Previous studies have shown that metformin (MET) prevents experimental pulmonary arterial hypertension (PAH) and that activation of autophagy is involved in the development of pulmonary vascular remodeling. However, the mechanism of how MET inhibits autophagy and reverses pulmonary vascular remodeling is still unclear. The objective of the present study was to investigate the role of autophagy in MET-induced hypoxia PAH protection and the underlying mechanisms. To examine the effects of MET on hypoxia, we treated rats with MET (100 mg/kg/day) after 3 weeks of hypoxia. Hemodynamic changes, weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and lung morphological features were examined after 3 weeks. In addition, alpha smooth muscle actin (α-SMA), p62, and PCNA were assessed by immunofluorescence and immunohistochemistry staining. BECN-1, LC3B, p62, and activation of adenosine monophosphate-activated protein kinase (AMPK) were analyzed by Western blotting. Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) and the 5-ethynyl-2′-deoxyuridine staining kit assay. Hypoxia induced increases in right ventricular systolic pressure and the RV/LV+S ratio, which were attenuated by MET treatment. MET also inhibited hypoxia-induced pulmonary vascular remodeling, collagen deposition, proliferation of pulmonary arterial smooth muscle cells, elevation of BECN-1 and the LC3B-II/-I ratio, and downregulation of p62. Further studies found that this process was mediated by inhibition of autophagy and activation of the AMPK signaling pathway.

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodelling (PVR). The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important cause of PVR leading to PAH. Mitochondria play a key role in the production of hypoxia‐induced pulmonary hypertension (HPH). However, there are still many issues worth studying in depth. In this study, we demonstrated that NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 like 2 (NDUFA4L2) was a proliferation factor and increased in vivo and in vitro through various molecular biology experiments. HIF‐1α was an upstream target of NDUFA4L2. The plasma levels of 4‐hydroxynonene (4‐HNE) were increased both in PAH patients and hypoxic PAH model rats. Knockdown of NDUFA4L2 decreased the levels of malondialdehyde (MDA) and 4‐HNE in human PASMCs in hypoxia. Elevated MDA and 4‐HNE levels might be associated with excessive ROS generation and increased expression of 5‐lipoxygenase (5‐LO) in hypoxia, but this effect was blocked by siNDUFA4L2. Further research found that p38‐5‐LO was a downstream signalling pathway of PASMCs proliferation induced by NDUFA4L2. Up‐regulated NDUFA4L2 plays a critical role in the development of HPH, which mediates ROS production and proliferation of PASMCs, suggesting NDUFA4L2 as a potential new therapeutic target for PAH.