Ang-(1-7)及Mas受体拮抗剂对低氧诱导小鼠肺动脉平滑肌细胞增殖及PKCα/Akt信号分子表达的影响  

Effects of Ang-(1-7)and Mas receptor antagonists on the proliferation of pulmonary artery smooth muscle cells and the expression of PKCα/Akt signaling molecule in hypoxia-induced mouse

作　　者：李杰娜 王晓亮 张亚萍 施熠炜[2] Li Jiena;Wang Xiaoliang;Zhang Yaping;Shi Yiwei(First Clinical Medical School,Shanxi Medical University,Taiyuan 030001,China;Department of Respiratory and Critical Care Medicine,First Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一临床医学院,太原030001 [2]山西医科大学第一医院呼吸与危重症医学科,太原030001

出　　处：《国际呼吸杂志》2022年第7期519-525,共7页International Journal of Respiration

基　　金：山西省回国留学人员科研资助项目(2020-167)。

摘　　要：目的探讨Ang-(1-7)及Mas受体拮抗剂A779对低氧诱导小鼠肺动脉平滑肌细胞(PASMCs)增殖及PKCα/Akt信号分子在低氧诱导小鼠PASMCs中表达的影响。方法本研究为实验研究。选取C57BL/6J健康成年小鼠,使用磁性分离法原代培养小鼠PASMCs,选取第3代细胞进行实验。使用三气培养箱进行低氧处理,给予Ang-(1-7)及Mas受体拮抗剂A779进行药物干预。实验共分为6组,即常氧组、常氧+Ang-(1-7)组、低氧组、低氧+Ang-(1-7)组、低氧+Ang-(1-7)+A779组和低氧+A779组。使用CCK-8检测各实验组小鼠PASMCs增殖情况;免疫荧光、蛋白质印迹法检测各实验组PKCα/Akt蛋白分子表达变化情况。结果经免疫荧光染色鉴定原代培养PASMCs纯度约90%。低氧24 h后,与常氧组比较,低氧组、常氧+Ang-(1-7)组与低氧+Ang-(1-7)组间细胞增殖能力差异均无统计学意义(P值均>0.05);低氧+Ang-(1-7)+A779组和低氧+A779组细胞增殖能力增强(P值均<0.05)。随着低氧时间延长至48、72 h,各低氧组细胞增殖能力呈逐渐下降趋势。与常氧组比较,低氧组、低氧+Ang-(1-7)组、常氧+Ang-(1-7)组的PKCα蛋白表达均降低,低氧+Ang-(1-7)+A779组和低氧+A779组的PKCα蛋白表达均升高,差异均有统计学意义(P值均<0.01)。与常氧组比较,常氧+Ang-(1-7)组、低氧组Akt蛋白分子表达差异无统计学意义(P值均>0.05),低氧+Ang-(1-7)组、低氧+Ang-(1-7)+A779组和低氧+A779组Akt蛋白表达均升高,差异均有统计学意义(P值均<0.05)。结论抑制Ang-(1-7)特异性Mas受体可促进低氧环境下小鼠PASMCs增殖,该过程可能通过提高PKCα、Akt信号分子的表达实现。Ang-(1-7)可抑制该过程中Akt信号分子表达,但尚未发现对PKCα信号分子表达及细胞增殖的影响。Objective To investigate the effects of angiotensin(Ang)-(1-7)and Mas receptor antagonist A779 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)and the expression of PKCα/Akt signaling molecules on PASMCs of hypoxia-induced mouse.Methods This is an experimental study.Third-generation cells form the primary culture of PASMCs of C57BL/6J healthy adult rats by magnetic separation techniques were conducted for experiments.After Hypoxic treatment in tri-gas incubator,Ang-(1-7)and Mas receptor antagonist A779 were performed for drug intervention.The subjects were divided into six groups:Normoxic group,Normoxic+Ang-(1-7)group,Hypoxia group,Hypoxia+Ang-(1-7)group,Hypoxia+Ang-(1-7)+A779 group and Hypoxia+A779 group.The proliferation of PASMCs was detected by Cell Counting Kit-8(CCK-8)assay and the protein expression of PKCα/Akt signaling molecular was recorded by immunofluorescence staining and Western-blot analysis between group.Results Immunofluorescence staining showed the purity of third-generation PASMCs was about 90%.After 24h of hypoxia,there was no significant difference in cell proliferation between Hypoxia group,Normoxic+Ang-(1-7)group,Hypoxia+Ang-(1-7)group and Normoxic group(all P>0.05),but cell proliferation was significantly enhanced in Hypoxia+Ang-(1-7)+A779 group and Hypoxia+A779 group(all P<0.05).After 48h and 72h hypoxia,cell proliferation of all Hypoxia groups tended to decrease gradually.Compared with Normoxic group,the expression of PKCαprotein molecules was decreased in Hypoxia group,Hypoxia+Ang-(1-7)group and Normoxic+Ang-(1-7)group;which was significantly increased in Hypoxia+Ang-(1-7)+A779 group and Hypoxia+A779 group with the statistical significance differences(all P<0.01).Meanwhile,the expression of Akt protein molecule showed no significant difference between Normoxic+Ang-(1-7)group,Hypoxia group and Normoxic group(all P>0.05);which were significantly increased in Hypoxia+Ang-(1-7)group,Hypoxia+Ang-(1-7)+A779 group and Hypoxia+A779 group(all P<0.05).Conclusions Inhibition

关 键 词：肺动脉高压 平滑肌 细胞低氧 蛋白激酶C 蛋白激酶B Ang-(1-7) 

分 类 号：R544.1[医药卫生—心血管疾病]