Differential Proteomics On Liver Of HBsAg Transgenic Mouse

Hepatitis B virus (HBV) causes world wide endemic or epidemic of viralhepatitis. HBV can cause acute or chronic hepatitis, liver cirrhosis and evenhepatocellular carcinoma (HCC). In chronic infections, hepatitis B surface antigen(HBsAg) as a serum marker of HBV infection persists more than six months. Theprolonged presence of HBsAg could be one of the major causes of chronic hepatitis Band other liver disorders. Many studies have focused on revealing the mechanisms ofthe chronic HBV infection, However, the pathogenesis initiated by HBsAg andmeasures to clear HBsAg have not been fully revealed.In the first part of this study, a transgenic mouse lineage of HBV establishedfrom an HBV strain isolated from a Chinese patient was explored by the differentialproteomics approach to investigate the global effects of persistent expression ofHBsAg on host cells. Compared to the control, 93 proteins identified weredifferentially expressed in the liver of HBsAg positive transgenic mice. After geneontology and biological analysis of the results, around 45% of the proteins fell into thecategory of enzymes related to metabolism. As a lipoprotein complex, HBV envelopecontained 25% lipid from the host cell. It was reported that the efficiency of HBsAgexpression was determined by the lipid level of host. Thus, we hypothesized thatHBsAg could lead to alteration of host lipid metabolism, which sequentially couldaffect metabolism of carbohydrate and amino acids. In the second part of this study,results acquired from the proteomics approach on proteins related to metabolism wereanalyzed and summarized. Combined with the results from microarray of the samemice livers, the alteration of metabolism pathways were summarized as followings: 1.Enhanced synthetic pathway of cholesterol and decreased synthesis of bile acid; 2.Decreased glycolysis but increased gluconeogenesis, 3. Enhanced catabolism ofamino acids. Sera from the transgenic mice and control mice were used to assess thealteration of liver metabolic pathway caused by HBsAg. Results indicated that totalcholesterol, triglyceride and low density lipoprotein were significantly decreased while amino acids were prone to catabolism due to the consumption of host lipid byHBV envelope. These findings implied that the physiological metabolism of the hostcell was impaired by HBsAg. However, these changes could favor the expression andpersistent secretion of HBsAg. In the third part of this study, cyclophilin A (CypA)was selected from the proteomics results for further study to elucidate its possible rolein the pathogenesis of HBsAg. The abundance of CypA was found decreased both inthe liver of HBsAg transgenic mice and in a HBsAg stably expressing cell line.Reduced CypA in HBsAg expressing Huh7 cells reverted to the normal level when theexpression of HBsAg was blocked by siRNA. Besides, HBsAg could interact withCypA in co-immunoprecipitation assay. To study the effect of HBsAg expression byCypA, blockage of CypA by siRNA enhanced HBsAg expression whileoverexpression of CypA resulted in decreased HBsAg expression. Preliminary studiesshowed that a decrease in CypA was found in the livers of some of the HBsAgpositive chronic HBV patients. To uncover the mechanism involved, transcriptionallevels of CypA were assayed between HBsAg positive and negative cells, but nochange was found. However, compared to the control cells, higher CypA was foundsecreted into culture supernatant of HBsAg positive cells. Increased secretion ofCypA was reported when cells were under stress such as during inflammation. In apreliminary study, neutrophils and lymphocytes were observed in only a few fields ofHBsAg transgenic mouse liver section by HE staining, which possibly implied thatHBsAg induced chronic liver inflammation via enhancing CypA secretion.In summary, this study revealed that HBsAg induced alteration of hostmetabolism to benefit its expression and secretion. During the persistent expression ofHBsAg, host physiological metabolic activities were impaired, namely, markedly inlipid metabolism, in carbohydrate metabolism and in amino acid metabolism. Besides,HBsAg enhanced extracellular CypA secretion, which could be chemotactic toinflammatory cells, and result in chronic inflammation of liver. It was hypothesizedthat HBsAg regulated intracellular CypA to facilitate expression of itself in a positivefeedback manner. The association between decrease in intracellular CypA andincrease of extracellular CypA with HBsAg expression provided a new field for thestudy of pathogenesis of HBsAg.