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。
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-αand IL-βincreased 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-OHDAinduced cell damage and inhibited the release of pro-inflammatory cytokines.
作者
李梦一
吴安婷
许泽婷
张庭
李军伟
周鹏
崔怀瑞
孙臣友
LI Meng-yi;WU An-ting;XU Ze-ting;ZHANG Ting;LI Jun-wei;ZHOU Peng;CUI Huai-rui;SUN Chen-you(Department of Anatomy,School of Basic Medical Sciences,Wenzhou Medical University,Zhejiang Wenzhou 325035,China;Institution of Neuroscience,School of Basic Medical Sciences,Wenzhou Medical University,Zhejiang Wenzhou 325035,China;School of Basic Medical Sciences,Wenzhou Medical University,Zhejiang Wenzhou 325035,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2023年第5期521-530,共10页
Acta Anatomica Sinica
基金
浙江省自然科学基金(LY22H090004)
温州市科技局基础性医疗卫生科技项目(Y20190059)。
