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目的:比较硼替佐米和几种天然药物体外对HBV的抑制作用,并探索硼替佐米抑制HBV的分子机制。方法:采用MTT法观察不同浓度药物对HBV-Tg小鼠原代肝细胞的毒性,确定最大无毒浓度,用ELISA方法检测培养24h后细胞上清中HBsAg的变化,比较不同药物对HBsAg的抑制率。经1μmol/L硼替佐米处理原代肝细胞24h后,提取蛋白质,采用双向凝胶电泳分离总蛋白,得到差异表达的蛋白质点进行分析比对。以RT-PCR及West blot验证选定的差异蛋白,分析其与HBV抑制作用的关系。结果:在8种天然药物中以扁蒴藤素对HBV作用最强,IC50为0.43μmol/L,EGCG的IC50是24.9μmol/L,且安全性较好。硼替佐米作用原代肝细胞蛋白质组分析发现了21个差异斑点,其中包括HSP60异构体(gi/26353954)。RT-PCR检测发现经硼替佐米作用后HSP60、HSP90表达下调,HSP70表达上调,而West blot鉴定HSP60变化不大。但是经EGCG作用后HSP90表达的下调与对HBsAg的抑制是一致的。结论:调节宿主细胞的蛋白质降解与折叠将有利于寻找新型抗HBV药物。
OBJECTIVE: To compare the inhibitory effect of bortezomib and several natural drugs on HBV in vitro and to explore the molecular mechanism of bortezomib inhibiting HBV. Methods: MTT method was used to observe the toxicity of different concentrations of drugs on primary hepatocytes of HBV-Tg mice. The maximum non-toxic concentration was determined. The changes of HBsAg in supernatant of cells were detected by ELISA. The inhibitory effect of different drugs on HBsAg rate. The primary hepatocytes were treated with 1 μmol / L bortezomib for 24 h, then the proteins were extracted and the total proteins were separated by two-dimensional gel electrophoresis. The differentially expressed protein spots were obtained for analysis and comparison. The selected proteins were verified by RT-PCR and Western blot, and their relationship with HBV inhibition was analyzed. RESULTS: Ampicillin had the strongest effect on HBV in 8 kinds of natural medicines with IC50 of 0.43μmol / L and EGCG with IC50 of 24.9μmol / L, which was safe. Bortezomib Effects Primary liver cell proteome analysis revealed 21 differential spots, including the HSP60 isoform (gi / 26353954). The results of RT-PCR showed that the expression of HSP60 and HSP90 was down-regulated and the expression of HSP70 was up-regulated after treated with bortezomib, while HSP60 was not changed by West blot. However, the down-regulation of HSP90 expression by EGCG is consistent with the inhibition of HBsAg. Conclusion: Regulation of host cell protein degradation and folding will be helpful in finding novel anti-HBV drugs.