盐酸哈尔酚骆驼蓬碱对人肝癌细胞生长的影响与自噬诱导作用的研究

Effects of harmol hydrochloride dihydrateon the growth of human hepatoma cells and study on the activation of autophagy

目的探讨低剂量盐酸哈尔酚骆驼蓬碱(harmol hydrochloride dihydrate,HHD)对肝癌细胞株HepG2生长的影响及其自噬诱导作用。方法用不同浓度的HHD作用于肝癌细胞株HepG224 h后四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测细胞活力,用流式细胞仪检测细胞凋亡,用Hoechst和PI双重染色方法观察细胞凋亡,用蛋白质印迹(western blotting,WB)的方法检测LC-3Ⅱ表达水平。结果MTT检测结果显示不同浓度的HHD作用于HepG2细胞24 h后,细胞活力随HHD浓度升高而降低,差异均有统计学意义(P<0.05)。0、0.005、0.01 mg/mL HHD作用于HepG2细胞24 h后,细胞凋亡率均明显高于空白对照组(P<0.05)。0.01 mg/mL的HHD处理HepG2细胞24 h后,凋亡小体的形成明显增多(P<0.05)。Western blotting法检测发现0.01 mg/mL HHD作用细胞24 h后自噬标记蛋白LC-3Ⅱ表达显著升高。结论HHD抑制肝癌细胞株HepG2在体外生长,其诱导细胞自噬可能是抑制作用机制之一。

Objective To investigate the effect of harmol hydrochloride dihydrate(HHD)on the growth of human hepatoma cells HepG2 and its mechanism of inducing autophagy.Methods Different concentrations of HHD were treated to HepG2 cells for 24 hrs.Cell viability was measured by MTT assay and apoptosis was detected by flow cytometry.Hoechst and PI were used to observe apoptosis by double staining and LC3-II expression level was detected by Western blotting.Results After treated with different concentrations of HHD in HepG2 cells for 24hrs,MTT assay results showed that cell viability decreased with increasing of the drug concentration(P<0.05).After treated with 0,0.005,0.01 mg/mL HHD in HepG2 cells,the apoptosis rate was significantly higher when compared with the control group(P<0.05).When treated with 0.01 mg/mL HHD of in HepG2 cells,it can significantly increase the formation of apoptotic bodies(P<0.05).Western blotting showed that the expression of autophagy marker protein LC3-II was significantly increased in HepG2 cells when treated with 0.01 mg/mL HHD for 24 hrs.Conclusion HHD can inhibit the growth of human hepatoma cell HepG2 in vitro and its induction of autophagy in cells may be one of the inhibitory mechanisms.