Effect Of Metformin On Proliferation Of Pulmonary Arterial Smooth Muscle Cells Induced By Nicotine And Its Underlying Mechanism

BackgroundPulmonary hypertension(PH) is a common complication of chronic obstructive pulmonary disease(COPD),which is associated with increased risk of exacerbation and reduced survival. However, nowadays, there is no reliable curative effect of prevention and treatment measures. It is well known that the main feature of the pathophysiology of PH in COPD patients is represented by pulmonary vascular remodeling, which is caused by the combined effects of hypoxia, inflammation and loss of capillaries in severe emphysema. However, based on these theory, the current treatment for PH in COPD including long term oxygen therapy(LTOT) and other medical treatment including calcium channel blockers and prostacyclin analogues, endothelin-1 receptor antagonists cannot reverse the progression of PH complicating COPD. More research on the underlying mechanisms that regulate the development of PH in COPD is needed to identify novel therapeutic targets.80-90% of COPD is associated with cigarette smoking. Much evidence has shown that cigarette smoking induces pulmonary vascular remodeling in patients with mild-to-moderate COPD and even in smokers with normal lung function. Although the precise mechanisms underlying this process remain unclear, recent several studies have shown that inflammation in COPD appears to increase the risk for developing PH in COPD. The circulating levels of inflammatory cytokines including interleukin(IL-6), tumor necrosis factor(TNF-а), monocyte chemotactic protein 1(MCP 1) have strong linear correlation with mean pulmonary arterial pressure. Additionally,the high-sensitivity c-reactive protein(hs-CRP)level can be used as an independent predictor of pulmonary hypertension. These data supported the hypothesis that chronic inflammation caused by cigarette smoking contributed to development of pulmonary vascular remodeling and pulmonary hypertension in COPD patients.There is much evidence that AMPK(AMP-activated protein kinase) is an new target of regulating oxidative stress and inflammation in addition to its traditional role as an energy sensor and regulator. The key mechanism in the pathogenesis of COPD is thought to be an abnormal inflammatory response and imbalance between oxidants and antioxidants in the lungs to the inhalation of toxic particles especially from tobacco smoke. A growing evidence showed that decreased AMPK activity was closely related to high oxidative stress and inflammation in tissues, and that activated AMPK inhibited inflammation-related gene expression and reduced tissue damage Metformin, the first-line drug of choice for the treatment of type 2 diabetes, has been recently described as an AMPK activator. A study by Agard et al, showed that Metformin can inhibit Monocrotaline and hypoxia-induced pulmonary hypertension, the underlying mechanism by which might be dependent on the activation of AMPK. Isoda found that metformin can inhibit the activation of NF-κB and release of IL-6 by activating AMPK in endothelial cells, smooth muscle cells and macrophages. On the basis of these observations, we reasoned that AMPK might be involved in COPD pathogenesis, and that metformin, the activator of AMPK, might inhibit the progression of pulmonary vascular remodeling andThis study aimed to investigate the effect of Metformin on the proliferation of nicotine-induced PASMCs, and explore the underlying mechanism,which contributed to the identification of metformin as an effective therapeutic agent in PH associated with smoking-related COPD.Objectives:1.To identify the optimal concentration of nicotine to induce proliferation of PASMCs2.To observe the effect of metformin on the nicotine-induced PASMCs proliferation.3.To observe the effect of metformin on the proliferation of PASMC, release of IL-6,TNF-α, production of ROS and explore the underlying mechanism.Materials and methods1. The primary PASMCs were cultured and identified2. CCK-8 assay was used to detect the proliferation of PASMCs induced by nicotine3. ELISA assay was used to detect the level of IL-6 and TNF-α in PASMCS4. ROS production was detected by flow cytometry.5. The expression of AMPK and phosphorylated AMPK proteinin PASMCs was detected by western blot.Results1.Immunofluorescence staining showed FITC labeled α-actin distributed in cytoplasm of primarily cultured PASMCs, which is parallel to long axis of cells.2.0.1μmol/ L nicotine induced proliferation of PASMCs significantly(P < 0.05), after 2mmol/L metformin treatment, the proliferation of PASMC induced by nicotine was inhibited significantly.3.Nicotine induced of the release of IL-6, TNF-α in PASMCs, which reached peak at the 6h.However, after 6h treatment with metformin, the release of IL-6 and TNF-α was inhibited significantly( P < 0.05).4. 0.1μmol/L nicotine induced significantly the production of ROS, which reached the peak(P < 0.05) in 60 min, then it began to decline. After 60 min treatment with metformin, the level of ROS decreased significantly(P < 0.05).5.The phosphorylated level of AMPK increased significantly at 1h exposure to 0.1μmol/L nicotine, then decreased gradually, while the expression levels of AMPK protein had no significant difference at different time point. Metformin maintained phosphorylation of AMPK at 1h, 6h, 12 h, 24 h exposure to nicotine, and the phosphorylated level of AMPK increased significantly after 12 h treatment with metfomin(P < 0.05).Conclusion1. Nicotine induced proliferation of PASMCs and release of IL-6, TNF-α and ROS, accompanied by the transient phosphorylation of AMPK.2. Metformin inhibited proliferation of PASMCs and release of IL-6, TNF-α and ROS induced by Nicotine.3. Metformin inhibited nicotine-induced inflammation,oxidative stress and proliferation of PASMC, which might be dependent on the activation of AMPK.