喹啉酸对PC12细胞自噬发生及相关信号通路蛋白表达的影响

Effects of quinolinic acid on autophagy and protein expressions of related signaling pathway in PC12 cells

目的探讨喹啉酸(QA)是否引起大鼠肾上腺嗜铬细胞瘤PC12细胞自噬的发生及其与糖原合酶激酶-3β(GSK-3β)/β-联蛋白相关信号通路的关系。方法用不同浓度QA(2.5,5和10 mmol·L^(-1))处理PC12细胞24 h,MTT法测定细胞存活,免疫荧光法观察微管相关蛋白1轻链3(LC3)荧光斑点标记的自噬体形成,Western蛋白印迹法检测细胞GSK-3β、β-联蛋白、LC3和Beclin 1的表达。结果 QA作用24 h,可浓度依赖性地降低PC12细胞存活率,IC_(50)为8.7 mmol·L^(-1)。QA作用24 h,与正常对照组比较,PC12细胞内LC3荧光斑点标记的自噬体增多,自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的比值和Beclin 1表达增高(P<0.01);同时PC12细胞内GSK-3β的表达增加(P<0.05,P<0.01),β-联蛋白的表达降低(P<0.05,P<0.01)。结论 QA可诱导PC12细胞自噬的发生,其机制可能与GSK-3β/β-联蛋白相关的信号通路激活有关。

OBJECTIVE To investigate whether quinolinic acid（QA） induces autophagy in PC12 cells and its relationship with glycogen synthase kinase-3β（GSK-3β）/β-catenin related signaling pathways.METHODS PC12 cells were treated with QA 2.5,5.0 and 10.0 mmol·L^（-1） for 24 h.The cell viability was determined by MTT assay.Autophagy fluorescent spots labelled form of microtubule-associated protein 1 light chain 3（LC3） was examined by LC3 immunostaining.The expressions of GSK-3β,β-catenin,LC3 and Beclin 1 were determined by Western blotting.RESULTS QA inhibited PC12 cell survival in a concentration-dependent manner,and IC_（50） was 8.7 mmol·L^（-1）.Compared with normal control group,QA 2.5,5.0 and 10.0 mmol·L^（-1） increased autophagic intracellular LC3 fluorescence spots,elevated the expression ratio of LC3- Ⅱ/LC3- Ⅰ and expression of Beclin 1 in PC12 cells（P〈0.05）.In addition,QA enhanced GSK-3β expression and decreased β-catenin expression（P〈0.05,P〈0.01）.CONCLUSION QA induces autophagy in PC12 cells.This mechanism may be associated with the activation of GSK-3β/β-catenin related signaling pathways.