解剖学报

解剖学报 ›› 2023, Vol. 54 ›› Issue (5): 521-530.doi: 10.16098/j.issn.0529-1356.2023.05.004

• 神经生物学 • 上一篇    下一篇

哺乳动物雷帕霉素靶蛋白复合物2/Akt信号通路对6-羟基多巴胺处理的SH-SY5Y细胞模型的作用及分子机制

李梦一1,2 吴安婷1,2 许泽婷1,2 张庭1,3 李军伟1,2 周鹏1,2 崔怀瑞1,2 孙臣友1,2*    

  1. 1.温州医科大学基础医学院解剖学教研室，浙江 温州 325035; 2.温州医科大学基础医学院神经科学研究所，浙江 温州 325035; 3.温州医科大学基础医学院2020级，浙江 温州 325035
  • 收稿日期:2022-06-14 修回日期:2022-07-13 出版日期:2023-10-06 发布日期:2023-12-25
  • 通讯作者: 孙臣友 E-mail:sunchenyou1972@aliyun.com
  • 基金资助:
    Notch/RBP-J作用于Prominin-1调控CD133（+）室管膜细胞再生对PD小鼠干预研究;mTORC2/AKT信号通路通过调控成体SVZ-NPCs再生干预PD小鼠的实验研究;激活mTORC2/Akt信号通路在改善6-羟基多巴胺模型小鼠多巴胺能神经元数目和行为学缺陷中的作用及机制研究

Effect and molecular mechanism of mammalian target of rapamycin complex 2/Akt signaling pathway on 6-hydroxydopamine-treated SH-SY5Y cell model

LI  Meng-yi1,2  WU  An-ting1,2  XU Ze-ting1,2  ZHANG  Ting1,3  LI  Jun-wei1,2  ZHOU  Peng1,2  CUI  Huai-rui1,2  SUN  Chen-you1,2* 
  

  1. 1.Department of Anatomy, School of Basic Medical Sciences, Wenzhou Medical University, Zhejiang Wenzhou 325035,China; 
    2.Institution of Neuroscience, School of Basic Medical Sciences, Wenzhou Medical University, Zhejiang Wenzhou 325035, China;   3.2020 Grade School of Basic Medical Sciences, Wenzhou Medical University,Zhejiang Wenzhou 325035, China
  • Received:2022-06-14 Revised:2022-07-13 Online:2023-10-06 Published:2023-12-25
  • Contact: Chen-You SUN E-mail:sunchenyou1972@aliyun.com

PDF

摘要：

目的  探讨调控哺乳动物雷帕霉素蛋白复合物2（mTORC2）/Akt信号通路对6-羟基多巴胺（6-OHDA） 损伤的SH-SY5Y细胞系是否具有保护作用，并阐明其分子作用机制。    方法  经维甲酸（RA）处理的 SH-SY5Y细胞分别给予6-OHDA、mTORC2信号通路抑制剂PP242和激动剂A-443654，通过免疫荧光染色观察各组细胞数目的变化；提取细胞总蛋白进行Western blotting和免疫共沉淀（CoIP）实验，明确mTORC2信号通路关键蛋白的表达水平及其相互作用；采用流式细胞术检测各组细胞的凋亡率。同时制备SH-SY5Y与Bv-2细胞系共培养的帕金森病（PD）模型，运用MTT及ELISA方法检测各组细胞活力及培养上清液中肿瘤坏死因子α（TNF-α）和白细胞介素β（IL-1β）的含量。    结果  6-OHDA损伤的PD细胞模型组酪氨酸羟化酶（TH）/增殖细胞核抗原（PCNA）/Hochest-、TH/5-溴脱氧尿嘧啶核苷（BrdU）-单标或双标阳性细胞数较正常组明显减少，凋亡率升高；Rictor、p-Akt、发育及DNA损伤反应调节蛋白1（REDD1）的表达均上升，且Rictor与p-Akt或REDD1存在相互作用；共培养模型中细胞活力明显降低且上清液中TNF-α和IL-β含量上升。A-443654组随着上述蛋白表达的进一步上调，细胞存活和凋亡以及炎性因子水平均有明显改善，而PP242组则呈现相反的变化。    结论  A-443654通过使Akt磷酸化而激活mTORC2信号通路，引起Rictor和REDD1蛋白的表达增加，继而提高细胞存活率并降低凋亡率，促进SH-SY5Y细胞的增殖分化，改善了6-OHDA引起的细胞损伤以及抑制了炎症因子的释放。 

关键词:  帕金森病, 哺乳动物雷帕霉素靶蛋白复合物2, PP242, A-443654, 免疫荧光, SH-SY5Y细胞系 

Abstract:

Objective To study whether the regulation of mammalian target of rapamycin complex 2(mTORC2)/Akt signaling pathway has a protective effect on SH-SY5Y cell line damaged by 6-hydroxydopamine (6-OHDA), and to clarify its molecular mechanism.    Methods  SH-SY5Y cells treated with retinoic acid (RA) were given 6-OHDA, mTORC2 signaling pathway inhibitor PP242 and agonist A-443654 respectively. The changes of cell number in each group were investigated by immunofluorescent staining; The total protein was extracted and the expression level and interaction of key proteins in mTORC2 signaling pathway were determined by Western blotting and co-immunoprecipitation (CoIP); The apoptosis rate of cells in each group was detected by flow cytometry. At the same time, the co-culture Parkinson’s disease(PD) model was made using SH-SY5Y cell line and Bv-2 cell line; MTT colorimetric method  was used to detect the cell viability of each group; ELISA was used to detect the content of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in cell culture supernatant.    Results  The number of tyrosine hydroxylase(TH)/proliferating cell nuclear antigen(PCNA)/hochest-, TH/5-bronmo-2’-deoxyuridine(BrdU)-labeled positive cells in 6-OHDA-lesioned PD cell model group was significantly lower than that in the normal group; The apoptosis rate was higher; The expression of Rictor, p-Akt and regulated in DNA damage and development 1(REDD1) was increased; There was an interaction between Rictor and p-Akt or REDD1; The cell viability was significantly reduced in the co-culture model; the content of TNF-α and IL-β  increased in the cell culture supernatant. With further up-regulation of the abovementioned protein expressions, the cell survival, apoptosis and pro-inflammatory cytokine levels in A-443654 group were significantly ameliorated, while PP242 group showed the opposite changes.    Conclusion  A-443654 activates mTORC2 signaling pathway by p-Akt, which increases the expression of Rictor and REDD1 protein. These changes contribute to the amelioration in cell survival rate, apoptosis rate, and the proliferation and differentiation and decreasion of apoptosis rate of SH-SY5Y cells. These result  improved 6-OHDA-induced cell damage and inhibited the release of pro-inflammatory cytokines.

Key words:

 

中图分类号: