目的 通过体外实验评价板蓝根Isatidis Radix及活性成分4-羟基-1H-吲哚-3-甲醛对乙型肝炎病毒（hepatitis B virus，HBV）的抑制作用，并探索其基于调控视黄酸诱导基因蛋白I-线粒体抗病毒信号蛋白（retinoic acid-inducible gene I-mitochondria antiviral signaling protein，RIG-I-MAVS）信号通路的抗病毒机制。方法 采用CCK-8方法在HBV稳定复制细胞系HepG2.2.15细胞分析板蓝根毒性，选择安全药物浓度进行抗HBV实验研究，计算药物对HBV DNA、乙型肝炎E抗原（HBeAg）、乙型肝炎表面抗原（HBsAg）的抑制率，评价板蓝根抗病毒药效；采用Western blotting方法检测板蓝根对宿主免疫相关RIG-I-MAVS信号通路中关键信号蛋白RIG-I、MAVS、干扰素调节因子7（interferon regulated-factor 7，IRF7）、磷酸化IRF7（phosphorylated IRF7，p-IRF7）表达水平的影响。基于TCMSP数据库检索板蓝根中的活性成分，使用Schr?oumldinger软件将板蓝根活性成分与RIG-I、MAVS、IRF7蛋白进行分子对接，初步筛选板蓝根中的抗HBV潜在活性成分，选定分子对接结果中与靶标蛋白结合能力较强的活性成分进行抗HBV活性评价并对其作用机制进行探索。结果 板蓝根对HepG2.2.15细胞中HBV DNA、HBeAg、HBsAg均具有明显的抑制作用（P＜0.05、0.01、0.001），抑制率分别为54.67%、23.29%、66.62%；板蓝根可上调RIG-I、MAVS、IRF7、p-IRF7蛋白的表达，对RIG-I的上调作用最为显著（P＜0.001）；分子对接结果显示，4-羟基-1H-吲哚-3-甲醛与RIG-I、MAVS、IRF7蛋白具有较强的亲和力，结合能分别为—5.218、—6.525、—6.813 kJ/mol；4-羟基-1H-吲哚-3-甲醛对HepG2.2.15细胞的HBV DNA、HBeAg、HBsAg的抑制率分别为33.87%、28.14%、46.14%，可促进RIG-I、MAVS、IRF7、p-IRF7蛋白的表达。结论 板蓝根及活性成分4-羟基-1H-吲哚-3-甲醛均可抑制HBV DNA、HBeAg、HBsAg的分泌，可能通过上调RIG-I-MAVS信号通路发挥抗HBV作用。

Objective To evaluate the inhibitory effect of Banlangen (Isatidis Radix) and its active component 4-hydroxy-1H-indole-3-carbaldehyde on hepatitis B virus (HBV) by in vitro experiments, and explore the antiviral mechanism based on regulation of retinoic acid-inducible gene I-mitochondria antiviral signaling protein (RIG-I-MAVS) signaling pathway. Methods The toxicity of Isatidi Radixs was analyzed in HBV stably-replicating cell line HepG2.2.15 cells by CCK-8 method, and anti-HBV experimental study was conducted under the safe drug concentrations. The inhibition rate of drugs on HBV DNA, HBeAg and HBsAg was calculated, and the antiviral efficacy of Isatidi Radixs was evaluated. Western blotting was used to detect the effects of Isatidi Radixs on expressions of RIG-I, MAVS, interferon regulated-factor 7 (IRF7) and phosphorylated IRF7 (p-IRF7), which were the key signaling proteins in host immune-related RIG-I-MAVS signaling pathway. Active compounds in Isatidi Radixs were retrieved based on TCMSP database, and were molecular docked with RIG-I, MAVS and IRF7 proteins using Schr?oumldinger software, so as to screen the potential anti-HBV active compounds in Isatidi Radixs. The active components with strong binding ability to target proteins in molecular docking results were selected to evaluate their anti-HBV activity and their mechanism were explored. Results Isatidi Radixs could significantly inhibit HBV DNA, HBeAg and HBsAg levels in HepG2.2.15 cells with inhibition rates of 54.67%, 23.29% and 66.62%, respectively. Isatidi Radixs could up-regulate RIG-I, MAVS, IRF7 and p-IRF7 protein expressions, and the up-regulation of RIG-I was the most significant (P＜0.001). Molecular docking results showed that 4-hydroxy-1H-indole-3-carbaldehyde had a strong affinity with RIG-I, MAVS and IRF7 proteins, and the binding energies were—5.218,—6.525 and—6.813 kJ/mol, respectively. The inhibition rates of 4-hydroxy-1H-indole-3-carbaldehyde on HBV DNA, HBeAg and HBsAg of HepG2.2.15 cells were 33.87%, 28.14%, 46.14%, respectively, which could promote RIG-I, MAVS, IRF7 and p-IRF7 protein expression.Conclusion Isatidi Radixs and its active ingredient 4-hydroxy-1H-indole-3-carbaldehyde could inhibit HBV DNA, HBeAg and HBsAg levels, suggesting that they play anti-HBV roles by up-regulating RIG-I-MAVS signaling pathway.

R285.5

国家自然科学基金重点项目（81930110）；国家自然科学基金创新群体项目（81721002）；河南省中医药科学研究专项（2023ZY3040）