雷公藤红素对肝内胆管癌细胞凋亡的影响

Effects of celastrol on apoptosis of intrahepatic cholangiocarcinoma cells

目的研究雷公藤红素诱导人肝内胆管癌细胞RBE凋亡的作用。方法将细胞分为对照组和低、中、高剂量实验组。对照组常规培养,低、中、高3个剂量实验组分别给予0.75,1.5,3μmol·L^(-1)雷公藤红素。用流式细胞术检测雷公藤红素对细胞凋亡的影响;Hoechst33342实验观察雷公藤红素诱导细胞凋亡的形态变化;以蛋白质印迹法检测雷公藤红素对降解的聚腺苷二磷酸核糖聚合酶(cleaved PARP)、胱天蛋白酶3(caspase3)、裂解的胱天蛋白酶3(cleaved caspase3)、胱天蛋白酶9(caspase9)、裂解的胱天蛋白酶9(cleaved caspase9)、B淋巴细胞瘤-2(Bcl-2)和存活蛋白(Survivin)表达影响。结果对照组和低、中、高剂量实验组的总凋亡率分别为(14.29±0.68)%,(17.11±0.75)%,(19.27±1.02)%,(27.28±1.64)%,细胞凋亡率随着雷公藤红素浓度的增加而升高。与对照组相比,中、高剂量实验组凋亡相关蛋白caspase3、caspase9的表达水平降低(P<0.05或P<0.01),cleaved PARP、cleaved caspase3、cleaved caspase9的表达水平升高(P<0.01),抗凋亡蛋白Bcl-2和Survivin表达水平降低(P<0.01)。结论雷公藤红素能诱导肝内胆管癌细胞的凋亡,其作用机制可能与抑制Bcl-2和Survivin表达,促进caspase3和caspase9的活化有关。

Objective To study the effect of celastrol on apoptosis of human intrahepatic cholangiocarcinoma cells RBE.Methods The cells were divided into control group and low,medium,high dose experimental groups.Control group was routinely cultured;low,medium,high dose experimental groups were given 0.75,1.5,3μmol·L^(-1)celastrol,respectively.The effect of celastrol on apoptosis was detected by flow cytometry.Hoechst33342 was used to observe the morphological changes of apoptosis induced by celastrol.Western Blotting experiment was used to detect the effect of celastrol on expression of apoptotic proteins cleaved poly-ADP-ribose polymerase(cleaved PARP),cysteine aspartic acid specific protease(caspase)3,cleaved caspase3,caspase9,cleaved caspase9,B cell lymphoma-2(Bcl-2)and Survivin.Results The total apoptosis rate of control group and low,medium,high dose experimental groups were(14.29±0.68)%,(17.11±0.75)%,(19.27±1.02)%,(27.28±1.64)%,respectively.The apoptosis rate increased with the increase of celastrol concentration.Compared with control group,the expression levels of apoptotic proteins caspase3 and caspase9 in medium,high dose experimental groups were decreased(P<0.05,P<0.01);expression levels of cleaved PARP,caspase3,and caspase9 were increased(P<0.01),and expression levels of anti-apoptotic proteins,Bcl-2 and Survivin were decreased(P<0.01).Conclusion Celastrol can induce the apoptosis of intrahepatic cholangiocarcinoma cells,and its mechanism may be related to inhibiting the expression of Bcl-2 and Survivin and promoting the activation of caspase3 and caspase9.