花生四烯酸对TNF-α/放线菌素D诱导内皮细胞凋亡的影响及作用机制

Effect of arachidonic acid on apoptosis of endothelial cells induced by TNF-α and ActD

目的探讨花生四烯酸对TNF-α/放线菌素D(Act D)诱导内皮细胞凋亡的影响及作用机制。方法将体外培养的人脐静脉内皮细胞随机分成空白组、模型组、环氧二十碳四烯酸(EET)组及EET+3-甲基腺嘌呤(3MA)组。空白组常规培养;后三组均加入TNF-α(10 ng/m L)和放线菌素D(Act D,5 ng/m L)诱导凋亡,EET组同时加入14,15-EET(100 nmol/L),EET+3MA组同时加入14,15-EET(100 nmol/L)、3MA(2 mmol/L)。各组培养24 h时采用流式细胞仪检测细胞凋亡率;采用Western blotting法检测微管相关蛋白轻链3-Ⅰ(LC3-Ⅰ)及微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ),计算LC3-Ⅱ/LC3-Ⅰ;采用ELISA法检测Caspase-8、Caspase-9活性。结果空白组、EET组及EET+3MA组细胞凋亡率均较模型组减少(P均<0.01),EET+3MA组细胞凋亡率较EET组增加(P<0.05)。EET组及EET+3MA组LC3-Ⅱ/LC3-Ⅰ较模型组、空白组增加(P<0.05或<0.01);EET+3MA组LC3-Ⅱ/LC3-Ⅰ较EET组降低(P<0.01)。模型组、EET组、EET+3MA组Caspase-8、Caspase-9活性均较空白组升高,模型组均较EET+3MA组、EET组升高,EET+3MA组较EET组升高,组间比较P<0.05或<0.01。结论花生四烯酸可抑制TNF-α/Act D诱导的内皮细胞凋亡;通过抑制Caspase-8、Caspase-9活性诱导自噬可能是其作用机制。

Objective To investigate the effect and mechanism of arachidonic acid on the apoptosis of endothelial cells induced by TNF-αand actinomycin D(ActD).Methods The human umbilical vein endothelial cells cultured in vitro were randomly divided into the blank group,model group,EET group,and EET+3MA group.The blank group was cultured normally.In the latter three groups,TNF-α(10 ng/mL)and ActD(5 ng/mL)were added to induce apoptosis,meanwhile,the EET group was added with 14,15-epoxyeicosatrienoic acids(EET)(100 nmol/L),and the EET+3MA group was added with 14 15-EET(100 nmol/L)and 3-methyl adenine(3-MA)(2 mmol/L).The apoptosis rate was detected by flow cytometry at 24 h.Western blotting was used to detect the microtubule-related protein light chain 3-Ⅰ(LC3-Ⅰ)and 3-Ⅱ(LC3-Ⅱ),and the LC3-Ⅱ/LC3-Ⅰwas calculated.The activity of caspase-8 and 9 was detected by ELISA.Results The apoptosis rates in the blank group,EET group,and EET+3MA group were lower than that in the model group(P<0.01),and the apoptosis rate in the EET+3MA group was higher than that in the EET group(P<0.05).The LC3-Ⅱ/LC3-Ⅰratios in the EET group and EET+3MA group were higher than those in the model group and the blank group(P<0.05 or P<0.01).The LC3-Ⅱ/LC3-Ⅰratio in the EET+3MA group was lower than that in the EET group(P<0.01).The activity of caspase-8 and caspase-9 in the model group,EET group,EET+3MA group was higher than that in the blank group,the model group was higher than EET+3MA group and EET group,and EET+3MA group was higher than EET group(P<0.05 or P<0.01).Conclusion Arachidonic acid can inhibit the apoptosis of endothelial cells induced by TNF-α,and the induced autophagy by inhibiting caspase-8 and caspase-9 may be its mechanism.