黄芪甲苷通过调控宿主核糖体翻译开关抑制HBV复制的机制

Mechanism of astragaloside IV inhibits hepatitis B virus replication by regulating host ribosome translation process

目的分析黄芪甲苷抑制HBV复制的宿主调控机制。方法使用不同浓度的黄芪甲苷处理人正常肝细胞(L-02),根据黄芪甲苷浓度的不同,分为0、5、10和20μg/mL 4组。应用CCK-8检测细胞活性,流式细胞法检测细胞凋亡,化学发光和生化方法检测AFP、ALT、AST和ALP外泌水平,评估黄芪甲苷对正常细胞的影响。用黄芪甲苷处理携带HBV的肝癌细胞Hep3B,应用qPCR方法检测HBV DNA、pgRNA、MTIF2、RPL10基因表达量,ELISA测法检测HBsAg和HBeAg,评估对HBV复制的影响。应用TCGA和GEO数据库结合R语言资料包对RPL10和MTIF2在临床样本中的预后影响进行分析验证。绘制Kaplan-Meier生存曲线,log-rank检验用于分析比较两组或多组之间的生存差异,进行了time ROC分析以比较RPL10和MTIF2基因的预测准确性和风险评分。计量资料多组间比较和同一组内不同时间段比较均采用单因素方差分析,进一步分析两两组间比较采用Bonferroni方法。结果20μg/mL组处理24 h和48 h相较未处理组细胞生长活性均显著提高(P值均<0.05),20μg/mL组处理72 h相较10μg/mL组生长活性提高(P<0.05),5μg/mL组处理72 h相较未处理组生长活性提高(P<0.05)。5、10和20μg/mL 3个处理组AFP水平均较未处理组显著增高(P值均<0.05),10和20μg/mL 2个处理组ALT水平分别均较未处理和5μg/mL 2组显著降低(P值均<0.05),20μg/mL组ALT水平较10μg/mL组显著降低(P<0.05)。5、10和20μg/mL 3个处理组AST水平较未处理组均显著增高(P值均<0.05)。5、10和20μg/mL 3个处理组HBV DNA、pgRNA、HBsAg、HBeAg、RPL10和MTIF2水平与未处理组比较差异均有统计学意义(P值均<0.05)。生物信息学分析结果显示,HBV感染的肝癌患者中RPL10和MTIF2基因较高水平表达的患者预后较差,而在无HBV感染的肝癌患者中不存在此现象。结论黄芪甲苷能够抑制翻译起始蛋白MTIF2和核糖体大亚基成分RPL10,通过调控宿主核糖体翻译开关降低HBV复制。

Objective To investigate the host regulatory mechanism of astragaloside IV in inhibiting hepatitis B virus(HBV)replication.Methods Normal human hepatocytes L-02 were treated with different concentrations of astragaloside IV,and according to the concentration of astragaloside IV,the cells were divided into 0,5,10,and 20μg/mL groups.CCK-8 assay was used to measure cell viability,flow cytometry was used to measure cell apoptosis,and chemiluminescence and biochemical methods were used to measure the levels of alpha-fetoprotein(AFP),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and alkaline phosphatase(ALP),so as to evaluate the influence of astragaloside IV on normal cells.Hepatoma cells Hep3B carrying HBV were treated with astragaloside IV;quantitative PCR was used to measure the mRNA expression levels of HBV DNA,pgRNA,MTIF2,and RPL10,and ELISA was used to measure the levels of HBsAg and HBeAg,so as to evaluate the effect of astragaloside IV on HBV replication.TCGA and GEO databases combined with R language package were used to analyze the prognostic effect of RPL10 and MTIF2 in clinical samples.The Kaplan-Meier method was used for survival analysis,and the log-rank test was used for comparison of survival between two or multiple groups;the time-dependent ROC curve analysis was performed to compare the predictive accuracy and risk score of RPL10 and MTIF2 genes.A one-way analysis of variance was used for comparison of continuous data between multiple groups and within each group at different time points,and the Bonferroni method was used for further comparison between two groups.Results Compared with the untreated group,the 20μg/mL group had a significant increase in cell growth activity at 24 and 48 hours of treatment(both P<0.05);compared with the 10μg/mL group,the 20μg/mL group had a significant increase in cell growth activity at 72 hours of treatment(P<0.05);compared with the untreated group,the 5μg/mL group had a significant increase in cell growth activity at 72 hours of treatment(P<0.05).Compared with the untreated group,the 5,10,and 20μg/mL groups had a significant increase in AFP(all P<0.05);compared with the untreated group and the 5μg/mL group,the 10 and 20μg/mL groups had a significant reduction in ALT(all P<0.05),and compared with the 10μg/mL group,the 20μg/mL group had a significant reduction in ALT(P<0.05).Compared with the untreated group,the 5,10,and 20μg/mL groups had a significant increase in AST(all P<0.05).There were significant differences in the levels of HBV DNA,pgRNA,HBsAg,HBeAg,RPL10,and MTIF2 between the 5/10/20μg/mL groups and the untreated group(all P<0.05).The bioinformatics analysis showed that among the liver cancer patients with HBV infection,the patients with high mRNA expression levels of RPL10 and MTIF2 genes tended to have a poor prognosis,while this phenomenon was not observed in liver cancer patients without HBV infection.Conclusion Astragaloside IV can inhibit the translation initiation factor MTIF2 and the large ribosomal subunit RPL10 and reduce HBV replication by regulating the initiation of host ribosome translation.