Role of Nrf2 regulated oxidative stress in inhibition of hypoxia-induced pulmonary artery smooth muscle cell proliferation by Adropin
Objective To determine the effect of Adropin on hypoxia-induced proliferation and oxidative stress in pulmonary artery smooth muscle cells(PASMCs)of SD rats,and its possible mechanisms.Methods The 1％O2 was used to induce the proliferation and oxidative stress of PASMCs,and then the reactive oxygen species(ROS)activator H2O2,antioxidant N-acetylcysteine(NAC)and different concentrations of Adropin(100,300,and 1000 nmol·L-1)were used for PASMCs for 24 h.The cell proliferation and levels of ROS were measured to determine the optimal concentration of Adropin for inhibiting the proliferation and oxidative stress.To explore the molecular mechanisms of how Adropin suppressed the proliferation and oxidative stress of PASMCs,1000 nmol·L-1Adropin and/or Nuclear factor erythroid 2-related factor 2(Nfr2)inhibitor ML385 were used to treat PASMCs for 24 h under hypoxic conditions.Nfr2 activator dimethyl fumarate(DMF)was used for the positive control group.The cell proliferation was analyzed by the CCK-8 kit;the intracellular ROS level was measured by DCFH-DA with fluorescence microplate reader.Intracellular superoxide dismutase(SOD),glutathione peroxidase(GPx),catalase(CAT),and malondialdehyde(MDA)were determined with the kits.Cell cycle was detected by flow cytometry;the expressions of Nrf2,Cyclin D1 and Cyclin E were measured by Western blot.Results H2O2 and hypoxia-induced the proliferation and ROS production in PASMCs,while different concentrations of Adropin(100,300,and 1000 nmol·L-1)decreased the proliferation and ROS production in hypoxia-exposed PASMCs in a concentration-dependent manner.In addition,the effects of antioxidant NAC on the inhibition of proliferation and ROS production were comparable with those of 1000 nmol·L-1 Adropin.Specifically,Adropin and DMF pretreatment downregulated the level of ROS and MDA,and upregulated the expression of antioxidant-related factors(SOD,GPx and CAT)via Nrf2 activation(P＜0.05).Adropin and DMF blocked the proliferation of hypoxia-exposed PASMCs by arresting the cell cycle progression in G0/G](P＜0.05).In accordance with these findings,DMF and Adropin induced inhibition of the cell cycle was associated with the inhibition of Cyclin D1 and Cyclin E(P＜0.05).Moreover,ML385 reversed the beneficial effects of Adropin on hypoxia-stimulated PASMCs(P＜0.05).Conclusion Adropin suppresses the proliferation of hypoxia-induced PASMCs via inhibition of the oxidative stress,whose mechanism may be related to the activation of Nrf2.
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