姜黄素通过抑制PI3K/Akt/mTOR通路增强自噬保护帕金森病细胞模型的研究

Study on Curcumin Enhances Autophagy by Inhibiting PI3K/Akt/mTOR Pathway to Protect Cell Models of Parkinson’s Disease

目的观察姜黄素对帕金森病(Parkinson’s disease,PD)细胞模型中磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)通路的影响,并探讨其是否通过抑制此通路来增强自噬而发挥神经保护作用。方法采用1-甲基-4-苯基-四氢吡啶离子处理人神经母细胞瘤SH-SY5Y细胞建立PD细胞模型,设立对照组、模型组、姜黄素组、姜黄素+胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)组、姜黄素+LY294002组,分别应用姜黄素、姜黄素联合PI3K通路激活剂IGF-1、姜黄素联合PI3K通路抑制剂LY294002进行干预处理。各组细胞在药物处理24 h后分别于光学显微镜下观察细胞形态,细胞计数试剂盒检测细胞活力,酪氨酸羟化酶免疫荧光染色观察多巴胺(dopamine,DA)能神经元存活数目,Western blotting检测PI3K、磷酸化-Akt(phospho-Akt,p-Akt)、磷酸化-mTOR(phospho-mTOR,p-mTOR)、α-突触核蛋白(α-synuclein,α-Syn)和微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3 beta,LC3B)的蛋白表达。结果与模型组比较,姜黄素组细胞皱缩、空泡变性的状态得到改善,细胞存活率提高(P<0.05),DA能神经元存活数目增加(P<0.01),LC3B-II/LC3B-I比值增加(P<0.05),α-Syn蛋白表达减少(P<0.01),p-Akt、p-mTOR的蛋白表达显著下调(P<0.01)。添加PI3K通路激活剂IGF-1,姜黄素的上述作用基本被抵消;添加PI3K通路抑制剂LY294002后,与单一姜黄素干预比较,虽然LC3B-II/LC3B-I比值进一步增加(P<0.05),但α-Syn蛋白表达增加(P<0.05),DA能神经元存活数减少(P<0.01)。结论在PD细胞模型中姜黄素可抑制PI3K/Akt/mTOR信号通路的活化,从而增强细胞自噬功能,继而促进α-Syn的清除是其发挥神经保护作用的主要机制之一。

OBJECTIVE To explore the effect of curcumin on phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway in cell models of Parkinson’s disease(PD)and discuss whether it can enhance autophagy by inhibiting this pathway to protect cell models of PD.METHODS SH-SY5Y human neuroblastoma cell was dealt with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to build cell models of PD.Control group,model group,curcumin group,curcumin and insulin-like growth factor 1(IGF-1)group,curcumin and LY294002 group were set.Curcumin,curcumin combined with IGF-1 which was PI3K pathway activator and curcumin combined with LY294002 which was PI3K pathway inhibitor were respectively used for intervention treatment.After 24 h of drug treatment,cell morphology was observed under a light microscope,cell counting kit-8 assay was used to detect cell activity,the number of surviving dopamine neurons was detected by immunofluorescence staining with tyrosine hydroxylase,Western blotting was used to detect the protein expression of PI3K,phospho-Akt(p-Akt),phospho-mTOR(p-mTOR),α-synuclein(α-Syn)and microtubule-associated protein 1 light chain 3 beta(LC3B)which was autophagy-related protein.RESULTS Compared with model group,the state of cell shrinkage and vacuolar degeneration was improved,the cell survival rate was increased(P<0.05),the survival number of dopaminergic neurons was increased(P<0.01),LC3B-II/LC3B-I ratio was increased(P<0.05),the protein expression ofα-Syn was decreased(P<0.01),and the protein expression of p-Akt and p-mTOR was significantly decreased(P<0.01)in curcumin group.When the PI3K pathway activator IGF-1 was added,the above effects of curcumin were basically offset.When the PI3K pathway inhibitor LY294002 was added,compared with single curcumin intervention,although the LC3B-II/LC3B-I ratio was increased(P<0.05),the expression ofα-Syn protein was increased(P<0.05),and the survival number of DA neurons was decreased(P<0.01).CONCLUSION Curcumin can inhibit the activation of PI3K/Akt/mTOR signaling pathway in cell models of PD,thereby enhance the autophagy function of cells and promote the clearance ofα-Syn,which is one of the main mechanisms of its neuroprotective effect.