基于斑马鱼模型的杠柳毒苷肝毒性评价

Evaluation of hepatotoxicity of periplocin based on zebrafish model

目的基于斑马鱼模型初步探讨杠柳毒苷的毒性靶器官和潜在作用机制。方法将斑马鱼(4dayspostfertilization,4 dpf)暴露于不同浓度(低浓度1μg·mL^(-1),高浓度1.5μg·mL^(-1))的杠柳毒苷溶液中24 h,统计杠柳毒苷对斑马鱼的致死率;10%致死剂量下(<LC10)暴露24 h后,基于肝脏面积变化,吖啶橙染色,肝脏组织病理切片,谷丙转氨酶(ALT)与谷草转氨酶(AST)活性变化等指标评价杠柳毒苷的肝毒性;通过网络药理学与分子对接技术预测杠柳毒苷肝毒性潜在作用机制,并通过实时荧光定量PCR(real-time PCR,RT-PCR)对预测靶点进行验证。结果杠柳毒苷对斑马鱼的亚致死浓度(LC10)为1.6126μg·mL^(-1),亚致死浓度暴露条件下,与空白组相比,杠柳毒苷能够引起斑马鱼肝脏面积减小和明显的肝脏细胞凋亡,组织病理切片结果显示杠柳毒苷能够诱导肝细胞出现结构排列松散紊乱以及明显空泡化等病理特征。此外,杠柳毒苷能够显著升高斑马鱼ALT、AST活性,证明杠柳毒苷对斑马鱼模型有显著的肝毒性;网络药理学结果表明杠柳毒苷诱导肝毒性与21个潜在作用靶点相关,PPI蛋白互作网络分析排名前5位的靶点为信号转导与转录激活因子3(STAT3)、热休克蛋白(HSP)90AA1、缺氧诱导因子1A(HIF1A)、一氧化氮合酶3(NOS3)、雷帕霉素靶蛋白(MTOR),KEGG结果显示杠柳毒苷肝毒性可能与缺氧诱导因子-1(HIF-1)信号通路、磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路、钙(Calcium)信号通路等信号通路相关,RT-PCR进一步验证以上预测结果。结论杠柳毒苷可能是通过影响HIF-1信号通路与PI3K-Akt信号通路,进而引发肝毒性。

Objective To explore the toxic target organs and potential mechanism of periplocin based on a zebrafish model.Methods Zebrafish(4 days post fertilization,4 dpf)were exposed to different concentrations of periplocin solution for 24 h(the low concentration was 1μg·mL^(-1)and the high concentration was 1.5μg·mL^(-1)),and the fatality rate of periplocin for zebrafish was calculated.After twenty-four hours of exposure to a sub-lethal dose,the hepatotoxicity of periplocin was evaluated based on changes of areas of the liver,aeridine orange staining,liver histopathologic sections,and changes in alanine aminotransferase(ALT)and aspartic aminotransferase(AST)activities.Furthermore,the potential mechanism of periplocin hepatotoxicity was predicted via network pharmacology and molecular docking techniques before being verified by real-time PCR.Results The sublethal dose of periplocin for zebrafish was 1.6126μg·mL^(-1).Under sub-lethal dose exposure,periplocin treatment could result in reduced liver areas of zebrafish and obvious apoptosis in the liver compared with the control group.Histopathological section results showed that periplocin exposure induced loose and disordered arrangement of liver cells as well as obvious vacuoles.In addition,periplocin could significantly increase the activities of ALT and AST in zebrafish,which proved the significant hepatotoxicity of periplocin in zebrafish.Network pharmacology indicated that periplocin-induced hepatictoxicity was related to the 21 potential targets and the top five targets of PPI protein interaction network were STAT3,HSP90AA1,HIF1A,NOS3 and MTOR.KEGG results showed that periplocin hepatotoxicity might be related to HIF-1 signaling pathway,PI3K/Akt signaling pathway,and calcium signaling pathway,as was confirmed by RT-PCR.Conclusion Periplocin-induced hepatotoxicity is associated with the disturbance of HIF-1 and PI3K/Akt signaling pathways.