Infectious Disease Department of the First Affiliated Hospital of Xinxiang Medical University, Weihui, 453100, China.
Gastroenterology Department of the First Affiliated Hospital of Xinxiang Medical University, Weihui, 453100, China.
J Ethnopharmacol. 2020 Jul 15;257:112782. doi: 10.1016/j.jep.2020.112782. Epub 2020 Mar 23.
Hepatitis B virus (HBV) infection frequently results in both acute and chronic hepatitis and poses serious threats to human health worldwide. Despite the availability of effective HBV vaccine and anti-HBV drugs, apparently inevitable side effects and resistance have limited its efficiency, thus prompt the search for new anti-HBV agents. The traditional Chinese medicine Radix Isatidis has been used for thousands of years, mainly for the treatment of viral and bacterial infection diseases including hepatitis.
In this study, antiviral activities of a Radix Isatidis (Isatis indigotica Fortune) polysaccharide (RIP) were evaluated in vitro model using the HepG2.2.15 cell line and the underlying mechanism was elucidated with the aim of developing a novel anti-HBV therapeutic agent.
Structure features of the purified polysaccharide RIP were investigated by a combination of chemical and instrumental analysis. Drug cytotoxicity was assessed using the MTT assay. The contents of HBsAg, HBeAg, intracellular and extracellular IFN-α level were measured using respective commercially available ELISA kit. The HBV DNA expression was evaluated by real-time quantitative polymerase chain reaction (PCR) and the relevant proteins involved in TFN/JAK/STAT signaling pathways were examined by western blot assay.
MTT assay showed that RIP had no toxicity on HepG2.2.15 cell line below the concentration 400 μg/ml at Day 3, 6 and 9. Furthermore, RIP at the concentration of 50, 100 and 200 μg/ml significantly reduced extracellular and intracellular level of HBsAg, HBeAg and HBV DNA in HepG2.2.15 cells in a time and dose-dependent manner. Moreover, RIP also enhanced the production of IFN-α in HepG2.2.15 cell via activation of JAK/STAT signal pathway and induction of antiviral proteins, as evidenced by the increased protein expression of p-STAT-1, p-STAT-2, p-JAK1, p-TYK2, OAS1, and Mx in HepG2.2.15 cells. In addition, the over expression of SOCS-1 and SOCS-3 was significantly abolished under same conditions.
These results suggested that the HBV inhibitory effect of RIP was possibly due to the activation of IFN-α-dependent JAK/STAT signal pathway and induction of the anti-HBV protein expression.
ETHNOPHARMACOLOGICAL 相关性：乙型肝炎病毒（HBV）感染常导致急性和慢性肝炎，对全球人类健康构成严重威胁。尽管有有效的 HBV 疫苗和抗 HBV 药物，但明显不可避免的副作用和耐药性限制了其效率，因此促使人们寻找新的抗 HBV 药物。中药板蓝根已使用了数千年，主要用于治疗病毒性和细菌性感染疾病，包括肝炎。
本研究通过 HepG2.2.15 细胞系体外模型评价板蓝根多糖（RIP）的抗病毒活性，并阐明其作用机制，旨在开发新型抗 HBV 治疗药物。
采用化学和仪器分析相结合的方法研究纯化多糖 RIP 的结构特征。采用 MTT 法测定药物细胞毒性。采用相应的商业 ELISA 试剂盒测定 HBsAg、HBeAg、细胞内和细胞外 IFN-α水平。采用实时定量聚合酶链反应（PCR）评价 HBV DNA 表达，采用 Western blot 法检测 TFN/JAK/STAT 信号通路相关蛋白。
MTT 法显示，RIP 在第 3、6 和 9 天，浓度低于 400μg/ml 时对 HepG2.2.15 细胞无毒性。此外，RIP 浓度为 50、100 和 200μg/ml 时，可显著降低 HepG2.2.15 细胞中外源和内源 HBsAg、HBeAg 和 HBV DNA 的水平，呈时间和剂量依赖性。此外，RIP 还通过激活 JAK/STAT 信号通路和诱导抗病毒蛋白，增强 HepG2.2.15 细胞 IFN-α的产生，这可通过 HepG2.2.15 细胞中 p-STAT-1、p-STAT-2、p-JAK1、p-TYK2、OAS1 和 Mx 蛋白表达的增加来证明。此外，在相同条件下，SOCS-1 和 SOCS-3 的过度表达明显被消除。
这些结果表明，RIP 的 HBV 抑制作用可能是由于激活了 IFN-α 依赖性 JAK/STAT 信号通路和诱导了抗 HBV 蛋白的表达。
