基于AMPK/mTOR信号通路的细胞自噬对大鼠肺动脉平滑肌细胞增殖和凋亡的影响  

Effects of autophagy on proliferation and apoptosis of rat pulmonary ar⁃tery smooth muscle cells based on AMPK/mTOR signaling pathway

作　　者：宋正阳 施晓倩[2] 田云娜 王新雨 张赛[1] 王肖婷 张聪聪 王万铁[1] SONG Zheng-yang;SHI Xiao-qian;TIAN Yun-na;WANG Xin-yu;ZHANG Sai;WANG Xiao-ting;ZHANG Cong-cong;WANG Wan-tie(Institute of Ischemia/Reperfusion Injury,Wenzhou Medical University,Wenzhou 325035,China;Hangzhou Medical College,Hangzhou 310052,China;College of Pharmacy,Zhejiang Pharmaceutical College,Ningbo 315000,China)

机构地区：[1]温州医科大学缺血/再灌注损伤研究所,浙江温州325035 [2]杭州医学院中医教研室,浙江杭州310052 [3]浙江医药高等专科学校药学院,浙江宁波315000

出　　处：《中国病理生理杂志》2022年第8期1416-1423,共8页Chinese Journal of Pathophysiology

基　　金：浙江省中医药科学研究基金计划项目(No.2022ZA097);浙江省介入肺脏病重点实验室建设项目(No.2019E10014);宁波市自然科学基金资助项目(No.2019A610350)。

摘　　要：目的:探讨在低氧高二氧化碳(hypoxia and hypercapnia,HH)条件下,基于AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路的细胞自噬的变化及其对大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)增殖和凋亡的影响。方法:以大鼠PASMCs为研究对象,饥饿24 h后随机分为5组:正常对照(normal control,N)组、模型组(HH组)、溶剂二甲基亚砜(dimethyl sulfoxide,DMSO)组(D组)、AMPK激动剂阿卡地新(acadesine;5-aminoimidazole-4-carboxamide riboside,AICAR)组(AI组)和AMPK抑制剂dorsomorphin(compound C,CC)组(CC组)。N组置于常氧环境(21%O_(2)、5%CO_(2)和74%N2)下培养24 h,其余4组预先加入相对应的干预药物置于HH条件(5%O_(2)、6%CO_(2)和89%N2)下培养24 h进行造模。造模结束后用CCK-8法测各组细胞活力;5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)法检测细胞增殖;TUNEL检测细胞凋亡;qPCR法检测微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、p62及caspase-3的mRNA表达;免疫印迹法检测AMPK、p-AMPK、mTOR、p-mTOR、LC3、p62、caspase-3和增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白水平;透射电镜观察自噬小体。结果:与N组相比,HH组PASMCs活力增强,EdU阳性率增加,凋亡率降低;LC3的mRNA表达上调,p62和caspase-3的mRNA表达下调;p-AMPK/AMPK比值、LC3-II/LC3-I比值和PCNA蛋白表达上调,p-mTOR/mTOR比值及p62和caspase-3蛋白表达下调;电镜下观察到细胞内有少量自噬小体。与HH组相比,AI组PASMCs活力进一步增强,EdU阳性率增加,凋亡率降低;LC3的mRNA表达上调,p62和caspase-3的mRNA表达下调;p-AMPK/AMPK比值、LC3-II/LC3-I比值和PCNA蛋白表达上调,p-mTOR/mTOR比值及p62和caspase-3蛋白表达下调;电镜形态表现为细胞自噬小体数量增加。而CC组较HH组PASMCs活力和EdU阳性率显著降低,凋亡率显著升高;LC3的mRNA表达下调AIM:To investigate the changes of autophagy based on AMP-activated protein kinase(AMPK)mammalian target of rapamycin(mTOR)signaling pathway and its effects on proliferation and apoptosis of rat pulmonary artery smooth muscle cells(PASMCs)under hypoxia and hypercapnia(HH)conditions.METHODS:Rat PASMCs were randomly divided into 5 groups after 24 h starvation:normal control(N)group,model group(HH group),solvent dimethyl sulfoxide(DMSO)group(D group),AMPK activator acadesine(5-aminoimidazole-4-carboxamide riboside,AICAR)group(AI group)and AMPK inhibitor dorsomorphin(compound C,CC)group(CC group).The cells in N group was cul-tured for 24 h under normal oxygen environment(21%O_(2),5%CO_(2)and 74%N2),and those in the other 4 groups were pre-added with corresponding intervention drugs and cultured for 24 h under HH conditions(5%O_(2),6%CO_(2)and 89%N2)for modeling.CCK-8 method was used to measure the cell viability after modeling.Cell proliferation was detected by 5-ethy-nyl-2´-deoxyuridine(EdU)assay.Apoptosis was detected by TUNEL.The mRNA expression levels of microtubule-associ-ated protein 1 light chain 3(LC3),p62 and caspase-3 were detected by qPCR.The protein levels of AMPK,p-AMPK,mTOR,p-mTOR,LC3,p62,caspase-3 and proliferating cell nuclear antigen(PCNA)were measured by Western blot.The autophagosomes were observed by transmission electron microscopy.RESULTS:Compared with N group,the viabili-ty and EdU positive rate of PASMCs in HH group were increased,while the apoptosis rate was decreased.The mRNA ex-pression of LC3 was up-regulated,while p62 and caspase-3 were down-regulated.The ratios of p-AMPK/AMPK and LC3-II/LC3-I,and the protein expression of PCNA were up-regulated,while the ratio of p-mTOR/mTOR,and p62 and caspase-3 protein expression levels were down-regulated.A few autophagosomes were observed under electron microscope.Com-pared with HH group,the viability and EdU positive rate of PASMCs in AI group were further increased,while the apopto-sis rate was further decreased.The mRNA expression of LC3 was furth

关 键 词：细胞自噬 脱氧尿苷 哺乳动物雷帕霉素靶蛋白 增殖细胞核抗原 信号通路 免疫印迹法 乙炔基 mTOR 

分 类 号：R544.1[医药卫生—心血管疾病]