黄连碱上调miR-146a-5p后通过PI3K/AKT通路减轻由MPP+诱导的帕金森病细胞模型损伤  

Coptisine up-regulates miR-146a-5p expression to attenuate injury of Parkinson's disease cell models induced by 1-methyl-4-phenylpy ridinium via PI3K/AKT pathway

作　　者：陈红利[1] 梁会娟[2] 李燕 Chen Hongli;Liang Huijuan;Li Yan(Department of Pharmacy,Xinxiang Central Hospital,Xinxiang 453000,China;Department of Clinical Pharmacy,Third Affiliated Hospital,Xinxiang Medical College,Xinxiang 453000,China)

机构地区：[1]新乡市中心医院药学部,新乡453000 [2]新乡医学院第三附属医院临床药学室,新乡453000

出　　处：《中华神经医学杂志》2020年第1期2-8,共7页Chinese Journal of Neuromedicine

摘　　要：目的探讨黄连碱对1-甲基-4-苯基吡啶离子(MPP+)诱导的帕金森病(PD)细胞损伤的影响及其机制。方法用0.3 mmol/L的MPP+处理SK-N-SH细胞作为PD细胞模型,记为MPP+组,以正常培养的细胞作为空白对照组。用浓度分别为10μmol/L、20μmol/L、40μmol/L的黄连碱预处理4h后再用0.3 mmol/L的MPP+处理作为不同浓度黄连碱处理组。将miR-con、miR-146a-5p转染至SK-N-SH细胞后再用0.3 mmol/L的MPP+处理记为MPP++miR-con组、MPP++miR-146a-5p组;将anti-miR-con、anti-miR-146a-5p转染至SK-N-SH细胞后用20μmol/L的黄连碱预处理4h及0.3 mmol/L的MPP+处理记为MPP++Cop+anti-miR-con组、MPP++Cop+anti-miR-146a-5p组。四甲基偶氮唑盐比色法(MTT)检测细胞存活率;Western blotting实验检测活化的半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、细胞周期蛋白D1(Cyclin D1)、磷酸化蛋白激酶B(p-AKT)、磷酸化磷脂酰肌醇3激酶(p-PI3K)蛋白表达水平;流式细胞术检测细胞凋亡;实时荧光定量PCR(RT-qPCR)检测miR-146a-5p表达水平。结果与空白对照组比较,MPP+处理后SK-N-SH细胞存活率显著降低,活化caspase-3表达水平显著升高,细胞凋亡率显著升高,CyclinD1、miR-146a-5p表达水平显著降低,差异均有统计学意义(P<0.05)。黄连碱处理及miR-146a-5p过表达后MPP+诱导的SK-N-SH细胞中细胞存活率显著升高,活化caspase-3表达水平显著降低,细胞凋亡率显著降低,CyclinD1、miR-146a-5p表达水平显著升高,差异均有统计学意义(P<0.05)。低表达miR-146a-5p逆转了黄连碱对SK-N-SH细胞增殖促进和凋亡抑制的作用。黄连碱处理后MPP+诱导的SK-N-SH细胞中p-AKT、p-PI3K表达水平显著升高,低表达miR-146a-5p逆转了黄连碱对p-AKT、p-PI3K表达水平的促进作用。结论黄连碱可促进细胞存活,抑制MPP+诱导的细胞凋亡,其机制可能与miR-146a-5p及PI3K/AKT信号通路有关。Objective To investigate the effect of coptisine on 1-methyl-4-phenylpy ridinium(MPP+)induced Parkinson's disease(PD)cell injury and its mechanism.Methods SK-N-SH cells were treated with 0.3 mmol/L MPP+to induce PD cell models(MPP+group);normal cultured cells were used as blank controls(NCs);pretreatment with coptisine at concentrations of 10,20,and 40μmol/L for 4 h was performed after giving 0.3 mmol/L MPP+,different concentration coptisine treatment groups were named.And miR-con and miR-146a-5p were transfected into SK-N-SH cells and treated with 0.3 mmol/L MPP+,and named MPP++miR-con group and MPP++miR-146a-5p group;anti-miR-con and anti-miR-146a-5p were transfected into SK-N-SH cells and pretreated with 20μmol/L coptisine for 4 h,and then,treated with 0.3 mmol/L MPP+,and named MPP++Cop+anti-miR-con group and MPP++Cop+anti-miR-146a-5p group.Cell viability was determined by MTT assay;Western blotting was used to detect the protein expressions of cleaved cysteine-containing aspartate-specific proteases-3(caspase-3),CyclinD1,phosphorylated protein kinase B(p-AKT),p-phosphoinositide 3 kinase(p-PI3K);apoptosis was detected by flow cytometry;real-time quantitative PCR(RT-qPCR)was used to detect the miR-146a-5p expression.Results As compared with the NCs,the MPP+induced SK-N-SH cells had significantly decreased viability and CyclinD1 and miR-146a-5p expressions,and significantly increased cleaved caspase-3 expression and apoptosis rate(P<0.05).After coptisine treatment and miR-146a-5p overexpression,MPP+induced SK-N-SH cells had significantly increased viability and expressions of CyclinD1 and miR-146a-5p,and significantly decreased cleaved-caspase-3 expression and apoptosis rate(P<0.05).Low expression of miR-146a-5p reversed the effect of coptisine on proliferation promotion and apoptosis inhibition of SK-N-SH cells.The expression levels of p-AKT and p-PI3K in MPP+-induced SK-N-SH cells were significantly increased after coptisine treatment.Low expression of miR-146a-5p reversed the effect of coptisine on expressi

关 键 词：黄连碱 miR-146a-5p PI3K/AKT信号通路 帕金森病 细胞损伤 

分 类 号：R28[医药卫生—中药学]