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目的研究内皮-间充质转化在低氧肺动脉高压的作用及积雪草苷(AS)对内皮-间充质转化的影响。方法 (1)体内实验:雄性SD大鼠分为常氧对照组、低氧模型组、AS 25和50 mg·kg~(-1)给药组。间歇低氧法(每天低氧8 h,每周6 d,持续4周)制备HPH大鼠模型,AS组用AS 25或50 mg·kg~(-1)·d~(-1)治疗4周。(2)体外实验:将人肺动脉内皮细胞(HPAEC)分为常氧组和低氧模型组,低氧模型组在低氧条件下(5%O_2,5%CO_2)培养,用AS 0,25,50,100或200 mg·L~(-1)作用72 h,CCK-8法检测HPAEC存活情况;再将HPAEC分为常氧对照组、常氧AS 100 mg·kg~(-1)组、低氧模型组和低氧AS 100 mg·kg~(-1)组,细胞培养5 d后用Transwell法检测HPAEC迁移能力,免疫荧光及Western蛋白印迹检测CD31和α平滑肌肌动蛋白(α-SMA)蛋白的表达水平。结果在体内和体外实验中,免疫荧光和Western蛋白印迹结果均显示,与常氧对照组相比,低氧模型组的CD31表达下降(P<0.01),α-SMA表达升高(P<0.01);与低氧模型组相比,AS给药组CD31表达升高,α-SMA表达减少(P<0.05,P<0.01)。HPAEC的增殖及迁移能力结果显示,与常氧对照组相比,低氧模型组HPAEC的增殖及迁移能力升高;AS 100 mg·L~(-1)能抑制低氧培养72 h后的HPAEC的增殖和迁移能力(P<0.05)。结论 HPH可能与内皮-间充质转化有关,AS对此有一定的抑制作用。
Objective To investigate the role of endothelial-mesenchymal transition in hypoxic pulmonary hypertension and the effect of asiaticoside (AS) on endothelial-mesenchymal transition. Methods (1) In vivo experiments: Male Sprague-Dawley rats were divided into normoxic control group, hypoxia model group, AS 25 and 50 mg · kg -1 groups. HPH rat model was established by intermittent hypoxia (hypoxia 8 h per day, 6 d per week for 4 weeks). AS group was treated with AS 25 or 50 mg · kg -1 d -1 week. (2) In vitro experiments: Human pulmonary artery endothelial cells (HPAEC) were divided into normoxia group and hypoxia model group. The hypoxia model group was cultured in hypoxia (5% O2, 5% CO2) , 50, 100 or 200 mg · L -1 for 72 h. The survival of HPAEC was detected by CCK-8 assay. HPAEC was divided into normoxic control group, normoxia AS 100 mg · kg -1 group Oxygen group and hypoxia AS 100 mg · kg -1 group. After cultured for 5 days, the migration ability of HPAEC was detected by Transwell method. The expressions of CD31 and α-smooth muscle actin (α-SMA) were detected by immunofluorescence and Western blot. Protein expression levels. Results The results of immunofluorescence and Western blot showed that the expression of CD31 in hypoxia model group was significantly lower than that in normoxic control group (P <0.01) ). Compared with hypoxia model group, the expression of CD31 and α-SMA in AS group were significantly decreased (P <0.05, P <0.01). The results of proliferation and migration of HPAEC showed that compared with normoxia control group, the proliferation and migration ability of HPAEC in hypoxia model group increased; AS 100 mg · L -1 could inhibit HPAEC Proliferation and migration ability (P <0.05). Conclusion HPH may be related to endothelial-mesenchymal transition, and AS may have a certain inhibitory effect on this.