Resveratrol Suppresses High Glucose-induced Vascular Smooth Muscle Cell Proliferation:Role Of Nuclear Factor-kB Pathway

Objective: Epidemiologic data of clinical events in patients with diabetes mellitus indicate that macrovascular complications, which is based on atherosclerosis, is the major cause of death. It has been estimated that patients with diabetes have about threefold to fourfold risk of death from atherosclerosis disease when compared with age-matched populations. However, atherosclerosis appears to proceed at a more rapid rate and is more extensive in diabetic than in nondiabetic patients. Therefore, diabetes mellitus is considered as an independent risk factors for atherosclerosis. The fact that diabetic patients have an increased risk of atherosclerotic vascular disease has been well documented. However, the reason for this risk is less well understood. The proliferation of vascular smooth muscle cells are important steps in the formation and developmentResveratrol (Res), a polyphenol compound found in grapes, peanuts, polygonum cuspidatum and other plants, is a phytoalexin produced naturally by several plants when under attack by pathogens such as bacteria or fungi. Res exerts several health benefits including anti-inflammatory, anti-oxidant stress, anti-aging and anti-cancer effects. However, it is possible that Res can reduce the risk of cardiovascular disease through multiple mechanisms including inhibit the proliferation of VSMCs. In the present study, the proliferation of vscular smooth muscle cells were induced by high glucose. In order to elucidate whether resveratrol can inhibit the proliferation of vscular smooth muscle cells and its related mechanisms.Methods:1. Vascular smooth muscle cell lines A7r5were purchased from CCTCC (China Center for Type Culture Collection). The cryopreserved A7r5cells were quickly put into37℃warm water, shaked as soon as possible to thaw, centrifugated in800rpm for5minutes. Then the supernatant was removed by adding and inoculated with5ml DMEM culture medium containing20%fetal bovine serum(FBS) and putted in the37℃,5%CO₂incubator. The cell of exponential phase of growth were inoculated in50ml culture bottles and on96pore culture boards for24hours. The above cells were replaced by DMEM media with0%FBS for24hours to make the cells in phase G0/G1and then divided into seven groups:(1) Low glucose contorl group:5.5mmol/L glucose(2) High glucose group:25mmol/L glucose(3) Mannitol contorl group:5.5mmol/L low glucose and19.5mmol/L mannitol(4) Res-treated group:25mmol/L high glucose and25μmol/L Res(5) Res-treated group:25mmol/L high glucose and50μmol/L Res(6) Res-treated group:25mmol/L high glucose and100μmol/L Res(7) NF-kB-inhibitor group:25mmol/L high glucose and30μmol/L PDTC2. The cells of exponential phase of growth were harvested for the following experiment. The effect of Res on the cell proliferating viability in high glucose was observed by MTT assay3. Flow cytometry was preformed to analyze the influences of Res on VSMCs cell cycle distribution in high glucose.4. The effect of Res on NF-kB, cyclinD1protein expression in VSMCs incubated in high glucose was detected by Western blot.Results:1. MTT assays were used to characterize the proliferation of VSMCs. The proliferation of VSMCs induced by high glucose was notably increased, comparing with the low glucose group (P<0.05). Meanwhile, the proliferative characters of VSMCs were not changed after treatment with mannitol control. Res, at25,50,100μmol/L respectively, can inhibit the proliferation of VSMCs induced by high glucose in a dose-dependent manner (P<0.05). 2. To detect the VSMCs cell cycle progression, flow cytometric analysis was performed. High glucose can induce cell cycle progression from G0/G1phase to S phase, and the progression was not affected by mannitol. Res (25,50,100μmol/L) can inhibit VSMCs cell cycle progression from G0/G1phase to S phase, the cell number remarkably increased in G0/G1phase and decreased in S phase(P<0.05).3. Western blot demonstrated that high glucose significantly up-regulated the expressions of NF-kB and cyclinD1; expression of target protein had no change when treated with mannitol; the up-regulating effects were attenuated when pre-treating with NF-kB inhibitor PDTC. Res (25,50,100μmol/L) significantly decreased the expressions of NF-kB p65protein and cyclinD1protein in a dose-dependent manner.Conclusions:1. High glucose can promote the cell cycle progression from Go/G1to S phase and the proliferation of VSMCs through the activation of NF-kB signaling pathway and inductiong of downstream target gene cyclinD1expression, indicating that high glucose condition may represent one of the mechanisms for atherosclerosis observed in T2DM.2. By interfering in NF-kB/cyclinD1pathway, resveratrol can inhibit the cell cycle progression from Go/Gi phase to S phase and the proliferation of VSMCs cultured in high glucose in a dose-dependent manner. These findings suggested that resveratrol may play a protective role against diabetic macrovascular complications through the intervention of proliferation in VSMCs.