Investigating The Role Of TLR3/TRIF Signaling And Dendritic Cells In The Pathogenesis Mechanisms Of Hepatitis B Virus Associated Glomerulonephritis In Mouse

ObjectiveTo investigate the expression of innate immune factor — toll-like receptor3(TLR3)and it’s related signals as well as dendritic cell(DC)in the mice model with hepatitis B virus associated glomerulonephritis(HBV-GN),further analysis their possible innate immune mechanisms in the pathogenesis of HBV-GN,which may provide a new target of treating HBV-GN.Methods(1)Model establishment and group dispatching: 21 pure BALB/c mice,weighing 18 g to 20 g,were established into HBV-GN model,then we divided the model mice(the remaining 18)into 3 groups(n=6),model group(group B),entecavir group(group C),entecavir combined with mycophenolate mofetil group(group D).At the same time,we chose 9 healthy male BALB/c mice as control group(group A).Group A and B were given normal saline,group C was given entecavir by gavage,group D was treated with entecavir and mycophenolate mofetil by gavage.All mice were sacrificed after one month.(2)Judging whether the model established was successful: during the time of modeling,we detected the urine protein of mice with test strips 2 times a week,which approved the renal damage in mice.Finally we took renal tissues from 3 model mice and 3 nomal mice stained with HE and PAS respectively,meanwhile,we detected HBsAg in renal tissue,which confirmed the mice suffering from HBV-GN.(3)After successful modeling and intervention,the expression of Toll-like receptor 3(TLR3),TIR-domain containing adaptor inducing interferon-β(TRIF),Interferon regulatory factor 3(IRF3),Interferon-β(IFN-β)proteins and mRNA were detected by Western blot and real-time fluorescent quantitative PCR.(4)Detecting the relative expression of DC in renal tissues via Flow cytometry.Results(1)From the second week after modeling,mice’s urine began to appear urine protein,at the end of the fourth week,the result of urine protein were up to + + +,andhealthy male BALB/c mice’s urinary protein were always negative.In the model group,a large number of inflammatory cells were observed in the renal tissue by HE satining,and the glomerular mesangial cells and mesangial matrix proliferation were observed by PAS staining.In addition,in the model group,HBsAg was found to be granular deposition along the glomerular mesangial area via immunofluorescence staining,and there was no HBsAg deposition in the renal tissue of healthy mice.(2)Compared with the group A,the expressions of protein TLR3,TRIF,IRF3,IFN-β and m RNA in renal tissue in group B,C,D were up-regulated(P<0.05).In group B,the levels of expressing proteins and mRNA increased compared with the other three groups(P<0.05).There was no statistically significant differences between group C and group D in the expression of each protein and mRNA(P>0.05).(3)The change of DC was consistent with the relative expression of proteins and mRNA above,the result of group B was higher than the other three groups(P<0.05),group A was lower than other three groups(P<0.05),there was no significant difference between group C and group D(P>0.05).Conclusion(1)The animal model of HBV-GN can be constructed successfully by intraperitoneal injecting Hepatitis B serum into BALB/c mice.(2)After infecting our body,HBV can produce dsRNA during its replication,which may bind TLR3 of DC that activated TLR3 / TRIF signaling pathway and stimulated the maturation of DC,leading to a series of immune inflammatory response and immune imbalance,eventually causing renal damage.(3)These findings may provide new strategy from the point of innate immune system for the treatment of HBV-GN.