自噬参与香烟烟雾提取物诱导的肺动脉内皮细胞凋亡

Autophagy is involved in pulmonary artery endothelial cell apoptosis induced by cigarette smoke extract

目的:探讨自噬在香烟烟雾提取物(cigarette smoke extract,CSE)诱导人肺动脉内皮细胞(human pulmonary artery endothelial cells,HPAECs)凋亡中的作用。方法:常规培养HPAECs,分为对照组、CSE组、3-甲基腺嘌呤(3-methyladenine,3-MA)组和3-MA+CSE组,应用Hoechst 33342染色和Annexin V/PI流式细胞术检测细胞凋亡,单丹磺酰尸胺(monodansylcadaverine,MDC)染色观察细胞自噬泡形成,Western bolt测定自噬相关蛋白beclin-1、LC3与cleaved caspase-3的水平。结果:MDC染色示CSE处理可以诱导细胞产生自噬泡,Western blot结果示自噬相关蛋白LC3及beclin-1表达升高,3-MA预处理后抑制上述蛋白的表达。Hoechst 33342染色和Annexin V/PI流式结果显示CSE组细胞凋亡率较对照组明显增加,在3-MA+CSE组,细胞凋亡率较CSE组进一步升高;同时,CSE组细胞cleaved caspase-3蛋白水平较对照组明显升高(P<0.05),3-MA+CSE组的caspase-3表达较CSE组进一步升高。结论:CSE能诱导HPAECs发生自噬和凋亡,抑制自噬能够进一步促进CSE对HPAECs的凋亡作用,这种作用可通过激活caspase-3实现。

AIM： To investigate the role of autophagy in the apoptosis of human pulmonary artery endothelial cells（ HPAECs） induced by cigarette smoke extract（ CSE）. METHODS： HPAECs were cultured routinely. HPAECs were treated with CSE at different concentrations,and the cell viability was detected by MTT assay. HPAECs were divided into control group,CSE group,3-methyladenine（ 3-MA） group and 3-MA＋CSE group. The autophagy was observed under fluorescence microscope with monodansylcadaverine（ MDC） staining. Annexin V/propidium iodide staining and Hoechst 33342 staining were employed to detect apoptosis. In addition,the protein levels of LC3,beclin-1 and cleaved caspase-3 were determined by Western blot. RESULTS： MDC staining showed the increased production of autophagic vacuoles was observed in CSE group. The results of Western blot showed that the expression levels of autophagy-related proteins LC3 and beclin-1 were increased,while 3-MA pretreatment inhibited the expression of these proteins and the production of autophagic vacuoles. Observation with Annexin V/propidium iodide staining and Hoechst 33342 staining showed that the apoptotic rate in CSE group was significantly higher than that in control group,and pretreatment with 3-MA induced further increase in the cell apoptosis. The protein level of cleaved caspase-3 in CSE group was significantly higher than that in control group（ P〈0. 05）,and 3-MA＋CSE treatment induced the further increase in the protein level of cleaved caspase-3. CONCLUSION： CSE induces autophagy and apoptosis in the HPAECs. Inhibition of autophagy promotes the apoptosis induced by CSE in HPAECs,which can be achieved through activation of caspase-3.