Signal Mechanisms And The Effects Of Resveratrol On MMP-2and MMP-9Expression In Rat Thoracic Aorta Smooth Muscle Cells Induced By High Glucose

Objective: Diabetic cardiovascular disease, one of the most commonchronic vascular complications of diabetes, has also been the leading cause ofdeath in patients with diabetes. There is a strong correlation between theoccurrence of cardiovascular disease and long-term hyperglycemic stateinduced atherosclerosis (AS). The migration of vascular smooth muscle cells(VSMCs) into the intima and their subsequent proliferation (intimalhyperplasia) plays a key role in the development of AS.Matrix metalloproteinases (MMPs), a family of metal ion-dependentproteolytic enzymes, have the ability to degrade almost all components of theextracellular matix, which play an important role in the formation anddevelopment of the atherosclerotic plaque. Currently, the study of MMP-2andMMP-9has become a hot topic. No matter the clinical research of pateintswith diabetes, diabetic animal model, or cultured vascular smooth muscle cellsunder high glucose condition, were confirmed MMP-2and MMP-9expressionand activity increased. Obviously, MMPs play a significant role in theoccurrence and development of diabetic angiopathy. However, the molecularmechanisms of activation of MMPs under high glucose conditions are not yetclear.Reseveratrol (Res), a promising natural phytoalexin, has been widelystudied over the recent years. It is regarded commonly that Resveratrol has awide range of biological and pharmacological effects, including anti-inflammatory, anti-cancer, anti-oxidant stress, anti-aging, inhibition of plateletaggregation, regulation of lipid metabolism, immune regulation and also hascardiovascular protective effect, such as inhibiting proliferation and migrationof VSMCs to reduce the risk of AS. At present, not many studies were to observe whether AS could be prevented by reseveratrol through adjusting theexpression level and activity of MMP-2and MMP-9.In this study, different concentrations of resveratrol and Ly294002effectcultured rat thoracic aorta smooth muscle cells in high glucose, which isdesigned to detect the expression level and activity of MMP-2and MMP-9and the protein expression of p-Akt, Akt. We sought to investigate whether themechanisms of changes in expression and activity of MMPs were associatedwith PI3K signaling pathway and to explore the possible mechanism ofresveratrol. It is possible to provide new targets for early prevention andtreatment of diabetic macroangiopathy, which also to provide experimentalbasis for the clinical application of resveratrol.Methods：1Vascular smooth muscle cell lines A7r5were purchased from ChinaCenter for Type Culture Collection (CCTCC). 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 and putted in the37℃,5%CO2incubator. The cells ofexponential phase of growth were inoculated in50ml culture bottles for48hours. The above cells were replaced by DMEM media with0%FBS for24hours to make the cells in phase G0/G1and then divided into some groups:①Normal glucose (NG) group:5.5mmol/L glucose②High glucose (HG) group:25mmol/L glucose③NG and Ly294002-treated (10μmol/L,20μmol/L,30μmol/L) group④HG and Ly294002-treated (10μmol/L,20μmol/L,30μmol/L) group⑤NG and Res-treated (25μmol/L,50μmol/L,100μmol/L) group⑥HG and Res-treated (25μmol/L,50μmol/L,100μmol/L) group2The proliferating viability of VSMCs was observed by MTT assay.3Immunohistochemistry (SABC) was used to examine the expression ofMMP-2and MMP-9.4The activity of MMP-2and MMP-9was measured by SDS-PAGE zymography.5The protein expression of p-Akt and Akt was detected by Western Blot.Results:1MTT assay was used to characterize the proliferating viability ofVSMCs. Compared with the NG group (0.357±0.009)，the proliferatingviability of VSMCs induced by the high glucose was notably increased (0.513±0.007, P<0.05). The proliferation of VSMCs in the NG plus Ly294002(10,20,30μmol/L) group were0.382±0.010,0.382±0.013,0.382±0.012separately, and the proliferation of VSMCs in the NG plus Res (25,50,100μmol/L) group were0.380±0.024,0.380±0.027,0.381±0.022separately.From these data it can be seen that the NG plus Ly294002(10,20,30μmol/L)group and the NG plus Res (25,50,100μmol/L) group had no significantproliferative effect on VSMCs (P>0.05), no toxic reaction was foundbasically.The proliferation of VSMCs induced by high glucose in Ly294002(10,20,30μmol/L) were0.421±0.016,0.402±0.017,0.373±0.022separately，and the proliferation of VSMCs induced by high glucose in Res(25,50,100μmol/L) were0.474±0.023,0.438±0.031,0.386±0.013separately. From these data it can be seen that Ly294002(10,20,30μmol/L)and Res (25,50,100μmol/L) can inhibit the proliferation of VSMCs inducedby high glucose in a dose-dependent manner (P<0.05).2Immunohistochemistry (SABC) was used to detect the expression ofMMP-2and MMP-9in each group. Compared with the NG group (0.240±0.015,0.204±0.021), the expression of MMP-2and MMP-9induced by thehigh glucose was notably increased (0.380±0.024,0.363±0.052, P<0.05).The expression of MMP-2induced by high glucose in Ly294002(10,20,30μmol/L) were0.323±0.044,0.282±0.026,0.248±0.013separately, andthe expression of MMP-9induced by high glucose in Ly294002(10,20,30μmol/L) were0.302±0.040,0.256±0.024,0.245±0.014separately. Theexpression of MMP-2induced by high glucose in Res (25,50,100μmol/L)were0.301±0.011,0.270±0.019,0.252±0.017separately, and theexpression of MMP-9induced by high glucose in Res (25,50,100μmol/L) were0.322±0.030,0.280±0.019,0.247±0.009separately. From these datait can be seen that Ly294002(10,20,30μmol/L) and Res (25,50,100μmol/L)can inhibit the expression of MMP-2and MMP-9induced by high glucose in adose-dependent manner (P<0.05). In addition to Res (100μmol/L) groupinduced by high glucose, which the expression of MMP-2had no significantdifference with the NG group (P>0.05), the expression of MMP-2and MMP-9for the rest of any two groups were still higher than the NG group (P<0.05).3SDS-PAGE zymography demonsrated that the activity of MMP-2andMMP-9induced by the high glucose (8969.000±201.968,9339.667±128.469) was notably increased as compared with the NG group (2599.000±88.476,2677.667±146.302, P<0.05). The activity of MMP-2induced by thehigh glucose in Ly294002(10,20,30μmol/L) were5962.667±211.316,4997.000±117.758,3792.333±137.012separately， and the activity ofMMP-9induced by the high glucose in Ly294002(10,20,30μmol/L) were7574.333±101.041,6233.000±158.206,4741.667±80.432separately. Theactivity of MMP-2induced by the high glucose in Res (25,50,100μmol/L)were7435.333±163.369,6209.667±195.372,5502.667±70.316separately,and the activity of MMP-9were8440.667±148.008,7889.333±155.828,6541.000±55.018separately. From these data it can be seen that Ly294002(10,20,30μmol/L) and Res (25,50,100μmol/L) can inhibit the activity ofMMP-2and MMP-9induced by high glucose in a dose-dependent manner(P<0.05), however, the activity of MMP-2and MMP-9of any two groupswere still higher than the NG group (P<0.05).4Western blot demonstrated that the expression of total Akt protein inNG group, HG group and HG plus Ly294002(10,20,30μmol/L) group were0.701±0.013,0.705±0.015,0.678±0.021,0.682±0.015,0.674±0.012separately，which had no significant difference among these groups (P>0.05).However, compared with NG group and HG group, Res (25,50,100μmol/L)can inhibit the expression of total Akt protein induced by high glucose with nodose-dependence (0.530±0.016,0.534±0.016,0.532±0.017, P<0.05).Compared with the NG group (0.085±0.009), high glucose significantly up-regulated the expression of p-Akt protein (0.583±0.017, P<0.05). Theexpression of p-Akt protein induced by high glucose in Ly294002(10,20,30μmol/L) were0.404±0.017,0.244±0.016,0.126±0.017separately, andthe expression of p-Akt protein induced by high glucose in Res (25,50,100μmol/L) were0.500±0.018,0.449±0.016,0.361±0.016separately. Fromthese data it can be seen that Ly294002(10,20,30μmol/L) and Res (25,50,100μmol/L) down-regulated the expression of p-Akt protein induced by highglucose in a dose-dependent manner (P<0.05), but the expression of p-Aktwas still higher than the NG group (P<0.05).Conclusions:1High glucose can increased the expression and activity of MMP-2andMMP-9in cultured VSMCs through activating the PI3K/Akt signalingpathway, which may play an important role in the proliferation of VSMCs.2Resveratrol may inhibit the high experssion of MMP-2and MMP-9incultured VSMCs activated by high glucose through suppressing PI3K/Aktsignaling pathway, while reduce the proliferation of VSMCs induced by highglucose in a dose-dependent manner within a certain range.