The Role Of Low Density Lipoprotein Receptor LDLR And Bile Acid Membrane Receptor TGR5 In HBV Infection

BackgroundHepatitis B is an infectious disease caused by HBV virus infection.After infection with hepatitis B virus,the body can gradually develop into a series of diseases such as hepatitis,liver cirrhosis,liver cancer and so on.Nearly 2 billion people worldwide are infected with HBV.Nearly 1 million people die each year from liver cirrhosis and liver cancer caused by chronic HBV infection.At present,interferon and nucleosides analogues used in the treatment of HBV can not completely eliminate HBV virus.Therefore,the search for more reasonable treatment is an important challenge for us.Low density lipoprotein receptor LDLR is an important receptor of lipid metabolism,which regulates the stability of lipid metabolism in the liver,while bile acid membrane receptor TGR5,as an important receptor of bile acid metabolism,modulates liver inflammation and regeneration and bile secretion in the liver.Cell proliferation and gallbladder filling.HBV infection is often accompanied by abnormal lipid metabolism and bile acid metabolism,but the effects of abnormal lipid and bile acid metabolism on HBV replication,expression and clearance are not clear.In this paper,LDLR and TGR5 were used as the starting point to explore the role of lipid and bile acid metabolism in HBV infection,which will provide a new way of thinking and direction for the treatment of viral hepatitis B.ObjectiveTo study the effects of lipid metabolism and bile acid metabolism on HBV replication,expression and clearance,so as to provide theoretical basis for the treatment and prognosis of hepatitis B.Methods1.The effects of HBV infection on lipid metabolism.（1）Correlation analysis between HBV infection and liver fat change.The paraffin sections and medical records of liver tissue of HBV infected patients were collected,HE staining and immunohistochemical HBsAg staining were collected to analyze the relationship between HBsAg and the degree of hepatic steatosis in HBV infected patients;（2）Effect of HBV infection on human serum lipoprotein.Serum lipoprotein was isolated from healthy people and HBV infected persons by rapid protein liquid chromatography（Fast protein liquid chromatography,FPLC）.The effect of HBV on VLDL-C,LDL-C and HDL-C was analyzed;（3）Effect of HBV infection on lipid metabolism in mice.The mouse model of HBV infection was established,the expression of HBV in serum was detected by ELISA;the serum lipoprotein was isolated by FPLC,and the effect of HBV infection on VLDL-C,LDL-C and HDL-C in mice was analyzed;Detection of the expression of Lipid Metabolism related genes in liver tissue of mice by mRNA,Real-Time PCR extraction;Detection of HMGCR and LDLR by extracting liver protein,Western Blot from mice;（4）Effects of HBV infection on free cholesterol and low density lipoprotein uptake.PAAV and pAAV/HBV1.2 plasmid were transferred into HepG2 cells.Fluorescence labeled NBD-cholesterol and Dil-LDL,were added to detect the uptake of free cholesterol and low density lipoprotein by HBV infection;2.Effects of LDLR^-/-on HBV replication,expression and immune response.The C57BL/6 and LDLR^-/-mice models of HBV infection were established.（1）Detection of HBsAg,HBeAg expression and HBcAb production in serum and liver of HBV mice by ELISA;（2）Quantitative determination of viral DNA in serum by extracting viral DNA,Real-Time PCR from mouse serum;（3）Expression of HBcAg in liver of mice in two groups by Immunohistochemical method;（4）Changes of HE staining and serum transaminase in liver;3.Effect of HBV infection on bile acid metabolism.（1）the effect of HBV infection on genes related to bile acid metabolism in mice.The mouse model of HBV infection was established,and the expression of genes related to bile acid metabolism was detected by mRNA,Real-Time PCR extracted from liver tissue of mice;The expression of TGR5 in mouse liver was detected by immunohistochemical staining in paraffin sections of mouse liver;（2）Effect of HBV infection on human liver TGR5.The expression of TGR5 in liver of patients with and without HBV infection was detected by immunohistochemical staining;4.Effects of TGR5 on HBV replication,expression and immune response.（1）the effects of TGR5^-/-on HBV replication,expression and immune response.C57BL/6 and TGR5^-/-mouse models of HBV infection were established.The serum HBsAg,HBeAg,HBcAb level of HBV mice was detected by Elisa,and then the changes of serum transaminase and total bile acid were detected respectively;Quantitative determination of viral DNA in serum by extracting viral DNA,Real-Time PCR from serum;the liver protein was extracted and the expression of HBsAg,HBeAg was detected by ELISA.The paraffin sections of mouse liver tissue were taken and the expression of HBcAg in the liver of the two groups was detected by immunohistochemistry;Detection of Interferon I related Gene expression in liver tissue by mRNA,Real-Time PCR extraction;HE staining was used to observe the degree of liver injury in mice from the morphological point of view;（2）the effect of TGR5 agonist INT-777 on HBV virus in cell model.In HepG2 cells,pAAV/HBV1.2 and phTGR5 were transferred into DMSO,cells.24 hours later,the cells in the control group were added with TGR5 agonist INT-777,24 for 24 hours,and the culture fluid was collected.The expression of HBsAg and HBeAg was detected by ELISA,and the serum virus DNA,was extracted.Quantitative determination of viral DNA in serum by Real-Time PCR;5.Effect of TGR5 on interferon I production.C57BL/6 and TGR5^-/-male mice aged 6 weeks and 8 weeks old were divided into two groups.The control group was injected with PBS solution,the experimental group was intraperitoneally injected with interferon inducer Poly（I/C）,and the blood samples were collected 24 hours later.The serum IFN-βlevel of the mice was quantitatively detected by ELISA.Results1.The effect of HBV infection on lipid metabolism.（1）the hepatic tissue sections of patients with HBV infection were stained,and the results of HE staining were evaluated with 0-5 score of steatosis,and the results of immunohistochemical staining were evaluated by 0-5 score of HBsAg expression.The relationship between HBsAg and hepatic steatosis was analyzed in patients with HBV infection.The results showed that there was a negative correlation between HBsAg and the degree of hepatic steatosis in patients with HBV（P=0.028）;（2）in the serum of healthy people and HBV-infected persons,the results of FPLC test showed that the serum VLDL-C and LDL-C of HBV-infected persons increased and the HDL-C decreased;（3）In C57BL/6 mice infected with HBV:the results of FPLC showed that serum LDL-C increased and HDL-C decreased,while HMGCR,LDLR was up-regulated at mRNA level（P<0.05）.At the protein level,the expression of HMGCR and LDLR were significantly increased（P<0.05）;（4）In the HBV infection model constructed by HepG2 cells,Real-Time PCR results showed that the expression of LDLR and HMGCR was up-regulated after HBV infection（P<0.05）;The results showed that there was no difference in the uptake of free cholesterol after HBV infection,while the uptake of low-density lipoprotein decreased significantly;2.Effects of LDLR^-/-on HBV replication,expression and immune response.（1）the results of ELISA showed that the expression of HBsAg,HBeAg in serum and liver of LDLR^-/-mice decreased,while the expression of HBcAb increased compared with that of C57BL/6 mice;（2）the content of serum viral DNA in LDLR^-/-mice was lower than that in C57BL/6 mice（P<0.05）;（3）Immunohistochemical staining showed that the expression of HBcAg in the liver of LDLR^-/-mice was significantly lower than that of C57BL/6 mice;（4）HE staining of liver showed that there was no significant difference in morphology between the two groups after HBV infection,but there was no significant difference in serum transaminase between the two groups;3.Effect of HBV infection on bile acid metabolism.In C57BL/6 mice,CYP7A1,CYP27A1 was up-regulated after HBV infection（P<0.05）,TGR5 was up-regulated more significantly（P<0.01）,CYP8B1 was down-regulated（P<0.05）;The results of Immunohistochemical staining showed that the expression of TGR5 in mouse liver was increased,mainly in the small bile duct of mouse liver tissue and around the hepatic sinus,but the expression of TGR5 in liver of clinical patients was higher before and after infection,and there was no significant difference between the two groups;4.Effects of bile acid membrane receptor TGR5 on the replication,expression and immune response of HBV.（1）in HBV infected mice,the results of ELISA detection showed that compared with C57BL/6 mice,TGR5^-/-mice showed HBsAg,in serum and liver.The expression level of HBeAg was increased,but the expression of HBcAb was decreased（P<0.05）.Immunohistochemical staining showed that the expression of HBcAg in the liver of TGR5^-/-mice was significantly higher than that of C57BL/6mice.The content of viral DNA in serum of TGR5^-/-mice was higher than that of C57BL/6 mice（P<0.05）,and there was no significant difference between the two groups in morphology after HBV infection by HE staining in liver.The levels of serum transaminase and bile acid in C57BL/6 and TGR5^-/-mice were significantly increased after intravenous injection,but there was no significant difference.（2）After adding TGR5 agonist INT-777 into the HBV infection model of HepG2 cells,the results of ELISA detection in the supernatant showed that INT-777 inhibited the expression of HBsAg（P<0.01）,and the expression of HBsAg was inhibited by INT-777（P<0.01）.The results of Real-Time PCR showed that after adding INT-777,the content of viral DNA decreased（P<0.05）;5.The effect of TGR5 on the production of type I interferon.The serum levels of IFN-βin C57BL/6 and TGR5^-/-mice were significantly higher than those in TGR5^-/-mice after injection of Poly（I/C）（P<0 05）.Conclusion1.HBV infection interferes with the function of LDLR,resulting in the increase of serum VLDL-C and LDL–C and the decrease of HDL-C.2.LDLR knockout inhibits the replication and expression of HBV virus and promotes the clearance of HBV virus;3.The activation of TGR 5 inhibits HBV virus expression and facilitates virus clearance.