Indole-3-carbinol inhibits the proliferation of hypoxia-induced pulmonary arterial smooth muscle cells by blocking the mTOR/HIF-1α pathway
Objective To determine the effect of indole-3-carbinol(I3C)on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells(PASMCs).Methods PASMCs were isolated from the pulmonary arteries of SD rats and digested with 0.2％collagenase Ⅰ.PASMCs were treated with hypoxia(1％O2)to stimulate the proliferation,then intervened with different concentrations of I3C(25,50,100,and 200 μmol·L-1)for 24 h to determine the cell proliferation and viability.To explore the molecular mechanisms of how I3C suppressed the cell proliferation under hypoxic conditions,PASMCs were treated with 100 μmol·L-1 I3C,mammalian target of rapamycin(mTOR)agonist MHY1485 or hypoxia-inducible factor 1α(HIF-1α)stabilizer DMOG for 24 h,and groups treated with mTOR inhibitor rapamycin or HIF-1α inhibitor LW6,were used as positive controls.Cell proliferation was analyzed by the CCK-8 kit.The cell cycle progression was determined by flow cytometry.The expressions of HIF-1α,total and phosphorylated mTOR,and cell cycle regulatory proteins were examined by Western blot.Results The proliferation of hypoxia-induced PASMCs was inhibited with I3C(25～100 μmol·L-1)in a concentration-dependent manner(P＜0.05).There was no obvious difference in the inhibition of hypoxia-induced PASMC proliferation treated with 100 and 200 μmol·L-1 I3C,and I3C was not cytotoxic at the experimental concentration.Both I3C(100 μmol·L-1)and LW6 blocked the proliferation of PASMCs,arrested cell cycle progression in G0/G1 phase,downregulated the expression of HIF-1α,Cyclin D1,Cyclin E,Cyclin-dependent kinase(CDK)2,CDK4 and CDK6(P＜0.05),while DMOG reversed all the above effect of I3C on hypoxia-induced PASMCs(P＜0.05).In addition,I3C(100 μmol·L-1)exerted similar effects with rapamycin in inhibiting the proliferation and inactivated the mTOR/HIF-1α pathway in hypoxia-induced PASMCs.More specifically,MHY1485 reversed the effect of I3C on blocking cell proliferation and inhibiting the mTOR/HIF-1α pathway in hypoxia-induced PASMCs(P＜0.05).Conclusion I3C may inhibit the cell proliferation of hypoxia-induced PASMCs by blocking the mTOR/HIF-1α pathway.
hypoxiapulmonary artery smooth muscle cellindole-3-carbinolproliferationmammalian target of rapamycin/hypoxia-inducible factor 1α
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