氧化苦参碱黄芩苷组合物抗乙型肝炎病毒和抗肝纤维化的体外实验

The in vitro study of effects of oxymatrine-baicailin compound on hepatitis B virus expression and anti-hepatic fibrosis

目的研究氧化苦参碱黄芩苷组合物(OB)体外抗乙型肝炎病毒和抗肝纤维化的作用。方法培养HepG2.2.15细胞和大鼠HSC-T6细胞,分别使用OB或者氧化苦参碱处理。采用酶联免疫吸附法检测细胞培养上清中HBsAg、HBeAg含量。收集HSC-T6细胞抽提总RNA,使用半定量RT-PCR法检测α平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原基因表达量;抽提细胞总蛋白,10%聚丙烯酰胺凝胶电泳分离蛋白,使用Western blot检测α-SMA和Ⅰ型胶原蛋白表达量。结果OB对乙型肝炎病毒抗原分泌抑制作用显著优于单独使用氧化苦参碱(两者对HBsAg抑制率的比较为:P=0.043;HBeAg为:P=0.026)。OB处理组的α-SMA含量不仅在转录水平而且在翻译水平显著低于氧化苦参碱组(mRNA:P=0.013,蛋白:P=0.042);OB处理组的Ⅰ型胶原表达量同样显著低于氧化苦参碱组(mRNA:P<0.01;蛋白:P<0.01)。结论OB对HepG2.2.15细胞分泌乙型肝炎病毒抗原和HSC-T6细胞表达α-SMA和I型胶原有抑制作用,且显著优于氧化苦参碱。

Objective To study the anti-hepatitis B virus and anti-hepatic fibrosis effect of oxymatrine-baicailin compound （OB） in vitro. Methods The HepG2.2.15 cells and rat HSC-T6 ceils were cell cultured and treated with OB or oxymatrine, respectively. HBsAg and HBeAg contents in HepG2.2.15 cell culture supernatants were detected by en- zyme linked immunoabsorbent assay. Total RNA was extracted from HSC-T6 cells and the gene level of a smooth muscle actin （a-SMA） and collagen I were determined by semi-quantitative PCR. Total cellular proteins extracted from HSC-T6 cells were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein level of a-SMA and collagen I were determined by Western blot. Results OB compound had superior inhibitory effect on hepatitis B viral antigen secretion to oxymatrine （HBsAg;P=0. 043; HBeAg： P=0. 026） in HepG2.2.15 cells. In HSC-T6 cells, the mRNA and protein expression levels of a-SMA in OB group were significantly lower as compared with those in oxymatrine group （mRNA P= 0. 013, protein P= 0. 042, respectively） ; the mRNA and protein expression levels of collagen I ifi OB group were significantly lower as compared with those in oxymatrine group （mRNA P〈0.01, protein P〈0.01, respectively）. Conclusion OB compound has a better inhibitory effect against hepatitis B virus antigen secretion, a-SMA expression and collagen I synthesis in HepG2.2.15 cells and HSC-T6 cells than oxymatrine in vitro.