青蒿琥酯通过增加活性氧簇生成诱导人肝癌HepG2细胞凋亡

Artesunate induces apoptosis of human hepatocellular carcinoma HepG2cells by increasing generation of reactive oxygen species

目的:探讨青蒿琥酯诱导人肝癌Hep G2细胞凋亡的机制及活性氧簇(ROS)在青蒿琥酯诱导Hep G2细胞凋亡中的作用。方法:采用MTT法观查青蒿琥酯对人肝癌Hep G2细胞存活的影响,Hoechst 33258荧光染色法观察细胞凋亡形态的变化,流式细胞术检测Hep G2细胞的凋亡率,DCFH-DA检测细胞凋亡过程中ROS的变化。Western blot检测细胞内凋亡相关蛋白Bax、Bcl-2、cleaved caspase-3和细胞色素C(Cyt C)蛋白水平的变化。采用NADPH氧化酶抑制剂夹竹桃麻素(apocynin)预处理Hep G2细胞,Western blot检测NADPH氧化酶亚基p47^(phox)和p22^(phox)蛋白表达水平,流式细胞术检测ROS变化。结果:与对照组相比,青蒿琥酯作用于Hep G2细胞24 h后,细胞存活率明显减少(P<0.05);细胞核呈致密浓染色,细胞凋亡比例升高(P<0.05);ROS明显升高(P<0.05);Western blot结果显示,青蒿琥酯作用后细胞内Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,cleaved caspase-3和Cyt C蛋白水平升高。Apocynin预处理能降低青蒿琥酯给药组细胞内p47^(phox)和p22^(phox)蛋白表达及ROS的生成。结论:青蒿琥酯能诱导Hep G2细胞凋亡,其凋亡过程可能与ROS的生成增加相关。

AIM:To investigate the effect of reactive oxygen species(ROS)on the apoptosis of HepG2cells induced by artesunate.METHODS:The effect of artesunate on the viability of HepG2cells was measured by MTT assay.The morphological changes of the apoptotic cells were observed by the method of Hoechst33258fluorescence staining.The apoptosis of HepG2cells was analyzed by flow cytometry.DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis.The protein levels of Bax,Bcl-2,cleaved caspase-3and cytochrome C(Cyt C)were determined by Western blot.HepG2cells were pretreated with apocynin and then Western blot was used to detect the expression of p47phox and p22phox,and ROS changes were analyzed by flow cytometry.RESULTS:Compare with control group,the cell viability was obviously inhibited after treatment with artesunate for24h(P<0.05).The nuclei were densely stained,and the proportion of apoptotic cells was increased(P<0.05).ROS was increased significantly(P<0.05).The results of Western blot demonstrated that the expression level of Bax was increased,Bcl-2was decreased,the ratio of Bax/Bcl-2was increased,and the protein levels of cleaved caspase-3and Cyt C were increased.Pretreatment with apocynin reduced the expression of p47phox and p22phox and the generation of ROS in the artesunate treatment group.CONCLUSION:Artesunate induces the apoptosis of HepG2cells.The possible mechanism may be related to the increase in the generation of ROS.