The Study On Mechanism Of Treatment With Different Proportion Of YinChen GanCao Decoction To Cholestatic Liver Injury Based On “Bile Acids-TLR₄-NF-κB” Pathway

Objectives:The study aimed at exploring the effects of different ratios of Yin Chen Gan Cao decoction on hepatic bile acid concentration and modulation of TLR₄/NF-κB signaling pathway;thus affecting the secretion of inflammatory cytokines in intrahepatic cholestatic（IC）model mice.The study could provide evidence to the optimal proportional compatibility of Yin Chen-Gan Cao decoction in treatment of cholestatic liver injury in clinic,and reveal the relevant mechanisms.Methods:1.ANIT was used to induce IC model in C57/BL mouse.Administered with different proportions of Yin Chen Gan Cao（4:0,4:1,2:1,1:1,1:2,1:4,0:4）decoction for 10consecutive days,blood and liver samples were collected.2.Plasma alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,alkaline phosphatase（ALP）,total bile acid（TBA）and total bilirubin（TBIL）in all groups were determined by biochemical automatic analyzer;liver tissue pathology（HE staining）were observed on light microscope.3.The liver concentrations of CA,DCA,α-MCA,β-MCA,TCA,TCNCA,TUDCA,TDCA and LCA were detected by LC-MS/MS.4.TLR₄,My D88 and p-NF-κB p65 proteins expression in liver were detected by Western blot;IL-6,IL-1β,TNF-α,MCP-1 and MIP-2 levels in liver samples were detected by ELISA.Result:1.In the liver of model mouse induced by ANIT,liver cell structure was destructed,and severe liver necrosis,accompanied with apparent inflammatory cells infiltration in the liver sinus were observed.Compared with normal group,serum biochemical level elevated significantly（P<0.05）,and the expression of TLR₄/NF-κB pathway proteins,as well as levels of cytokines and inflammatory factors were significantly increased（P<0.05）in model group.2.Compared with model group,Yin Chen Gan Cao（1:4,0:4）decoction groups significantly down-regulated the levels of plasma TBA,TBIL,AST,ALT and ALP（P<0.05）.Microscopic examination results showed that hepatocyte necrosis was significantly reduced.Meanwhile,inflammatory cell infiltration was also declined in hepatic sinus.The Yin Chen Gan Cao（0:4）decoction group had the best treatment effect with the lowest necrosis score.While the Yin Chen Gan Cao（4:0,4:1,2:1,1:1）decoction groups didn’t show obvious improvement,with blood biochemical levels,necrosis and inflammatory cell infiltration almost as the same as the model group,.3.Compared with model group,Yin Chen-Gan Cao at proportions of 2:1,1:1,1:2,1:4and 0:4 all reduced the concentration of liver bile acids（P<0.05）,with Yin Chen Gan Cao（0:4）decoction group exhibiting the greatest difference.However,bile acid level in Yin Chen Gan Cao（4:0,4:1）decoction groups didn’t show significant change.4.Compared with model group,Yin Chen-Gan Cao at proportions of 1:4 and 0:4 greatly reduced the expression of TLR₄,My D88 and p-NF-κB p65（P<0.05）,and down-regulated the levels of inflammatory factors IL-6,IL-1β,TNF-αas well as chemokine MCP-1（P<0.05）.Yin Chen Gan Cao（1:4,0:4）decoction groups showed the best improvement.Neverthelss,Yin Chen Gan Cao（4:0,4:1,2:1,1:1）decoction groups didn’t differ significantly from model group in the expression of proteins in TLR₄/NF-κB signaling pathway（P>0.05）.Conclusion:Yin Chen Gan Cao decoction at proportions of 1:4 and 0:4 decreased the concentration of bile acids in liver and inhibited TLR₄/NF-κB signaling pathway significantly.Subsequently,the release of inflammatory factors was reduced,followed by less inflammatory reactions and inflammatory cell infiltration in the liver,thus liver injury was alleviated.