雷公藤多苷促进斑马鱼肝细胞凋亡的实验研究

An experimental study on promotion of hepatocyte apoptosis by tripterygium glycosides in zebrafish

目的:考察雷公藤多苷对斑马鱼肝脏损伤的表现及对肝细胞凋亡的影响。方法:采用斑马鱼为研究对象,考察不同浓度、不同给药作用时间下雷公藤多苷对斑马鱼肝脏大小、肝脏不透光度、卵黄囊面积的影响;AO荧光染色考察各实验组斑马鱼肝细胞凋亡情况;ELISA法考察肝脏组织中与凋亡相关酶Caspase-3和Caspase-9活性的变化;QRT-PCR考察凋亡相关蛋白Bax和Bcl-2的改变。结果:雷公藤多苷对斑马鱼的MNLC为383 g·mL^(-1),LC_(10)为417 g·mL^(-1)。雷公藤多苷在剂量为383,417 g·mL^(-1)时给药24～48 h即可显著性增加斑马鱼的卵黄囊面积。雷公藤多苷在剂量为42.5,128,383 g·mL^(-1)时连续给药48 h后可观察到斑马鱼肝细胞凋亡明显增加,Caspase-3活性增加,促凋亡分子Bax的表达量显著增加;但Caspase-9的活性以及Bcl-2的表达未见明显改变。结论:雷公藤多苷对斑马鱼的肝脏损伤主要表现为增加肝脏面积和延迟卵黄囊的吸收。它可通过激活Caspase-3,促进促凋亡蛋白Bax的表达的途径诱导肝细胞过度凋亡。

OBJECTIVE To study the effects of tripterygium glycosides(TG)on liver injury and hepatocyte apoptosis in zebra fish.METHODS Effects of TG on liver size and light obscuration and yolk sac area were examined at different concentrations and under different action time of TG,by utilizing zebrafish as the study object.Hepatocyte apoptosis caused by TG in each experimental group was assessed with AO fluorescence staining;the enzymic activity of Caspase-3 and Caspase-9 was detected with ELISA;and the expression of Bax and Bcl-2 was measured with QRT-PCR.RESULTS The MNLC of TG in zebra fish was 383 g·mL-1,and its LC10 was 417 g·mL-1.Yolk sac area was obviously increased 24-48 hours after administration at the doses of 383 and 417 g·mL-1.Hepatocyte apoptosis,Caspase-3 activity and expression of the apoptosis promoting molecule Bax were increased notably in the TG groups at the doses of 42.5,128 and 383 g·mL-1,respectively,but Caspase-9 activity and the expression of Bcl-2 had no distinct change in all the TG treatment groups.CONCLUSION Liver injury after treatment with TG is manifested mainly as delayed the yolk sac absorption and increased liver size.TG has the ability to induce excessive apoptosis of liver cells by activating Caspase-3 and promoting expression of the pro-apoptotic protein Bax.