Effects Of Resveratrol On P27^kip1 Expression And Mechanism In Rat Thoracic Aorta Smooth Muscle Cells Induced By High Glucose

Objective: Diabetes is a chronic disease with no cure. Diabeticmacrovascular complications includes hypertension, coronary artery disease,cerebrovascular disease, and peripheral vascular disease. Macrovasculardisease is the leading cause of death in patients with diabetes, causingseventy-five percent of the deaths in this population. Compared withnon-diabetic patients, Cardiovascular risk of death is increased three fold,cerebrovascular risk of death is increased two fold, and amputation risk isincrease five fold. Thus, it should be appreciated that the complications ofdiabetes can eventually become as problematic as the primary disease ofdiabetes, if not more so. Unfortunately, at the time of diabetes diagnosis, theprogression of long-term complications has already started in almost onefourth of diabetic patients. Atherosclerosis is the basic pathological changes ofthe macrovascular complications, in the development of diabetic atherogenesis,vascular smooth muscle cell proliferation is recognized as a key event.Epidemiological studies have shown that：Despite a high intake of dietarycholesterol and saturated fat in the southern France, because of its long-termconsumption of red wine rich in resveratrol, there is a low morbidity andmortality of coronary artery disease, compared to the other western countriesof the similar diet habits. This is the famous "French paradox". Resveratrol (3,4',5-trihydroxy-stilbene), high content in red wine and other plants, is onekind of non-flavonoid polyphenol compounds and a phytoalexin producednaturally by several plants, and have a wide range of biological andpharmacological effects, including oxidative stress, anti-inflammatory,cardiovascular protective effect, reducing blood glucose, improving insulinresistance, regulation of lipid metabolism, immune regulation, anti-tumor, anti-aging and estrogen-like activity. Resveratrol play a protective role inatherosclerosis through multiple mechanisms, such as inhibiting oxidation oflipoprotein and proliferation of vascular smooth muscle cells, aggregation ofplatelet and inflammation reaction.Recent studies have shown that, the effect of cell cycle progression withcell cycle kinase inhibitor (CKI)-p27^kip1is closely related to atherosclerosis.Cell cycle progression is controlled by a series of kinase complexes,composed of cyclins and cyclin-dependent kinases(CDKs) and CDK inhibitors(CKIs). p27^kip1is one of the CKIs and elimination of p27^kip1during the lateG1-phase is required for cell cycle progression from the G1to S phase, tocomplete the cells proliferation.In our research, by studying the effect of resveratrol to the proliferation ofrat aortic smooth muscle cells and expression of p27^kip1, p-p38MAPK protein,investigate the pathogenesis of diabetic macrovascular complications andpharmacological effects of resveratrol.Methods：1. Vascular smooth muscle cell lines A7r5were purchased from CCTCC(China Center for Type Culture Collection). The cryopreserved A7r5cellswere quickly put into37℃warm water, shaked as soon as possible to thaw,centrifugated in800rpm for5minutes. Then the supernatant was removed byadding and inoculated with5ml DMEM culture medium containing20%fetalbovine serum(FBS) and putted in the37℃,5%CO2incubator. The cell ofexponential phase of growth were inoculated in50ml culture bottles and on96pore culture boards for24hours. The above cells were replaced by DMEMmedia with0%FBS for24hours to make the cells in phase G0/G1and thendivided into seven groups:(1) Contorl group:5.5mmol/L glucose(2) High glucose group:25mmol/L glucose(3) Mannitol contorl group:5.5mmol/L low glucose and19.5mmol/Lmannitol(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) p38MAPK-inhibitor group:25mmol/L high glucose and10μmol/LSB2035802. The cells of exponential phase of growth were harvested for thefollowing experiment. The effect of Res on the cell proliferating viability inhigh glucose was observed by MTT assay.3. Flow cytometry was preformed to analyze the influences of Res onVSMCs cell cycle distribution in high glucose.4. The effect of Res on p-p38MAPK, p27^kip1protein expression inVSMCs incubated in high glucose was detected by Western blot.Results:1. MTT assays were used to characterize the proliferating viability ofVSMCs. Compared with the low glucose group,the viability of VSMCsinduced by the high glucose was notably increased (P<0.05). As an osmoticcontrol, mannitol had no significant proliferative effect on VSMCs; Res(25,50,100μmol/L)can inhibit the proliferation of VSMCs induced by highglucose in a dose-dependent manner (P<0.05).2. To detect the VSMCs cell cycle progression, flow cytometric analysiswas performed. High glucose can induce cell cycle progression from G0/G1phase to S phase, and the equal concentration of mannitol did not affect cellcycle progression. Res (25,50,100μmol/L) can inhibit VSMCs cell cycleprogression from G0/G1phase to S phase, the cell number remarkablyincreased in G0/G1phase and decreased in S phase(P＜0.05).3. Western blot demonstrated that high glucose significantly up-regulatedthe expression of p-p38MAPK protein and down-regulated the expression ofp27^kip1protein(P＜0.05); mannitol did not affect the expression of targetprotein; The expression of p-p38MAPK protein decreased and the expressionof p27^kip1protein increased with pre-treatment of p38MAPK inhibitorSB203580(P＜0.05). Res (25,50,100μmol/L) significantly decreased theexpression of p-p38MAPK protein and increased p27^kip1protein in a dose-dependent manner(P＜0.05).Conclusions:1. High glucose can promote VSMCs proliferation through activating thep38MAPK signaling pathway and inhibiting p27^kip1protein expression.2. Resveratrol can inhibit VSMCs proliferation induced by high glucose,through suppressing p38MAPK signaling pathway and up-regulating p27^kip1protein expression.In summary, resveratrol may play an important protective role against thediabetic macroangiopathy through inhibiting VSMCs proliferation.