Adiponectin Improves Hypoxia-induced Pulmonary Microvascular Endothelial Cells Dysfunction And Underlying Mechanisms

AIM:Pulmonary arterial hypertension(PAH)is a common concern of the global health problem,which is a kind of microvascular disease that affects the heart from the pulmonary artery.Hypoxic pulmonary hypertension is one of the common types of pulmonary hypertension,which is caused by the death of right ventricular volume overload,and finally lead to right heart failure and death.Therefore,it is urgent to clarify the pathogenesis of the disease,and to provide an effective experimental basis for clinical prevention and treatment.The purpose of this study is to explore the effects of recombinant human globular adiponectin(g Ad,APN)through the promotion of nitric oxide(NO)production and the inhibition of endothelin-1(ET-1)generation improve hypoxia induced rat pulmonary microvascular endothelial cells(PMVECs)dysfunction,and explore its potential molecular mechanism.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 O2,50ml/LCO2,740ml/L N2,37°C),hypoxia condition(20 ml/L O2,50ml/LCO2,930ml/L N2,37°C)or hypoxia condition(20 ml/L O2,50ml/LCO2,930ml/L N2,37°C)with treatment of APN.Supernatant and cell lysate were collected at different time intervals at 3h,6h,9h and 12 h.The concentration of NO was measured by nitrate reductive enzymatic method,and the concentration of ET-1 was measured by enzyme linked immunosorbent assay(ELISA)in supernatant,respectively.The expression of e NOS-m RNA and ET-1-m RNA were observed by RT-PCR.The protein levels of AMPK/p-AMPK,p-85-PI3 K,Akt/p-Akt,e NOS/p-e NOS and ERK1/2/p-ERK1/2 were respectively detected by Western blot from 12 h cells in each group.RESULTS:(1)Primary culture of PMVECs was successful.(2)At the same time interval,NO production and m RNA of e NOS in the hypoxia group decreased significantly compared with those in the normoxia group(P<0.01);No production,e NOS m RNA in the hypoxia+ANP groups were significantly higher than those in the hypoxia groups(P<0.01);(3)At the same time interval,compared with the normal group,the concentration of ET-1 and ET-1-m RNA gene expression induced by hypoxia were increased(P<0.05);compared with the hypoxia group,hypoxia+APN group concentration of ET-1 and ET-1-m RNA gene were significantly decreased(P<0.01).(4)There was no significant difference in total protein levels of AMPK,Akt,e NOS and ERK1/2 among the three groups.Phosphorylated AMPK,PI3 K,Akt and e NOS decreased significantly(P<0.05);however,the expression level of ERK1/2 phosphorylation was increased(P<0.05)in the hypoxia group compared with those in the normoxia group.Phosphorylated AMPK,PI3 K,Akt and e NOS increased significantly(P<0.05),nevertheless,phosphorylated ERK1/2 decreased remarkably(P<0.05)in hypoxia+ANP group compared with those in hypoxia group.CONCLUSION:At the cellular level for the first time confirmed that APN may promote pulmonary artery endothelial function of NO generated by the AMPK/PI3K/Akt/e NOS signal pathway,synthesis and secretion of ET-1 by inhibiting ERK1/2/ET-1 signaling pathway in pulmonary artery endothelial function;APN may be a potential adjuvant drug treatment of hypoxic pulmonary hypertension.