摘要
目的探讨miR-145-3p对1-甲基-4苯基吡啶离子(MPP^(+))诱导的帕金森病(PD)细胞模型线粒体自噬的影响及其机制。方法将人神经母细胞瘤细胞(SH-SY5Y)分为对照组、模型组、模拟物(mimics)组、钙调蛋白依赖性蛋白激酶激酶β(CaMkkβ)抑制剂(STO-609)组、mimics+STO-609组、环磷酸腺苷反应元件结合蛋白(CREB)抑制剂(KG-501)组、mimics+KG-501组和STO-609+KG-501组。流式细胞术检测细胞凋亡,透射电镜观察自噬体结构,Western blot检测凋亡、自噬和CaMkkβ/腺苷酸活化蛋白激酶(AMPK)/CREB通路相关蛋白的表达。结果与对照组相比,模型组细胞凋亡率、Bcl-2关联X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)和微管相关蛋白轻链3-I(LC3-Ⅰ)蛋白表达水平均升高(P<0.01),自噬体结构减少,B淋巴细胞瘤-2(Bcl-2)、自噬基因(Beclin-1)、微管相关蛋白轻链3-II(LC3-Ⅱ)、磷酸化钙调蛋白依赖性蛋白激酶激酶β(p-CaMkkβ)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、磷酸化环磷酸腺苷反应元件结合蛋白(p-CREB)蛋白水平均降低(P<0.01);与模型组相比,mimics组细胞凋亡率、Bax、Caspase-3和LC3-Ⅰ蛋白表达水平降低(P<0.05),自噬体结构增多,Bcl-2、Beclin-1、LC3-Ⅱ、p-CaMkkβ、p-AMPK、p-CREB蛋白水平升高(P<0.05),STO-609组和KG-501组趋势相同均与mimics组相反;与mimics组相比,mimics+STO-609组和mimics+KG-501组细胞凋亡率、Bax、Caspase-3和LC3-Ⅰ蛋白表达水平升高(P<0.01),自噬体结构减少,Bcl-2、Beclin-1、LC3-Ⅱ、p-CaMkkβ、p-AMPK、p-CREB蛋白水平降低(P<0.01);与STO-609组相比,STO-609+KG-501组细胞凋亡率、Bax、Caspase-3和LC3-Ⅰ蛋白表达水平升高(P<0.01),自噬体结构减少,Bcl-2、Beclin-1、LC3-Ⅱ、p-CaMkkβ、p-AMPK、p-CREB蛋白水平降低(P<0.05)。结论miR-145-3p能够抑制MPP^(+)诱导的PD细胞模型的凋亡,促进线粒体自噬,其机制可能与促进CaMkkβ/AMPK/CREB通路的激活有关。
Objective To investigate the effect and mechanism of miR-145-3p on mitophagy in 1-methyl-4-phenylpyridiniumion(MPP^(+))-induced Parkinson′s disease(PD)cell model.Methods Human neuroblastoma cells(SH-SY5Y)were divided into control group,model group,mimics group,calmodulin-dependent protein kinase kinaseβ(CaMkkβ)inhibitor(STO-609)group,mimics+STO-609 group,cyclic adenosine monophosphate response element-binding protein(CREB)inhibitor(KG-501)group,mimics+KG-501 group and STO-609+KG-501 group.Cell apoptosis was detected by flow cytometry,autophagosome structure was observed by transmission electron microscopy,and apoptosis,autophagy and CaMkkβ/adenylate activated protein kinase(AMPK)/CREB pathway related protein expression were detected by Western blot.Results Compared with control group,the apoptosis rate,Bcl-2-associated X protein(Bax),cysteine proteinase-3(Caspase-3)and microtubule-associated protein light chain 3-I(LC3-Ⅰ)protein expression levels in model group increased(P<0.01),and the autophagosome structure decreased.The protein levels of B cell lymphoma-2(Bcl-2),autophagy gene(Beclin-1),microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ),phosphorylated calmodulin-dependent protein kinase kinaseβ(p-CaMkkβ),phosphorylated cadenylate activated protein kinase(p-AMPK),and phosphorylated cyclic adenosine monophosphate response element-binding protein(p-CREB)decreased(P<0.01).Compared with model group,the apoptosis rate,Bax,Caspase-3 and LC3-Ⅰprotein expression levels in mimics group decreased(P<0.05),and the autophagosome structure increased.The protein levels of Bcl-2,Beclin-1,LC3-Ⅱ,p-CaMkkβ,p-AMPK,p-CREB increased(P<0.05).The trend of STO-609 group and KG-501 group was the same and opposite to mimics group.Compared with mimics group,the apoptosis rate,Bax,Caspase-3 and LC3-Ⅰprotein expression levels in the mimics+STO-609 group and the mimics+KG-501 group increased(P<0.01),and the autophagosome structure decreased.The protein levels of Bcl-2,Beclin-1,LC3-Ⅱ,p-CaMkkβ,p-AMPK,p-CREB protein levels decreased(P<0.01).Compared with STO-609 group,the apoptosis rate,Bax,Caspase-3 and LC3-Ⅰprotein expression levels of STO-609+KG-501 group increased(P<0.01),and the autophagosome structure decreased.The protein levels of Bcl-2,Beclin-1,LC3-Ⅱ,p-CaMkkβ,p-AMPK and p-CREB decreased(P<0.05).Conclusion miR-145-3p can inhibit the apoptosis of MPP^(+)-induced PD cell model and promote mitophagy,and its mechanism may be related to the activation of the CaMkkβ/AMPK/CREB pathway.
作者
文晓东
罗宁
周欣梅
卢建政
曾振
张艺
王春玲
Wen Xiaodong;Luo Ning;Zhou Xinmei;Lu Jianzheng;Zeng Zhen;Zhang Yi;Wang Chunling(Brain Disease Area 1,Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine,Nanning 530011;College of Pharmacy,Guangxi University of Traditional Chinese Medicine,Nanning 530000)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第11期1846-1853,共8页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:82060888)
广西自然科学基金(编号:2022JJA140430)
广西壮族自治区中医药管理局课题(编号:GZZC2020107)
广西中医药大学校级课题(编号:2020MS049)。
