The Mechanisms Of Bile Acid Receptor FXR In HBV Infection

BackgroundHepatitis B virus belongs to Hepadnaviridae with a partially double-stranded and circular DNA.Hepatitis B virus infection is a major public health problem worldwide,almost 2 billion people have been infected with HBV,more than 240 million patients of them are chronically infected with HBV.Hepatitis B virus can cause many liver disease including hepatitis,liver fibrosis and hepatocellular carcinoma after infection.Existing interferon drugs and nucleoside analogue can not completely eliminate HBV DNA,so treatment of HBV is still an important challenge for clinicians.Farnesoid X receptor(FXR)is the bile acid receptor,one of the members of the nuclear receptor superfamily,playing an important role in bile acid,cholesterol,triglyceride and glucose metabolism.HBV infection can lead to elevated bile acid levels in the body,but the pathophysiological role of bile acid and bile acid receptor FXR in HBV infection remains unclear.ObjectiveThe aim of the study is to investigate the mechanisms of bile acid and bile acid receptor FXR in HBV infection,thus providing experimental basis for the treatment of hepatitis B virus infection.Methods(1)Liver biopsy specimens were obtained from patients with choronic HBV infection.The expressions of HBs Ag,HBc Ag and CYP7A1 were studied using immunohistochemistry,which was used to analyze the relationship between bile acid and hepatic pathological changes;(2)Hep G2 cells transfected with p AAV/HBV1.2 plasmid and p FXR plasmid was cultivated for 24 hours,then CDCA and Z-Guggulsterone were also added for 24 hours.The expressions of HBs Ag and HBe Ag of intracellular proteins and supernatant were determined by ELISA;(3)The plasmids of p GL3-SP1 or p GL3-SP2,p FXR and p RL-TK were transfected into Hep G2 cells,and transfected cells were cultivated for 24 hours,then CDCA was added.At 2 days post-transfection,cells were lysed and assayed for luciferase activity by using a dual-luciferase reporter assay system;(4)The plasmids of p AAV/HBV1.2 or p AAV/HBV1.2 HBe Ag-null and p FXR were co-transfected into Hep G2 cells for 24 hours.Then CDCA was added for 48 hours.Total RNA of treated cells was extracted and RT-PCR was used to detect the expressions of related moleculars;(5)Ten micrograms of p AAV/HBV1.2 DNA was injected hydrodynamically into the tail veins of specific pathogen-free(SPF)C57BL/6 and FXR-/-male mice aged from 6 to 8 weeks.After injection,the mice were regularly bled to monitor the serum levels of HBs Ag,HBe Ag,HBc Ab,HBV DNA and IL-10.The liver proteins from mice injected with HBV were extracted to detect the expressions of HBs Ag and HBe Ag in these mice.Immunohistochemical technique was used to detect the expression of HBc Ag in the liver of mice infected with HBV.Liver tissues from mice were smashed by ultrasonic and total RNA were extracted,then RT-PCR technique was used to detect liver related innate immune molecules in level.Liver tissue sections were stained with hematoxylin and eosin(H&E).Serum alanine aminotransferase(ALT)levels were also determined;(6)The data were statistically analyzed by using SPSS 19.0 software for independent sample t test(t-test).The figure was made by Graph Pad Prism5.0 software.P < 0.05 is considered as statistical significance.Results(1)The liver of patients infected with HBV was collected for immunohistochemical staining to detect the expressions of HBs Ag and CYP7A1,0-5 points were scored according to the result of immunohistochemical staining,a correlation curve was made to analyze the relationship of HBs Ag and total bile acid levels in serum of patients infected with HBV.The result showed that the expression of HBs Ag in the liver of patients infected with HBV was positively correlated with the level of serum total bile acid,and HBs Ag was also positively correlated with CYP7A1.Ground-glass hepatocytes of patients infected with HBV were associated with bile acids;(2)p AAV/HBV1.2 and p FXR plasmid were co-transfected into Hep G2 cells for 24 hours,then CDCA and Z-Guggulsterone were added into medium for another 24 hours.The result showed that FXR antagonist Z-Guggulsterone could inhibit the expressions of HBs Ag and HBe Ag(P < 0.05);(3)The plasmids of p GL3-SP1 or p GL3-SP2,p FXR,p RL-TK were transfected into Hep G2 cells,transfected cells were cultivated for 24 hours,then CDCA was added.At 2 days post-transfection,cells were lysed and assayed for luciferase activity by using a dual-luciferase reporter assay system.The result showed CDCA increased the activity of the pre-S1 promoter by 1.7 fold through FXR,and CDCA increased the activity of the pre-S2 promoter by 1.4 fold through FXR(P < 0.05);(4)p AAV/HBV1.2 and p FXR plasmid were co-transfected into Hep G2 cells for 24 hours,then CDCA was added into medium for 48 hours.The result showed that the TLR2 expression of the group of FXR+CDCA decreased,meanwhile TNFα decreased and ATF6 increased(P < 0.05);The result of detection of virus DNA in intracellular and supernatant showed that after transfection of p FXR plasmid and stimulation with CDCA,the amount of viral DNA in the supernatant decreased,while the amount of intracellular viral DNA increased(P <0.05).p AAV/HBV1.2 HBe Ag-null and p FXR plasmid were also transfected into Hep G2 cells for 24 hours,then CDCA was added for 48 hours,the result demonstrated that the expression of innate immune molecule TLR2 was no longer declined,and TNFα decreased,ATF6 increased;(5)Serum and liver of mice were collected when p AAV/HBV1.2 DNA was injected hydrodynamically into the tail veins of specific pathogen-free(SPF)C57BL/6 and FXR-/-male mice aged from 6 to 8 weeks.The result showed that the expressions of HBs Ag and HBe Ag in serum and liver tissue of FXR-/-mice infected with HBV declined comparing with C57BL/6 mice.Immunohistochemical staining result showed that the expression of HBc Ag of FXR-/-mice also decreased.The production of HBc Ab in FXR-/-mice infected with HBV decreased too,however,there was no difference in cytokine IL-10 level between C57BL/6 mice and FXR-/-mice.The result of innate immune molecules and related inflammatory molecules in the liver of mice showed that the expression of TLR2 in the liver of FXR-/-mice increased,however,the expressions of TNFα and IFNγ decreased.In the meantime,the content of virus DNA in serum of FXR-/-mice was lower than that of C57BL/6 mice.Liver HE staining result showed that there was no significance in morphology between C57BL/6 mice and FXR-/-mice.There was no difference in level of serum transaminase between C57BL/6 mice and FXR-/-mice.Conclusion(1)In the model of FXR-/-mice infected with HBV,the expressions of HBs Ag,HBe Ag and HBc Ag decreased;(2)Bile acids promote the expression of HBe Ag through FXR,but inhibit the expression of innate immune molecule TLR2;(3)Bile acids promote the replication of HBV through FXR,but inhibit the release of HBV particles;(4)Ground-glass hepatocytes of patients infected with HBV were associated with bile acids.