Resveratrol Attenuates Endotoxin-induced The Inflammatory Response And NF-κB Activation Via Activation Of The Mammalian Target Of Rapamycin Pathway In H9c2Cardiomyocytes

Objective:The study was to investigate the protective and signal mechanism of resveratrol in myocardial ischemia-reperfusion injury with the lipopolysaccharide (LPS)-induced H9C2cardiomyocytes modle.Methods:H9C2cardiomyocytes were cultured and treated differently in vitro. During the experiments, H9C2cardiomyocytes were randomly divided into four groups, including the blank control group, the LPS-stimulated (1μg/ml,20min or24h) group, resveratrol (0.05,1, and10μM) was added1h before LPS treatment group and pretreatment with1μM Rapamycin for1h then added resveratrol and LPS treatment group. The MTT were used to detected H9C2cardiomyocytes vitality. The mRNA and protein levels of pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2), and pro-inflammatory cytokines, tumor necrosis factor-a (TNF-a) and interleukin-6(IL-6) were analysed by RT-PCR and double immunofluorescence labeling, respectively. NF-κB DNA-binding activity was determined by EMSA. Phosphorylation levels of mTOR、IκB and mitogen-activated protein kinases (MAPKs) cascades were measured by western blotting.Results:Resveratrol significantly attenuated the LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2), tumor necrosis factor-a (TNF-a) and interleukin-6(IL-6) in H9C2cardiomyocytes. Resveratrol increased mTOR phosphorylation in a time-dependent manner. Rapamycin, a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced H9C2cells inflammatory response. Meanwhile, mTOR inhibition markedly reversed resveratrol-inhibited the nuclear translocation and DNA binding ability of NF-κB in H9C2cardiomyocytes. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB and mitogen-activated protein kinases (MAPKs) cascades.Conclusions:This investigation demonstrates that Resveratrol and/or Rapamycin do not affect the viability of H9C2cardiomyocytes; mTOR activation is an important signaling in resveratrol-mediated inhibition of translocation and DNA binding ability of NF-κB, phosphorylation of IκB and MAPKs activation, pro-inflammatory mediators and cytokines production in response to LPS in H9C2cardiomyocytes.