Effects Of Resveratrol On SIRT1and NADPH Oxidase-p47Protein Expressions And Mechanisms In Rat Thoracic Aorta Smooth Muscle Cells Induced By High Glucose

Objective: Diabetes (diabetes mellitus, DM) is an incurable chronicdisease. Currently, the prevalence of DM continues to increase worldwide yearby year. Among the most prevalent macrovascular complications are highblood pressure, coronary heart disease, cerebrovascular disease and peripheralvascular disease. Diabetic chronic vascular complications are a major publichealth threat among which cardiovascular disease has become the highestmorbidity and mortality. The central pathological mechanism in diabeticmacrovascular disease is the process of atherosclerosis (AS) in which vascularsmooth muscle cells (VSMCs) change their phenotypes from quiescentcontractile state to the active synthetic state. Then they can migrate andproliferate from media to the intima. These changes may be mediated underhigh glucose concentration environment or inflammatory cytokinesstimulation. High glucose promotes the production of reactive oxygen species(ROS) in vascular endothelial cells, including NADPH oxidase (nicotinamideadenine dinucleotide phosphate oxidase, NOX), xanthine oxidase,mitochondrial respiratory chain enzyme complexes, endothelial nitric oxidesynthase and lipid oxygenase enzymes. NADPH oxidases are believed to bethe major source of vascular ROS. This multicomponent enzyme system iscomposed of cytosolic proteins (p47phox, p67phox, p40phox, and rac1/2). NADPHoxidase-derived ROS play a crucial role in the development of atherosclerosis,which further triggers macrovascular and microvascular disease, resulting indiabetes complications.In recent years, resveratrol (Resveratrol, Res) attracted much attentionbecause of its multiple biological activities. This natural polyphenoliccompound, named as3,4’,5-trihydroxy stilbene (trans-3,4’,5-trihybdroxy stilbene), present in variety of herbs and other plants. Many studies haveshown that resveratrol exerts many biological effects, such as anti-tumor,anti-oxidation, anti-inflammatory, lowing blood sugar, reducing plateletaggregation, vasodilation and cardiovascular protective role against type2diabetes and its related complications. In addition, Res can inhibit the in vitrocultured rat VSMCs proliferation. It has been reported that Res suppresseshigh glucose-induced proliferation of VSMCs, to a certain extent related to theexpression of SIRT1(NAD+-dependent deacetylase). Res is the strongestnatural activator of SIRT1. However, whether and how Res has effects on highglucose-induced VSMCs proliferation via SIRT1pathway is unclear. In thepresent study, we evaluated the effects of resveratrol on the proliferation of rataortic smooth muscle cells and protein expressions of SIRT1, NADPH-p47induced by high glucose, investigating its protective role and potentpharmacological effects on diabetic macrovascular diseases.Methods：1Vascular 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 duringexponential 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 eight groups:(1) Normal control group(control):5.5mmol/L glucose(2) High glucose group(25G):25mmol/L glucose(3) High glucose+SIRT1inhibitor group (25G+Sirtionl):25mmol/Lglucose and50μmol/L Sirtionl(4) High glucose+NADPH oxidase inhibitor group (25G+Apocynin):25mmol/L glucose and1mmol/L Apocynin (5) Mannitol contorl group (Mannitol):5.5mmol/L glucose and19.5mmol/L mannitol(6) Res-treated group:25mmol/L high glucose and25μmol/L Res(7) Res-treated group:25mmol/L high glucose and50μmol/L Res(8) Res-treated group:25mmol/L high glucose and100μmol/L Res2The cells of exponential phase of growth were harvested for thefollowing experiment. The effect of Res on the cell proliferating viabilityinduced by high glucose was observed by MTT assay.3Flow cytometry was preformed to analyze the influences of Res onVSMCs cell cycle distribution induced by high glucose.4The effects of Res on SIRT1, p47protein expressions in VSMCsincubated with high glucose were detected by Western blot.Results:1MTT assays were used to characterize the proliferating viability ofVSMCs. Compared with the normal 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 concentration-dependent manner (P <0.05).2To 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).3Western blot technology were used to investigate Res on VSMCsSIRT1, NADPH oxidase p47protein expressions, the results showed: highglucose can significantly increased NADPH oxidase-p47protein expression,the protein expression of SIRT1was simultaneously decreased (P<0.05);After pretreatment with Apocynin, NADPH oxidase was inhibited, theexpression of NADPH oxidase-p47protein reduced, and the expression of SIRT1protein increased (P <0.05); Pretreatment with Sirtionl can markedlyinhibited SIRT1protein expression, increased the expression of NADPHoxidase-p47protein (P <0.05); No changes were observed in hypertonicgroup; Res (25,50,100μmol/L) could significantly reduced the expression ofNADPH oxidase protein, increased the expression of SIRT1protein in aconcentration dependent manner (P <0.05).Conclusions:1High glucose can promote VSMCs proliferation through activatingthe p47signaling pathway and inhibiting SIRT1protein expression. Highglucose induced pathological proliferation of VSMCs may play a potentialrole in the pathogenesis of diabetic macrovascular diasese.2Res can inhibit VSMCs proliferation induced by high glucose,increase the percentage of cells in G0/G1phases and decrease the percentageof those in the S-phase. The inhibitory effects of Res on VSMCs proliferationinduced by high glucose may via up-regulating SIRT1signaling pathway andsuppressing p47protein expression.In summary, Res inhibits high glucose induced proliferation of VSMCsvia SIRT1/NADPH oxidase-p47pathway, indicating its potential protectiverole against the diabetic macroangiopathy.