Studies On The Mechanisms Of Yiguanjian Decoction And The Combination Of Active Ingredients In Treating Liver Fibrosis With Liver-kidney Yin Deficiency Syndrome

Objective:To explore the mechanism of Yiguanjian decoction in treating liver fibrosis with liver and kidney yin deficiency,to find out the active compounds and optimize the combination of active compounds to create a new anti-hepatic fibrosis formula.Methods:1)Gene-chip was used to detect and analyze specific genes of liver and kidney yin deficiency syndrome;combined with the Chinese medicine compounds and liver fibrosis gene database to construct a network of"Yiguanjian-Liver and Kidney Yin Deficiency syndrome-Liver Fibrosis",and identified the active compounds and their targets according to contribution of the network topology parameters,then predict their mechanism of action through KEGG and GO analysis;2)Assessed the activities of candidate compounds on the inhibition of hepatic stellate cells（HSC）proliferation,found out safe and effective active compounds consider of the drug toxicity.Used uniform design to find the best combination of compounds to inhibit HSC proliferation;3)Evaluated the anti-liver fibrosis effect of Yiguanjian and its new formula by CCl₄-induced rat fibrosis model;4)Constructed the“hepatitis B virus-stellate cells-immune cells”co-culture system to imitation the process of hepatitis B virus in vivo causing liver fibrosis,and explored the role of Yiguanjian and its new formula in the process.Results:The nodes of the core network of"Formula-syndrome-Disease"of Yiguanjian included 27 compounds,17 direct action targets,and 76 indirect action targets.The main signal pathways involved in TNF pathway,NF-κB pathway,PI3K-Akt pathway,TLR receptor pathway,cytokine receptor pathway,hepatitis B and hepatitis C related pathways,and cancer pathways;2)Among the 16 candidate compounds,methylophiopogonone A,levistolide A,8-geranyloxypsoralen and bergapten significantly inhibited the proliferation of HSC in rats and human cells,and also can reduce the expression ofα-smooth muscle actin（α-SMA）and collagen I protein level.The uniform design was used to investigate the inhibition of HSC proliferation by the combination of these five compounds,and the best compatible molar concentration ratio was calculated to be0.98:2.74:1.2:2.43:2.62;3)Yiguanjian and its new formula can effectively inhibit CCl₄inducted rat liver fibrosis,and can significantly reduce the serum ALT and AST,and also inhibit the TGFβ/SMAD and PI3K/AKT pathway signal pathway;4)In the co-culture system of“hepatitis B virus-immune cells-stellate cells”,the active compounds of Yiguanjian and its new formula also can inhibit the proliferation of stellate cells,decrease the production of inflammatory factors,and participate in the regulation of TGFβ/SMAD pathway,NF-κB pathway and PI3K/AKT pathway.Conclusion:Yiguanjian decoction treated liver fibrosis of liver and kidney yin deficiency through multi-components,multi-targets and multi-pathways.It and its new formula composed with quercetin、Methylophiopogonone A、Levistolide A、8-Geranyloxypsoralen and Bergaptin can inhibit HSC proliferation and activation,and decrease CCl₄-induced liver fibrosis in rats.Its mechanism of action on liver fibrosis maybe reduce phosphorylation of SMAD2/3,AKT,P65 and IKBαproteins,and regulate the signaling pathways,to alleviate liver fibrosis.