摘要
目的探讨富亮氨酸重复激酶2(LRRK2)干扰对1-甲基-4-苯基吡啶离子(MPP+)诱导的帕金森病(PD)细胞模型线粒体功能及CaMKK-β/AMPK通路的影响。方法采用MPP+诱导传代培养的SH-SY5Y细胞构建PD细胞模型。将造模的SH-SY5Y细胞随机分PD模型组、空载转染组(空载转染PD模型细胞)和siRNA转染组(siRNA-LRRK2转染PD模型),另设正常对照组,每组设3个复孔。采用实时定量PCR(qRT-PCR)检测各组细胞LRRK2 mRNA表达,采用免疫印迹(WB)法检测各组细胞LRRK2蛋白表达,采用JC-1探针法检测各组细胞线粒体膜电位,采用试剂盒检测各组细胞线粒体复合物I(Complex I)活性及ATP含量,采用WB检测各组细胞中CaMKK-β/AMPK通路相关蛋白CaMKK-β、AMPK和p-AMPK蛋白表达。结果与正常对照组相比,PD模型组LRRK2蛋白表达增加(P<0.05),线粒体膜电位下降,Complex I活性及ATP含量降低(P<0.05),CaMKK-β/AMPK通路蛋白CaMKK-β、AMPK、p-AMPKα(Thr 172)蛋白表达增加(P<0.05)。与PD模型组比较,siRNA转染组LRRK2 mRNA及蛋白表达水平降低(P<0.05),线粒体膜电位提升,Complex I活性及ATP含量增加(P<0.05),CaMKK-β、AMPK和p-AMPK蛋白表达降低(P<0.05)。结论LRRK2干扰可改善MPP+诱导的PD细胞模型中受损的线粒体功能,抑制CaMKK-β/AMPK信号通路。
Objective To explore the regulatory effect of leucine-rich repeat kinase 2(LRRK2)on mitochondrial function in MPP+induced SH-SY5Y cell.Methods The Parkinson's disease(PD)cell model was constructed by MPP+induced SH-SY5Y cells,and randomly divided into a PD model group,a null carrier transfection group(PD model cell transfected with null carrier),and a siRNA transfection group(PD model cell transfected with siRNA-LRRK2).Untreated SH-SY5Y cell was set as the normal group.Each group had three replicates.The mRNA expression of LRRK2 was detected by qRT-PCR.The protein expression of LRRK2 was detected by Western blot.JC-1 probe was employed to measure mitochondrial membrane potential.The detection kits were used to detect ATP content and mitochondrial complex I(complex I)activity.Western blot was used to evaluate the expression of CaMKK-β/AMPK pathway related proteins.Results Compared with the normal group,the PD model group showed higher protein expression of LRRK2(P<0.05),lower mitochondrial membrane potential,reduced ATP content and complex I activity,and higher expression levels of CaMKK-β,AMPK and p-AMPKα(Thr 172)in CaMKK-β/AMPK pathway(P<0.05).Compared with the PD model group,while LRRK2 was disturbed by the transfection of siRNA-LRRK2-1,the mitochondrial membrane potential was elevated,the impaired mitochondrial function was partially recovered,the expressions of CaMKK-β/AMPK pathway related proteins were lowered significantly(P<0.05),and the phosphorylation level of AMPK was reduced significantly(P<0.05).Conclusions LRRK2 interference can regulate the mitochondrial functions of SH-SY5Y cells induced by MPP+,and inhibit the CaMKK-β/AMPK signal pathway.
作者
罗宁
王春玲
蒙冰
周欣梅
冯文勇
李昌海
文晓东
LUO Ning;WANG Chunling;MENG Bing;ZHOU Xinmei;FENG Wenyong;LI Changhai;WEN Xiaodong(不详;Department of Neurology,The Affiliated Ruikang Hospital,Guangxi University of Chinese Medicine,Nanning 530011,China)
出处
《中国神经免疫学和神经病学杂志》
CAS
北大核心
2022年第1期6-10,16,共6页
Chinese Journal of Neuroimmunology and Neurology
基金
国家自然科学基金地区科学基金项目(82060888)
广西中医药大学2018年广西一流学科建设项目重点课题(2018XK089)。
