The Protective Effect Of Huangqi Decoction (HOD) On DDC-induced Chronic Hepatic Cholestatic Liver Injury And Its Mechanism In Mice

Object: Intrahepatic cholestasis is a clinical disease characterized by abnormal bile acid metabolism and impaired bile acid circulation.Chronic cholestasis could lead to fibrosis,cirrhosis,liver cancer,and cholangiocarcinoma.However,the treatment options for cholestatic liver disease are limited.HQD was first recored in the classic prescription of "Prescriptions People’s Welfare Pharmacy".HQD has the function of "supplementing qi and benefiting deficiency",and has a unique therapeutic effect on chronic liver diseases,such as liver fibrosis and cirrhosis.We have proved that HQD has the effect of anti-acute intrahepatic cholestasis in preliminary study.However,the effect of HQD against chronic intrahepatic cholestasis is still not clear.In this study,3,5-diethoxycarbonyl-1,4-dihydroxychollidine(DDC)was used to induce chronic intahepatic cholestasis(CIHC)in mice.Global/targeted metabolomics,16 Sr DNA sequencing and molecular biology were used to explore the protective mechanism of HQD on chronic intrahepatic cholestasis.This study not only provides an important theoretical basis for the application and development of HQD,but aslo gives a new insight for the prevention and treatment of chronic cholestatic liver disease.Method: 1.The effects of different dose,method,and time of DDC on mice serum biochemical index,liver injury indicators were observed.The CIHC mouse model was optimized.2.A 0.025% DDC containg fed diet induced CIHC mouse model was used to observe the effects of low and high doses of HQD(2,4 mg/kg)on chronic cholestasis.After treaeted with HQD for 4 weeks and 8 weeks,serum biochemical indexes,liver histological morphology,and hepatic fibrosis indexes of mice were investigated.The protective effect of huangqi decoction on CIHC were evaluated.(3)Based on UPLC-LTQ-Orbitrap,non-targeted metabonomics method were applied.The different metabolites that influenced by HQD with 4 weeks and 8 weeks treatment in CIHC mice liver was investigated.The influence of the differences between screening and identification of metabolites,and analysis of metabolic pathways were observed.Based on UPLC/MS/MS targeted metabolomic methods,the effect of HQD with 4 weeks and 8 weeks treatment on serum and liver bile acid levels in the CIHC mice was investigated.The study the effect of HQD on bile acid homeostasis in CIHC mice was explored.4.Real-time PCR and western blot were used to detect the effects of HQD on the expression of bile acid transporters,metabolic enzymes,nuclear receptors,membrane receptors,and nuclear transcription factor in CIHC mice after 4 and 8 weeks of administration.The effects of HQD on the bile acid homeostasis in mice with CIHC was investigated.Western blot methods was used to study the effect of HQD on NF-κB/IL-6/Stat3 pathway in liver tissues of mice.The protective mechanism of HQD on chronic cholestasis was explored.5.Using the method of 16 Sr DNA analysis,the effect of HQD on intestinal microbiota in CIHC mice was investigated.The corrolation among different microbiota and pharmacodynamics index and serum bile acid level was analysed.The possible mechanism of HQD on intestinal microbiota in CIHC mice was explored.Result: 1.The CIHC mice model was successfully induced by 0.025% DDC diet.The serum ALT,AST and TBA levels were increased as compared with normal control group.2.Serum biochemical analysis showed that treatment with HQD for 4 weeks could significantly reduce serum levels of ALT,AST and TBA in DDC model mice as compared with model control group.After 8 weeks administration of HQD,serum ALT and TBA levels in CIHC mice were also decreased.The results of HE staining showed that HQD treatment at 4 and 8 weeks could significantly improve DDC-induced bile duct cell injury and inflammatory cell infiltration in the portal area in mice.Hepatic fibrosis indexes and Sirius red staining showed that HQD administration can reduce the level of liver fibrosis caused by DDC.The results showed that HQD has the effect of anti-DDC-induced chronic cholestasis and inhibits liver fibrosis caused by cholestasis.3.The liver non-targeted metabolomics experiment showed that 13 different metabolites were screened out after 4 weeks of administration of HQD,and 12 different metabolites were screened out after 8 weeks of administration of HQD.Pathway analysis of these metabolites showed that these metabolites were related to the biosynthetic pathway affecting primary bile acid.BA-targeted metabolomics results showed that HQD could reduce the levels of various bile acids in serum and liver of cholestasis mice.5.Real-time PCR results showed that treatment with HQD for 4 weeks could induce the expression of bile acid effusion transporters Mrp2,Mrp3,Mrp4 and Ost-α.Treatment with HQD for 8 weeks could induce the expression of bile acid effusion transporter Mrp4,inhibit the expression of bile acid uptake transporter Ntcp,and induce the expression of bile acid metabolism enzyme Cyp2b10.Western blot results showed that HQD could significantly induce the liver expression of nuclear transcription factor Nrf2 at both 4 and 8 weeks treatment.The results showed that HQD can promote liver bile acid efflux,inhibit bile acid uptake,and promote bile acid metabolism.These effects may be related to the induction of HQD Nrf2 activiation.6.Real-time PCR results showed that HQD at 8 weeks could significantly improve the expression of inflammatory factors(IL-1β and TNF-α),cell adhesion factors(ICAM-1),chemokine(MIP-2 and MCP-1),and growth factor(TGF-β)in the liver of CIHC mice.Those result may be related to HQD inhibition of NF-κB inflammatory pathway.HQD can significantly reduce the expression of biomarker CK-19 in bile duct cells and the expression of Collagen I in liver tissues after 8 weeks of administration.Western blot analysis showed that HQD inhibited bile duct hyperplasia may be related to its inhibition of NF-κB /IL-6 /STAT3 pathway.7.According to 16 Sr DNA analysis,it was found that intestinal microbiota diversity of CIHC mice was significantly decreased as compared with normal mice.The flora richness was significantly reduced.HQD intervention for 8 weeks markedly improved the microflora diversity and richness.A total of 17 differential species were found at the OTU classification level.More over,Prevotellaceae＿NK3B31＿group(OTU433),Alistipes(OTU435),Parabacteroides(OTU430),Gordonibacter(OTU383)and Parabacteroides＿goldsteinii(OTU255)were positively correlated with pharmacodynamic indicators.Real-time PCR results showed that HQD could significantly reduce the expression of genes related to intestinal inflammation in DDC mice.Conclusion: 1.HQD alleviated liver injury and fibrosis in 0.025% DDC-induced CIHC mice.2.HQD regulated the expression of bile acid transporters and metabolic enzymes in CIHC mice,improved the outflow and metabolism of liver bile acid,and inhibited the uptake of bile.Moreover HQD decreased the content of serum and liver bile acid,and this effect may be related to its induction of nuclear transcription factor-E2-factor(Nrf2).3.HQD inhibited the liver inflammation and reduce the liver cell damage in CIHC mice,which may be related to its inhibitied effect on NF-κB pathway.Moreover HQD inhibitied liver fibrosis and bile duct proliferation in CIHC mice,which is related to its inhibition of IL6/STAT3 pathway.4.The intestinal microbiota diversity and richness of CIHC mice were reduced,and the microbiota structure was changed.After intervention of HQD,the intestinal microbiota diversity and richness of cholestasis mice could be improved.Additionally,after treatment with HQD,intestinal microbiota composition and intestinal microbita disorder of cholestasis mice was improved.The intestinal inflammatory reaction was also inhibited by HQD.