胆碱能M受体调控细胞周期蛋白依赖性激酶5及其在敌敌畏诱导SH-SY5Y细胞毒性损伤中的作用

Acetyl cholinergic M receptor regulates cyclin dependent kinase 5 and plays pivotal role in DDVP induced cytotoxic injury in SH-SY5Y cells

目的研究胆碱能M受体(主要为M1受体)参与SH-SY5Y细胞周期依赖性蛋白激酶5(Cdk5)调控及其在敌敌畏(DDVP)诱导SH-SY5Y细胞毒性损伤中的作用。方法①应用免疫荧光结合共聚焦显微镜检测胆碱能M1受体及Cdk5在SH-SY5Y细胞中的分布和表达。②细胞对照组、氧化震颤素(Oxo-M)组(1×10-4mol·L-1作用48 h)、罗考唯亭(Rosc)组(培养结束前1 h加入Rosc 1×10-4mol·L-1)、Rosc+Oxo-M组(Rosc 1×10-4mol·L-1预处理1 h后,再加入Oxo-M 1×10-4mol·L-1作用至48 h),应用MTT比色法检测细胞存活率,Western印迹法检测Cdk5表达。③细胞对照组、DDVP组(1×10-5mol·L-1作用48 h)、Rosc组(培养结束前1 h加入1×10-4mol·L-1)、Rosc+DDVP组(Rosc 1×10-4mol·L-1预处理1 h后,再加入DDVP1×10-5mol·L-1作用至48 h),用MTT比色法检测细胞存活率,Western印迹法检测Cdk5表达,流式细胞术膜联蛋白Ⅴ-碘化丙啶双染法检测细胞凋亡率,TUNEL法检测细胞凋亡。结果①激光共聚焦显微镜结果表明,SH-SY5Y细胞中M1受体与Cdk5均有分布和表达。②Oxo-M 1×10-4mol·L-1处理SH-SYSY细胞48 h,细胞存活率为(59.1±7.3)%,与细胞对照组相比显著降低(P<0.05),Cdk5表达显著升高(P<0.05);Rosc 1×10-4mol·L-1处理SH-SYSY细胞1 h,Cdk5表达显著降低(P<0.05);Rosc+Oxo-M组细胞存活率为(84.5±20.9)%,与Oxo-M组相比明显升高(P<0.05),Cdk5表达明显降低(P<0.05)。③DDVP1×10-5mol·L-1处理SH-SYSY细胞48 h,细胞存活率为(68.6±4.1)%,与细胞对照组相比显著降低(P<0.05),Cdk5表达显著升高(P<0.05);Rosc+DDVP细胞存活率为(84.4±8.8)%,与DDVP组相比显著升高,Cdk5表达显著降低(P<0.05)。流式细胞术结合TUNEL染色结果显示,DDVP处理SH-SYSY细胞48 h,细胞凋亡率为(64.1±8.4)%,与细胞对照组相比明显升高(P<0.05),TUNEL阳性细胞增多;而Rosc+DDVP组细胞凋亡率为(34.4±9.5)%,与DDVP组相比明显降低(P<0.05),TUNEL阳性细胞减少。结论 Oxo-M和DDVP可通过增强Cdk5活性引起SH-SY5Y细胞损伤,提示抑制Cdk5活性可显著降低由Oxo-M和DDVP引发的细胞毒作用。

OBJECTIVE To investigate the regulatory effect of muscarinic acetyl cholinergic M receptor(M1 subtype)on cyclin-dependent kinase 5(Cdk5)and the mechanism of neurotoxic injury induced by Cdk5-mediated dimethyl-dichlorovinyl-phosphate(DDVP)in SH-SY5Y cells.METHODS①The laser confocal was used to detect the expressions of M1 receptor and Cdk5 in SH-SY5Y cells.②The cell control,oxotremorine(Oxo-M)(Oxo-M 1×10-4 mol·L-1 was treated for 48 h),roscovitine(Rosc)(Rosc 1×10-4 mol·L-1 for 1 h),and Rosc+Oxo-M(Rosc 1×10-4 mol·L-1 pre-treated for 1 h followed by Oxo-M 1×10-4 mol·L-1 for 47 h)groups were detected by MTT assay to analyze cell viability while Western blotting used to evaluate the expression of Cdk5.③The cell control,DDVP(DDVP 1×10-5 mol·L-1 was treated for 48 h),Rosc(Rosc 1×10-4 mol·L-1 for 1 h),Rosc+DDVP(Rosc 1×10-4 mol·L-1 pre-treated for 1 h,followed by DDVP 1×10-5 mol·L-1 for 47 h)groups were detected by MTT assay to analyze cell viability,while Western blotting was used to evaluate the expression of Cdk5.AnnexinⅤ-FITC/PI staining and TUNEL staining were used to detect the rate of cell apoptosis.RESULTS①The results of laser confocal microscopy confirmed that M1 receptor and Cdk5 were expressed in SH-SY5Y cells.②When Oxo-M(1×10-4 mol·L-1)treated SH-SY5Y cells for 48 h,the cell survival rate was significantly reduced to(59.1±7.3)%,and the expression of Cdk5 was significantly increased(vs control,P<0.05).When Rosc 1×10-4 mol·L-1 treated SH-SY5Y cells for 1 h,the expression of Cdk5 was significantly increased(vs control,P<0.05).The cell viability of Rosc+Oxo-M significantly increased to(84.5±20.9)%,and the expression of Cdk5 was significantly decreased(vs Oxo-M,P<0.05).③When DDVP(1×10-5 mol·L-1)treated SH-SY5Y cells for 48 h,the cell survival rate was significantly reduced to(68.6±4.1)%,and the expression of Cdk5 was significantly increased(vs control,P<0.05).The cell viability of Rosc+DDVP significantly increased to(84.4±8.8)%,but the expression of Cdk5 was significantly decreased(vs DDVP,P<0.05).AnnexinⅤ-FITC/PI combined with TUNEL staining results showed that the apoptosis rate of DDVP increased to(64.1±8.4)%and TUNEL-positive cells were significantly increased(vs control,P<0.05),but the apoptosis rate of Rosc+DDVP was reduced to(34.4±9.5)%.Rosc significantly suppressed DDVP-induced TUNEL-positive cells of SH-SY5Y cells(vs DDVP,P<0.05).CONCLUSION The over-activation of cholinergic M receptor results in increasing Cdk5 activity as well as SH-SY5Y cytotoxicity.Furthermore,the enhancement of Cdk5 activity plays a critical role in DDVP induced injury of SH-SY5Y cells,and the inhibition of Cdk5 activity can significantly reduce the cytoxicity caused by DDVP and Oxo-M.