缺氧通过磷脂酰肌醇3激酶-蛋白激酶B-金属调节转录因子1信号通路诱导肺动脉高压大鼠肺动脉平滑肌细胞增殖及迁移  

Hypoxia induces pulmonary smooth muscle cells proliferation and migration in pulmonary hypertension rats via phosphatidylinositol 3-kinase(PI3K)-protein kinase B (Akt)-metal-regulatory transcription factor 1 (MTF-1) signaling pathway

作　　者：吴剑敏[1] 陈嫒 练桂丽 高谷丰 刘俊萍 谢良地[1] 罗莉[1] WU Jian-min;CHEN Ai;LIAN Gui-li;GAO Gu-feng;LIU Jun-ping;XIE Liang-di;LUO Li(Department of Geriatrics,Fujian Hypertension Research Institute,The First Affiliated Hospital of Fujian Medical University,Fuzhou,Fujian 350005,China)

机构地区：[1]福建医科大学附属第一医院老年科,福建省高血压研究所,福建福州350005

出　　处：《中华高血压杂志》2022年第12期1177-1185,共9页Chinese Journal of Hypertension

基　　金：中青年骨干人才培养项目(2020GGA048);国家自然科学基金面上项目(81873537,82170355)。

摘　　要：目的 探索磷脂酰肌醇3激酶(PI3K)-蛋白激酶B(Akt)-金属调节转录因子1(MTF-1)信号通路对缺氧诱导的肺动脉高压(PH)大鼠肺动脉平滑肌细胞(PASMC)增殖和迁移的影响。方法 动物实验:将16只雄性成年Sprague-Dawley(SD)大鼠随机均等分为对照组(Ctrl组)和缺氧诱导的PH组(hypoxia-PH组)。将hypoxia-PH组大鼠置于自制缺氧饲养玻璃箱内[氧浓度(10.0±0.2)%],连续缺氧3周后常氧2周建立慢性PH模型,Ctrl组大鼠在无特定病原体(SPF)级房间正常饲养。在造模5周后,右心导管法测定平均肺动脉压(mPAP),HE染色评估肺动脉形态学改变,组织免疫荧光观察MTF-1及胎盘生长因子(PlGF)的表达。Western-blot法检测组织中磷酸化Akt(p-Akt)、总Akt(T-Akt)、MTF-1及PlGF的表达。细胞实验:提取5只正常大鼠来源的PASMC,每只大鼠来源的PASMC分为对照-PASMC(Ctrl)、缺氧-PASMC(hypoxia)和缺氧+PI3K-Akt通路抑制剂LY294002-PASMC(hypoxia+LY294002)组。hypoxia组利用100μmol/L氯化钴干预24 h模拟缺氧条件,hypoxia+LY294002组在氯化钴干预前30 min加入LY294002,Ctrl组未行任何处理。Western-blot法检测细胞中p-Akt、T-Akt、MTF-1及PlGF的表达。通过5-乙炔基-2’-脱氧尿苷(EdU)、流式细胞术和划痕实验评估PASMC增殖及迁移情况。结果 在动物实验中,hypoxia-PH组大鼠出现mPAP升高,肺血管壁增厚,右心室肥厚(均P<0.05)。组织免疫荧光染色及Western-blot可观察到hypoxia-PH组大鼠肺组织中p-Akt、MTF-1及PlGF表达较Ctrl组升高(均P<0.05),而T-Akt差异无统计学意义(P>0.05)。在细胞实验中,Western blot可观察到hypoxia组中p-Akt、MTF-1及PlGF表达较Ctrl组升高(均P<0.05),而LY294002可逆转这一结果。同时,EdU、流式细胞术及划痕实验显示hypoxia组的增殖迁移水平较Ctrl组增高,而LY294002可抑制这一过程[EdU实验阳性细胞比例:Ctrl (0.33±0.06)%比hypoxia (0.86±0.06)%比hypoxia+LY294002(0.41±0.14)%,F=24.13,P<0.01;流式细胞术G2+M细胞比例:CtrlObjective To investigate the effect of phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt)-metal-regulatory transcription factor 1(MTF-1) signaling pathway on pulmonary artery smooth muscle cells(PASMC) proliferation and migration in hypoxia-induced pulmonary hypertension(PH) rats. Methods In animal experiments, 16 adult male Sprague-Dawley(SD) rats were randomly divided into two groups: control group(Ctrl group) and hypoxia-induced PH group(hypoxia-PH group). In hypoxia-PH group, rats were placed in a glass chamber [oxygen concentration(10.0±0.2)%], and chronic PH model was established by normoxia for 2 weeks after continuous hypoxia for 3 weeks. Rats in Ctrl group were normally fed in specific pathogen free(SPF) room. At the end of the 5thweek after modeling, mean pulmonary arterial pressure(mPAP) was measured by right heart catheterization and morphological changes of pulmonary artery were assessed by HE staining. Histoimmunofluorescence was used to observe changes of MTF-1 and placental growth factor(PlGF). Western-blot was used to detect the expressions of phospho-Akt(p-Akt), total-Akt(T-Akt), MTF-1 and PlGF in lung tissues. In cell experiments, PASMC were derived from 5 untreated rats, and PASMC from each rat were divided into three groups: control-PASMC(Ctrl), hypoxia-PASMC(hypoxia) and hypoxia+LY294002-PASMC(hypoxia+LY294002) group. Cells were treated with 100 μmol/L cobalt chloride for 24 h to mimic hypoxia conditions in hypoxia group and hypoxia +LY294002 group. PI3K-Akt pathway inhibitor LY294002 was administrated 30 min before cobalt chloride intervention in hypoxia +LY294002 group. Cells were untreated in Ctrl group. Western-blot was used to detect the expressions of p-Akt, T-Akt, MTF-1 and PlGF in PASMC. PASMC proliferation and migration were evaluated by 5-ethynyl-2’-deoxyuridine(EdU), flow cytometry and scratch assay. Results In animal experiments, hypoxia-PH rats showed elevated mPAP, thickened pulmonary vascular wall, and right ventricular hypertrophy(all P<0.05). Histoimmunofluorescen

关 键 词：肺动脉高压 缺氧 脂酰肌醇3激酶-蛋白激酶B信号通路 金属调节转录因子1 胎盘生长因子 

分 类 号：R544.1[医药卫生—心血管疾病]