivo model,the contents of TG,ALT and AST in liver decreased after administration,and H&E staining,oil red O staining,immunofluorescence staining and PSR staining showed that steatosis,lipid deposition,inflammatory cell infiltration and collagen deposition were reduced.Breviscapine administration also reduced the expression levels of inflammatory and fibrotic markers.4.cells and liver tissues of the transcriptome analysis results showed that the differences in sample cells gene enrichment of the main pathways have nine,five in liver tissue,including two common signaling pathways in cells and tissues,respectively MAPK signaling pathways and TGF-β signaling pathways,and further studies have found differences in gene expression of MAPK signal pathway more.Transcriptome,proteomics and phosphorylomics data combined showed that the expression of three phosphorylated proteins Mapk14,Hspa8 and Map3k7 in MAPK signaling pathway had the strongest correlation with differential genes and differential proteins.Literature review found that TAK1(Map3k7)may be a key regulatory protein in MAPK signaling pathway.Finally,Western blotting experiment verified that p-TAK1 and downstream p-p38 and p-JNK were significantly inhibited.Conclusion: Breviscapine can significantly improve lipid deposition,inflammation and fibrosis in liver under metabolic stress,and its mechanism may be related to MAPK signaling pathway,and its antiNASH effect can be brought into play by inhibiting TAK1 phosphorylation.