摘要
目的 在野百合碱诱导的肺动脉高压大鼠模型上抑制内质网应激,观察内质网应激-线粒体自噬通路在大鼠肺动脉高压中的作用。方法 选择45只SD大鼠,将其随机分为3组:分别为正常对照组、野百合碱诱导的肺动脉高压(pulmonary arterial hypertension, PAH)组及4-苯基丁酸(4-phenylbutyric acid, 4-PBA)药物干预组,每组15只。利用生物信息采集系统测定各组大鼠血流动力学指标平均肺动脉压力和平均右心室压力;采用苏木精-伊红染色观察肺血管重塑改变,并利用Image J病理分析软件测定肺血管重塑相关指标;在电镜下观察肺血管平滑肌细胞中线粒体状态改变;采用qPCR分子生物学手段测定内质网应激-线粒体自噬通路关键因子的mRNA表达水平。结果 (1)野百合碱诱导的PAH组平均肺动脉压力及平均右心室压力均升高。经过4-PBA干预后,与PAH组相比,平均肺动脉压力及平均右心室压力均下降(P<0.001)。(2)HE染色后发现PAH组肺小血管重塑明显,血管重塑指标测定提示肺小动脉中膜厚度增加,管腔狭窄(P<0.05)。(3)电镜观察肺血管平滑肌细胞超微结构发现,PAH组肺血管平滑肌细胞线粒体肿胀、线粒体嵴结构破坏,溶解消失,可见线粒体自噬现象增多。抑制内质网应激后,线粒体正常结构破坏减少,线粒体自噬减少。(4)内质网应激-线粒体自噬通路关键因子的mRNA水平测定结果发现,PAH组内质网应激相关因子PERK、ATF4、Bcl-2、CHOP的mRNA表达水平上调(P<0.001),线粒体融合关键因子Mfn2(mitofusin-2)表达水平下调,线粒体分裂关键因子Drp1(dynamin-related protein 1, Drp1)表达上调(P<0.001),自噬相关因子12 (autophagy related 12,Atg12)、微管相关蛋白1A/1B-轻链3 (microtubule-associated proteins 1A and 1B,LC3)、p62 (sequestosome-1,p62/SQSTM1)的mRNA表达上调(P<0.01)。抑制内质网应激后,内质网应激相关因子的mRNA表达水平下降(P<0.001),线粒体融合因子Mfn2表达上调(P<0.001),线粒体分裂因子Drp1的mRNA表达下调(P<0.001),Atg12、LC3和p62的mRNA表达水平降低(P<0.01)。结论 内质网应激可能通过线粒体自噬途径在肺动脉高压发生发展中发挥重要作用,干预该通路可能为肺动脉高压的防治提供治疗新思路。
Objective To investigate the role of the endoplasmic reticulum(ER) stress-mitochondrial autophagy pathway in pulmonary arterial hypertension(PAH) in rats by inhibiting ER stress induced by monocrotaline. Methods SD rats were randomly divided into three groups(15 rats per group): normal control group, group with PAH induced by monocrotaline, and 4-phenylbutyric acid(4-PBA) group. The mean pulmonary artery pressure(mPAP) and mean right ventricular pressure(mRVP) in the rats were measured by the Biological Information Acquisition System. The remodeling of pulmonary vessels was observed by hematoxylin-eosin(HE) staining, while ImageJ was used to measure indices related to pulmonary vascular remodeling. The ultrastructure of mitochondria in pulmonary artery smooth muscle cells was observed by electron microscopy. Quantitative real-time PCR analysis was used to measure the mRNA levels of the key factors in the ER stress-mitochondrial autophagy pathway. Results(1) mPAP and mRVP increased in the PAH group(P<0.001). However, after 4-PBA intervention, mPAP and mRVP decreased compared with those in the PAH group(P<0.001).(2) HE staining showed clear remodeling of pulmonary small vessels in the PAH group, and the measurement of vascular remodeling indices indicated increased thickness of pulmonary arteriole media and lumen stenosis(P<0.05).(3) The mitochondria of pulmonary artery smooth muscle cells in the PAH group were swollen, the mitochondrial crista structure had dissolved or disappeared, and mitochondrial autophagy was increased, as revealed by electron microscopy. After the ES stress was inhibited, destruction of the mitochondrial structure and mitochondrial autophagy were reduced.(4) Compared with those in the NC group, the mRNA expression levels of ERS-related factors PERK, ATF4, Bcl-2 and CHOP were increased in the PAH group(P<0.001), mitochondrial fusion factor Mfn2 decreased(P<0.001), mitochondrial fission factor Drp1 increased(P<0.001), and the expression of Atg12, LC3 and p62 increased in the PAH group(P<0.001). After rats had been administered 4-PBA, the mRNA expression levels of ERS-related factors decreased(P<0.001), the mRNA level of Mfn2 increased(P<0.001), while that of Drp1 decreased(P<0.001). Moreover, the mRNA expression levels of mitochondrial autophagy factors Atg12, LC3 and P62(P<0.01) decreased. Conclusions ERS may promote the establishment and progression of pulmonary hypertension through the mitochondrial autophagy pathway. This may provide a new concept for the treatment and prevention of pulmonary hypertension.
作者
武云
闫倩芝
王捷
张敏芳
王瑞
王世超
WU Yun;YAN Qianzhi;WANG Jie;ZHANG Minfang;WANG Rui;WANG Shichao(Department of General Medicine,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Department of Pharmacy,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054;Electron Microscope Lab,Xinjiang Medical University,Urumqi 830054)
出处
《中国比较医学杂志》
CAS
北大核心
2022年第5期53-59,共7页
Chinese Journal of Comparative Medicine
基金
国家自然科学基金地区科学基金项目(81860096)。
关键词
内质网应激
线粒体自噬
肺动脉高压
肺血管平滑肌细胞
4-苯基丁酸
endoplasmic reticulum stress
mitochondrial autophagy
pulmonary hypertension
pulmonary vascular smooth muscle cell
4-phenylbutyric acid
