Pathological Study On Gerbils And C57BL/6Mice Inoculated With Human Hepatitis B Virus

Hepatitis B virus (HBV) infection, characterized by liver injury, caused public health problem worldwide. The studies on HBV have long been hampered by the strict host specificity and lack of appropriate cell models or animal models. To explore the possibility of animals being infected by human HBV, Mongolian gerbils and C57BL/6mice were inoculated with human HBV. A variety of approaches including ELISA, PCR, Real-time PCR, immunohistochemistry staining and western blot were used for detection of HBV related antigens and antibodies in serum, faces and liver samples. Proteins involved in apoptosis and endoplasmic reticulum stress (ERS) were analyzed to explore the mechanism of liver injury caused by HBV inoculation. The results are as follows:1、After inoculated with human HBV, the liver and kidney tissues of gerbils were detected to be HBV DNA positive1d post inoculation (dpi), however only the livers were HBV DNApositive at2dpi and4w dpi. In addition, HBV DNA was detected positive in the liver tissues of C57BL/6mice but only at1dpi. However, HBV DNA were fail to be positive in serum, faeces, brain and salivary glands.ELISA results demonstrated that HBsAg and HBeAg were transiently existed in the serum of gerbils and C57BL/6mice within1w after inoculation, while the HBsAb and HBcAb were detected till8w. Immunohistochemical analysis showed the persistent presence of HBV antigens in livers of gerbils and C57BL/6mice after inoculation.The signals of HBV related antigens were increased with the inoculation time. Western blot analysis revealed the presence of PreS1in the liver tissues at7dpi of both gerbils and C57BL/6mice after inoculation with HBV.2、Obvious histopathological changes resembled viral hepatitis were observed in gerbils and C57BL/6mice after inoculated with human HBV. With dilated sinus and congestion in liver, swollen hepatocytes with extensive cytoplasmic vascuolation were obviously observed. Besides, lymphocytes infiltration around portal area and biliary hyperplasia proliferation were also observed.The lesions were aggravated with the time after inoculation. The hepatic ultrastructure observed by TEM demonstrated loose cytoplasmic matrixes in hepatocytes with marked changes in endoplasmic reticulum and mitochondria. The mitochondria were dilated with dissolved cristae, some of which formed concentric lamellar bodies in cytoplasm. Besides, spherical viral particles morphologically similar to HBV particles were observed in the nucleus of hepatocyte and the cytoplasm of epitheliums of renal tubules.3、Immunohistochemical staining results showed that the expression levels of PCNA and HSP70were increased at the initial stage and then slowly declined. The expression levels of the two proteins were significantly higher than that of the control group, suggesting that protective mechanisms were induced in response to the liver damage induced by HBV inoculation.4、The immunohistochemical staining results showed that the expressions of the apoptosis-related molecules Bax, Bcl-2, Caspase3and Fas/FasL were significantly higher in livers of HBV inoculated group than that of control (P<0.05), indicating the engagement of apoptotic pathways.5、The immunohistochemical staining results showed the expressions of the ERS associated GRP78and CHOP were significantly higher in livers of HBV inoculated group than that of control group (P<0.05). The expressions of JNK and Caspase-12in the inoculated group were also increased. Western blot analysis further confirmed the increased expressions of the ERS related proteins, suggesting that ERS-induced apoptosis in hepatocytes occurred after HBV inoculation.6、Mitochondria related genes were amplified and analyzed. One mutation in the D-loop of CytB and CytC mtDNA was revealed in the inoculated geibels, which might be associated with injuries of mitochondria structurally and functionally.In conclusion, the results showed that human HBV could induce hepatic pathological damage in gerbils and C57BL/6mice. ELISA and PCR analysis demonstrated the existence of HBV DNA and HBV related antigens and antibodies after inoculation. The expressions of apoptosis and ERS related proteins were increased after HBV inoculation, indicating apoptotic and ERS pathways were mobilized in response to HBV inoculation and liver injury.