Resveratrol Inhibits Oxidized Phospholipids-induced Cx43Phosphorylation And Vascular Smooth Muscle Cell Proliferation

Background:Atherosclerosis is defined as a chronic immuno-inflammatory disease. VascularSmooth Muscle Cell (VSMC) proliferation is a major feature in atherosclerosis. Thegap junction protein, connexin43(Cx43), might be involved in the development ofatherosclerosis. Changes in Cx43expression and function have been found inassociation with neointima formation, macrophage infiltration, VSMC proliferationand migration during the progression of atherosclerotic lesions and following injuryin large arteries. Therefore, suppression of VSMC proliferation and the Cx43activation can be useful therapeutic interventions for Atherosclerosis.OxPLs, active components in minimally modified low density lipoproteins(mmLDL), have been considered to be a risk factor in atherosclerotic lesions. There isa growing body of evidence that OxPLs enhances further lipid accumulation andoxidation, promotes key changes in VSMC phenotype, and triggers the disease state.Recent reports suggest that POVPC promotes VSMC proliferation correspondingwith enhanced Cx43S279/282phosphorylation.Resveratrol (Res) is a polyphenol compound possessing cardiac protectiveeffects．The cardiovascular protection and anti-inflammatory effects of resveratrolhave been documented but the mechanisms underlying these effects are still not fullyunderstood. In addition, our previous results have indicated that resveratrol inhibits AngII-induced VSMC proliferation. Whether there is an inhibitory effect ofresveratrol on POVPC-induced VSMC proliferation and Cx43-phosphorylation?What are the possible mechanisms if the inhibitory effect is present? There are noanswers for these questions; more studies need to be done.Objectives:1. To identify the inhibitory effect of resveratrol on POVPC-induced VSMCproliferation and Cx43phosphorylation.2. To investigate whether Src/MEK/ERK1/2signaling pathway are involved ininhibition of VSMC proliferation and Cx43phosphorylation by resveratrol.Methods:1. VSMCs were treated with various concentrations of resveratrol for24h, the celltoxicity of resveratrol was evaluated by Cell Counting Kit-8(CCK-8) assay. VSMCswere treated for24h with various concentrations of POVPC in DMEM containing10%FBS,the cell proliferation was evaluated by CCK-8assay. VSMCs were treated for30min with resveratrol and then incubated with POVPC for24h. To evaluate the cellviability of various resveratrol doses on POVPC-induced VSMCs with CCK-8assay.The proliferation of VSMCs was measured using EdU Cell Proliferation Assay Kit.The cell cycle of VSMCs was measured by flow cytometry (FCM).2. VSMCs were pretreated with resveratrol and then were cultured with POVPC for30min. To analyze the phosphorylation level of Cx43by western blot.3. VSMCs were cultured with POVPC for10min,20min and30min.To observe thephosphorylation level of ERK1/2, MEK, and Src by western blot. VSMCs werepretreated with resveratrol and then were cultured with POVPC for30min. Toanalyze the phosphorylation level of ERK1/2, MEK, and Src by western blot. 4. VSMCs were pretreated with Src inhibitor(PP2), MEK1inhibitor(PD98059) andthen cultured with POVPC for24h. The proliferation of VSMCs was measured usingEdU Cell Proliferation Assay Kit. The cell cycle of VSMCs was measured by flowcytometry (FCM).5. VSMCs were pretreated with Src inhibitor(PP2), MEK1inhibitor(PD98059) andthen were cultured with POVPC for30min. To analyze the phosphorylation level ofCx43by western blot.Results:1. Effects of resveratrol on OxPLs-induced VSMC proliferationResveratrol-induced cell toxicity was negligible at concentrations of0-50μM inVSMCs. POVPC dose-dependently increased VSMC proliferation, the effect wasgreatest at5μg/ml.POVPC (5μg/ml) significantly increased [3H] thymidine incorporation intoVSMCs, which was prevented by resveratrol in a dose-dependent manner. VSMCswith24-hour stimulation of POVPC increased [3H] thymidine incorporation by27.1%.10μM and50μM resveratrol inhibited POVPC-induced [3H] thymidineincorporation by21.0%and34.0%, respectively.In EdU assay, after stimulation with POVPC (5μg/ml) for24h,48.6%ofVSMCs showed high proliferation, which was1.7-fold higher than that of the controlcells.After24h treatment, FCM analysis showed a27.7%decrease in G1phase cellsand a26.9%increase in S-phase cells in POVPC-treated cells. Resveratrol increasedG1phase cells by20.3%and decreased S phase cells by24.0%compared withPOVPC-treated cells. There were no significantly differences for the G2phase cells.2. Effects of resveratrol on OxPLs induced phosphorylation of Cx43 VSMCs were pretreated with50μmol/L resveratrol for0.5h and were thenstimulated with5μg/ml POVPC for0.5h. The presence of resveratrol significantlyinhibited the POVPC-induced Cx43phosphorylation.3. Effects of OxPLs on phosphorylation levels of Src, MEK and ERK1/2VSMCs were cultured with POVPC for10min,20min and30min. Thephosphorylation levels of Src, MEK and ERK1/2were significantly enhanced at30min.4. Effects of Src, MEK and ERK1/2on OxPLs-induced VSMC proliferation andCx43phosphorylationSrc inhibitor(PP2) and MEK1inhibitor(PD98059) were incubated with VSMCsfor30min prior to POVPC stimulation. After24h stimulation, EdU and Flowcytometry assay were used to determine the cells proliferation. POVPC-inducedVSMC proliferation was strongly inhibited by PP2and PD98059. Stimulation withPOVPC caused a significant increase in VSMC proliferation, and thisPOVPC-induced increase was greatly diminished by the pre-treatment of PP2andPD98059. Flow cytometric analysis demonstrated that PD98059and PP2treatmentinduced cell cycle arrest at the G1phase with concomitant decrease of S phase, butthere were no significantly differences for the G2phase cells.POVPC (5μg/ml) induced Cx43phosphorylation at30min, which were inhibitedby PD98059and PP2.5. Effects of resveratrol on OxPLs-induced phosphorylation of Src, MEK andERK1/2VSMCs were pretreated with50μmol/L resveratrol for0.5h and were thenstimulated with5μg/ml POVPC for30min. The presence of resveratrol significantlyinhibited the POVPC-increased phosphorylation levels of Src, MEK and ERK1/2. Conclusions:1. Resveratrol inhibits OxPLs-induced VSMCs proliferation and Cx43phosphorylation.2. Signal pathways of Src/MEK/ERK1/2are involved in the inhibition of VSMCproliferation and Cx43phosphorylation by resveratrol.