七氟醚通过mTOR信号通路诱导人神经母细胞瘤细胞自噬的发生

Sevoflurane induces autophagy in human neuroblastoma cells via mTOR signaling pathway

目的:探讨七氟醚对人神经母细胞瘤细胞(SHSY-5Y细胞)自噬的影响及mTOR信号通路在其中的作用。方法:将培养的SHSY-5Y细胞随机分为4组,空白对照组(C组)、空白对照+雷帕霉素50 nmol/L组(C+R组)、4.8%七氟醚12 h组(S组)和4.8%七氟醚12 h+雷帕霉素50 nmol/L组(S+R组)。各组分别于处理结束时采用Western blot法测定各组自噬相关蛋白LC3-Ⅱ、LC3-Ⅰ、Atg5以及p-mTOR、t-mTOR、β-actin的表达水平;利用mRFP-EGFP-LC3质粒转染,共聚焦显微镜观察各组细胞内自噬小体以及自噬溶酶体的数量,从而判断七氟醚对SHSY-5Y细胞自噬流的影响及其具体机制。结果:与C组比较,C+R组、S组和S+R组自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值、Atg5表达水平均增高(P<0.05);与C+R组和S组比较,S+R组LC3-Ⅱ/LC3-Ⅰ比值、Atg5表达水平也增高(P<0.01)。与C组比较,C+R组、S组和S+R组p-mTOR/t-mTOR比值均降低(P<0.001);与C+R组和S组比较,S+R组p-mTOR/t-mTOR比值也降低(P<0.001)。与C组比较,C+R组、S组和S+R组自噬小体、自噬溶酶体数量均增加(P<0.001);与C+R组和S组比较,S+R组自噬小体、自噬溶酶体数量也增加(P<0.001)。结论:吸入麻醉药七氟醚通过mTOR信号通路诱导SHSY-5Y细胞自噬的发生。

Objective:To investigate the effect of sevoflurane on autophagy in human neuroblastoma SHSY-5Y cells and the role of mTOR signaling pathway in autophagy in human neuroblastoma cells.Methods:The cultured SHSY-5Y cells were randomized to blank control group(group C),blank control+rapamycin 50 nmol/L group(group C+R),4.8%sevoflurane 12 h group(group S),and 4.8%sevoflurane 12 h+rapamycin 50 nmol/L group(group S+R).Autophagy-related protein LC3-Ⅱ,LC3-Ⅰand Atg5 expression,p-mTOR and t-mTOR expression as well asβ-actin level were measured by Western blotting at the end of treatment.The effect and mechanism of sevoflurane on the autophagy flux of SHSY-5Y cells were measured through transfection of the mRFP-EGFP-LC3 plasmid in each group.The number of autophagosomes and autophagolysosomes were observed by confocal microscope in 40 cells of each group at the end of treatment.Results:Compared with group C,the expression of autophagy-related proteins LC3-Ⅱ/LC3-Ⅰand Atg5 was enhanced(P<0.05),the ratio of p-mTOR/t-mTOR was decreased(P<0.001),and the number of autophagosomes and autophagolysosomes were grown(P<0.001)in group C+R and group S.When compared with group C+R or group S,the ratio of LC3-Ⅱ/LC3-Ⅰand Atg5 expression were further increased(P<0.01),whereas the ratio of p-mTOR/t-mTOR tended to decrease in group S+R(P<0.001).The number of autophagosomes and autophagolysosomes were significantly increased in group S+R(P<0.001).Conclusion:Inhalation of anesthetic sevoflurane can induce autophagy in SHSY-5Y cells through the mTOR signaling pathway.