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

关键词:  帕金森病  姜黄素  自噬  α-突触核蛋白  PI3K/Akt/mTOR通路

Key words:  Parkinson's disease  curcumin  autophagy  α-synuclein  PI3K/Akt/mTOR pathway

摘要:

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。

Abstract:

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 of α-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 of α-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 of α-Syn, which is one of the main mechanisms of its neuroprotective effect.