基于骨膜素探讨黄芪甲苷对低氧诱导肺动脉高压小鼠的保护作用及机制

Study on the Protective Effect and Mechanism of Astragaloside IV on Hypoxia-Induced Pulmonary Arterial Hypertension in Mice Based on Periostin

目的 探讨黄芪甲苷对低氧诱导肺动脉高压(PAH)小鼠的保护作用及作用机制。方法 将60只昆明种小鼠随机分为空白组、模型组及黄芪甲苷低、中、高剂量组(40、80、120 mg·kg^(-1))。将小鼠置于氧浓度恒定为10%的常压低氧舱中持续饲养4周建立PAH模型,造模同时灌胃给药,每日1次。4周后检测各组小鼠的血流动力学指标[右心室收缩压(RVSP)、平均肺动脉压(mPAP)]及右心室肥厚指数(RVHI);采用HE染色法评估肺小动脉病理性重构;免疫荧光法检测肺组织α-平滑肌肌动蛋白(α-SMA)的表达;Masson染色法观察肺组织纤维化程度,计算胶原容积分数(CVF);ELISA法检测血清和肺组织匀浆中羟脯氨酸(HYP)的含量;免疫组织化学法检测肺组织中骨膜素(Periostin)表达情况;Western Blot法检测肺组织中纤维化相关蛋白表达水平。结果 与空白组比较,模型组小鼠的mPAP、RVSP、RVHI均显著升高(P<0.01);肺小动脉壁明显增厚,管腔狭窄,组织间隙变小,血管周围有明显炎性细胞浸润,有血管重塑表现,管壁厚度占外径的百分比(WT%)、管壁面积占血管总面积的百分比(WA%)显著升高(P<0.01);小鼠肺组织的α-SMA荧光强度明显增强,肺小动脉平滑肌层增厚,完全肌化肺小血管比例显著升高(P<0.01);肺组织的蓝色胶原沉积明显增多,CVF显著升高(P<0.01);血清和肺组织匀浆中HYP含量显著升高(P<0.01);小鼠肺组织中骨膜素阳性表达显著上调(P<0.01);肺组织中Periostin、MMP-9、TIMP-1、TGF-β1、Col-Ⅰ、Col-Ⅲ蛋白表达显著上调(P<0.01),Smad2/3磷酸化水平及MMP-9/TIMP-1比例显著升高(P<0.01)。与模型组比较,黄芪甲苷低、中、高剂量组小鼠的m PAP、RVSP、RVHI均显著降低(P<0.01);小鼠的血管壁增厚情况有显著改善,WT%、WA%均明显降低(P<0.01);小鼠肺组织的α-SMA荧光强度明显降低,肺小动脉平滑肌层变薄,完全肌化肺小血管比例显著降低(P<0.01);肺组织的蓝色胶原沉积明显减少,CVF明显降低(P<0.05,P<0.01);血清和肺组织匀浆中HYP含量明显降低(P<0.05,P<0.01);小鼠肺组织中骨膜素阳性表达明显下调(P<0.05,P<0.01);肺组织中Periostin、MMP-9、TIMP-1、TGF-β1、Col-Ⅰ、Col-Ⅲ蛋白表达明显下调(P<0.05,P<0.01),Smad2/3磷酸化水平及MMP-9/TIMP-1比例显著下降(P<0.01)。结论 黄芪甲苷能够改善低氧诱导的PAH小鼠的肺动脉压升高、血管重构以及纤维化,其机制可能与下调骨膜素及纤维化相关蛋白表达,调节细胞外基质平衡有关。

Objective To study the protective effect of astragaloside Ⅳ in mice with hypoxia-induced pulmonary arterial hypertension(PAH)and its potential mechanism.MethodsSixty Kunming mice were randomly divided into a blank group,a model group and astragaloside Ⅳ low-,medium- and high- dose groups(40,80 and 120 mg·kg^(-1)).The mice were kept in an atmospheric hypoxic chamber with a constant oxygen concentration of 10% for 4 weeks to establish the PAH model,meanwhile,the drug was administered by gavage once a day.The hemodynamics[right ventricular systolic pressure(RVSP),mean pulmonary artery pressure(mPAP)]and right ventricular hypertrophy index(RVHI)of each group of mice were measured after 4 weeks;HE staining was used to assess the pathological remodeling of small pulmonary arteries;immunofluorescence was used to detect the expression of α-smooth muscle actin(α-SMA) in lung tissue;Masson staining was used to observe the degree of pulmonary fibrosis,and the collagen volume fraction(CVF) was calculated;the content of hydroxyproline(HYP) in serum and lung tissue homogenate was detected by ELISA;the positive expression of periostin in lung tissue was detected by immunohistochemistry;the expression levels of fibrosis-related proteins in lung tissue were detected by Western Blot.ResultsCompared with the blank group,mPAP,RVSP and RVHI were significantly increased in the model group(P<0.01);the wall of small pulmonary arteries was significantly thickened,the lumen was narrowed,the tissue space was smaller,the perivascular inflammatory cells were significantly infiltrated,there was vascular remodeling,the percentage of wall thickness to outer diameter(WT%)and the percentage of wall area to total vessel area(WA%)were significantly increased(P<0.01);the α-SMA fluorescence intensity was significantly enhanced,the smooth muscle layer of small pulmonary arteries was thickened,and the percentage of completely mycotic small pulmonary vessels was significantly increased(P<0.01);α-SMA fluorescence intensity was significantly enhanced in lung tissue,the smooth muscle layer of small pulmonary arteries was thickened,and the proportion of completely muscularized small pulmonary vessels was significantly increased(P<0.01);blue collagen deposition was significantly increased in lung tissue,and CVF was significantly increased(P<0.01);HYP content in serum and lung tissue homogenates was significantly increased(P<0.01);periostin-positive expression in lung tissue was significantly up-regulated(P<0.01).The protein expressions of periostin,MMP-9,TIMP-1,TGF-β1,Col-Ⅰand Col-Ⅲ were significantly up-regulated in lung tissue(P<0.01),and the phosphorylation level of Smad2/3 and the ratio of MMP-9/TIMP-1 were significantly increased(P<0.01).Compared with the model group,the mPAP,RVSP and RVHI of mice in the astragaloside IV low-,medium-and high-dose groups were significantly decreased(P<0.01);the thickeness of blood vessel walls in mice was significantly improved,and the WT% and WA% were significantly decreased(P<0.01);the α-SMA fluorescence intensity of mice lung tissue was significantly decreased,the smooth muscle layer of small pulmonary arteries was thinned,and the percentage of completely muscularized small pulmonary vessels was significantly decreased(P<0.01);blue collagen deposition in lung tissue was significantly reduced and CVF was significantly decreased(P<0.05,P<0.01);HYP content in serum and lung tissue homogenate was significantly reduced(P<0.05,P<0.01);positive expression of periostin in lung tissue was significantly down-regulated(P<0.05,P<0.01);protein expressions of periostin,MMP-9,TIMP-1,TGF-β1,Col-Ⅰand Col-Ⅲ were significantly down-regulated in lung tissue(P<0.05,P<0.01),and Smad2/3 phosphorylation level and MMP-9/TIMP-1 ratio were significantly decreased(P<0.01).Conclusion Astragaloside Ⅳ improves pulmonary artery pressure elevation,vascular remodeling and fibrosis in mice with hypoxia-induced PAH.The mechanism may be related to the down-regulation of periostin and fibrosis-related proteins and the regulation of extracellular matrix homeostasis.