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

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.