脂联素改善低氧诱导的大鼠肺微血管内皮细胞功能障碍及机制研究

Adiponectin improves hypoxia-induced pulmonary microvascular endothelial cell dysfunction and its mechanism

目的研究重组人球状脂联素(APN)改善低氧诱导的大鼠肺微血管内皮细胞(PMVECs)功能障碍,探讨其潜在的分子机制。方法取SD雄性大鼠PMVECs原代培养,传至第三代行细胞鉴定;PMVECs分3组,常氧(210ml/L O_2,37℃)组;低氧(20 ml/L O_2,37℃)组;低氧(20 ml/L O_2,37℃)+APN(1μg/ml)处理组,分别培养3 h、6 h、9 h和12 h,收集细胞上清测NO浓度;3组PMVEC行RT-PCR测定eNOS基因表达;BCA法测定各组总蛋白含量;行Western blot检测AMPK/p-AMPK、PI3K/p-PI3K、Akt/p-Akt和eNOS/p-eNOS表达。结果 (1)鉴定培养细胞为PMVECs;(2)在相应时间点,与正常组比较,低氧诱导的NO浓度、eNOS mRNA表达水平、总蛋白表达显著下降(P<0.01);与低氧组对比,低氧+APN组显著增加了低氧诱导的NO浓度、eNOS mRNA表达水平、总蛋白表达(P<0.01);(3)3组AMPK、PI3K、Akt和eNOS总蛋白表达水平不变;与正常组比较,低氧诱导的AMPK、PI3K、Akt、eNOS磷酸化表达水平下降(P<0.05);与低氧组对比,低氧+APN组增加了低氧诱导的AMPK、PI3K、Akt、eNOS磷酸化表达水平(P<0.05)。结论在细胞水平上,APN能改善低氧条件下诱导的PMVECs功能障碍,促进肺血管具有舒张作用的NO产生,其可能机制可能是伴随着AMPK/PI3K/Akt/eNOS/NO信号通路的激活,APN可能成为潜在治疗低氧性肺动脉高压的辅助药物。

AIM To investigate the effect and molecular mechanism of adiponectin in improving hypoxia-induced dysfunction of rat pulmonary microvascular endothelial cells （PMVECs）. METHODS The third passage of primary cultured PMVECs from SD rats were identified by immunohistochemical method and were divided into three groups. The three groups were cultured respectively under normoxia condition （210 ml/L O2, 37℃）, hypoxia condition （20 ml/L O2, 37℃） or hypoxia condition （20 ml/L O2, 37℃） with treatment of APN （1 μg/ml）. Supernatant and cell lysate were collected at different time intervals at 3 h, 6 h, 9 h and 12 h. NO concentration in supernatant was determined by nitrate reductive enzymatic method, the expression of eNOS-mRNA was observed by RT-PCR, the total protein concentration was detected by the method of BCA, and protein levels of AMPK/p-AMPK, PI3K/p-PI3K,Akt/p-Akt and eNOS/p-eNOS were respectively detected by Western blot from 12 h cells in each group. RESULTS Primary culture of PMVECs was successful. At the same time interval, NO production, mRNA of eNOS and total protein levels in the hypoxia group decreased significantly compared with those in the normoxia group （P〈0.01）. No production, eNOS mRNA and total protein levels in the hypoxia＋ANP group were significantly higher than those in the hypoxia group （P〈0.01）. There was no significant difference in total protein levels of AMPK, PI3K,Akt and eNOS among the three groups. Phosphorylated AMPK, PI3K,Akt and eNOS decreased significantly（P〈0.05） in the hypoxia group compared with those in the normoxia group. Phosphorylated AMPK, PI3K,Akt and eNOS increased significantly（P〈0.05） in the hypoxia＋ANP group compared with those in the hypoxia group. CONCLUSION APN improves hypoxia-induced dysfunction of PMVECs at cellular levels. It can increase the production of NO possibly through the activation of AMPK/PI3K/Akt/eNOS/NO signaling pathway. APN is a potential ancillary drug for the treatment of hypoxia pulmonary hypertension.