Study On The Mechanism Of Warming Kidney And Dispersing Blood Stasis In The Treatment Of Liver Cirrhosis Based On PI3K/AKT/HIF-1α Signaling Pathway

Objective: To observe the efficacy and molecular mechanism of warming kidney and removing blood stasis in the treatment of liver cirrhosis through PI3K/Akt/ HIF-1α signaling pathway,and to explore the theoretical basis of TCM and the correlation of modern molecular biological mechanism of the Jiawei Jisheng Shenqi decoction(JWJSD)which can reduce the degree of fibrosis and improve liver function.Methods:(1)Network pharmacological analysis: The key components and targets of JWJSD were obtained from TCMSP,CNKI and Uniprot databases,disease targets of liver cirrhosis and hypoxia were obtained from Gene Card database,TCM component targets were matched with disease targets,and the network of "medicine-components-disease-targets" was constructed by Cytoscape software.PPI network was drawn by STRING database,and GO enrichment and KEGG signal pathway analysis were performed by DAVID database(2)45 SPF SD rats were induced to liver chirrosis by intraperitoneal injection of 40% carbon tetrachloride olive oil solution for 8 weeks.5 rats were selected randomly to verify modeling,and other rats were randomly divided into model group and JWJSD high-dose,medium-dose and low-dose group,with 10 rats in each group.10 healthy SD rats were divided into blank group.The blank group and model group were not given intervention,while the Chinese medicine treatment group was given high-dose,medium-dose and low-dose Jiawei Jisheng Shenqi suspension by intragastric administration,once a day,continue to 4 weeks.After the treatment,the histopathological changes of the rat liver were observed,the level of serum ALT,AST,ALB,TBIL and SOD were detected by automatic biochemical analyzer,the expression of TGF-β1and Smad3 in liver tissues of rats in each group was determined by immunohistochemistry,the level of serum EPO was detected by ELISA,the expression of HIF-1 α and VEGF in liver were detected by Westernblot,and the mRNA expression of PI3 K,AKT,HIF-1α and VEGF were detected by realtime quantitative PCR.Results:(1)The 13 key components and 17 effective tragets of JWJSD in the treatment of liver cirrhosis were predicted.Berberine,oleic acid,sitosterol were the most effective components,while INS,CASP3,HIF-1,IL-1 were the most effective tragets.The 43 signaling pathway including cancer pathway and HIF-1 pathway were obtained in enrichment analysis.(2)Compared with model group,the condition of nutrition,mental state and movement were improved to varying degrees.Liver histopathology: severe fiber deposition,shape of pseudolobule and infiltration of inflammatory cell could be seen in the liver tissue of model group rats;compared with model group,liver fibrosis,inflammatory cell infiltration and liver cell necrosis were improved in all treatment groups after TCM intervention,and the improvement effect was most obvious in the high-dose group.liver function index: compared with blank group,level of ALT,AST and TBIL in model group were significantly increased,while level of ALB were significantly decreased(P <0.05);compared with model group,ALT,AST and TBIL in all treatment groups decreased to varying degrees,while ALB level increased(P < 0.05),high dose had the best effect on lowering ALT,AST and TBIL while increasing ALB(P< 0.05).Comparison of SOD and EPO in serum of rats in each group:Compared with normal group,the levels of SOD and EPO in serum of rats in model group were significantly decreased(P < 0.05);Compared with model group,SOD and EPO in serum of all treatment groups increased in different degrees,and the effect of high dose of JWJSD was the most obvious(P <0.05).Comparison of TGF-β1 and Smad3 in liver tissues of rats in each group:compared with blank group,the expression of TGF-β1 and Smad3 in liver tissue of rats in model group was significantly up-regulated;after intervention,compared with model group,the expression of TGF-β1 and Smad3 in liver tissue of rats in all treatment groups was down-regulated to varying degrees,and high-dose JWJSD had the most significant down-regulation effect(P <0.05).Comparison of HIF-1α and VEGF in liver tissue of rats in each group:compared with blank group,the expression of HIF-1α and VEGF in liver tissue of rats in model group was significantly up-regulated;after intervention,compared with model group,the expression of HIF-1α and VEGF in liver tissue of rats in all treatment groups were down-regulated to varying degrees,among which high-dose JWJSD had the most significant down-regulation effect(P <0.05).Comparison of PI3 K,AKT,HIF-1α and VEGF mRNA in each group:compared with blank group,the mRNA expression of PI3 K,AKT,HIF-1α and VEGF in liver tissue of model group was significantly up-regulated(P <0.05);After intervention,compared with model group,mRNA expression of PI3 K,AKT,HIF-1α and VEGF in liver tissue of rats treated with JWJSD was down-regulated to varying degrees,and the expression in high-dose group was lower than medium-dose and low-dose groups’(P < 0.05).Conclusion: The study showed that,in the treatment of cirrhosis rats,JWJSD,as well as Ji Sheng Shenqi decoction combine with sanqi and biejia,can effectively improve the liver function and reduce inflammation and fibrosis.These effects may be related to the regulation of PI3K/AKT/HIF-1α signaling pathway,inhibition of pathological angiogenesis and correction of liver hypoxia microenvironment.