Effects Of Resveratrol On MCP-1/VCAM-1Expression And Mechanism In Rat Thoracic Aorta Smooth Muscle Cells Induced By High Glucose

Objective: Diabetic macrovascular complications includes hypertension,coronary artery disease, cerebrovascular disease, and peripheral vasculardisease, macrovascular disease is the primary cause of death in patients withdiabetes.Atherosclerosis (AS) is the main pathological basis of macrovasculardisease.Inflammation and vascular smooth muscle cells proliferation plays akey role in the development of atherosclerosis in the process.Resveratrol, high content in grapes, Polygonum cuspidatum, pine andother plants, is one kind of mere existence of non-flavonoid polyphenolcompounds and a strong antioxidant produced naturally by plant under stress,and have a wide range of biological and pharmacological effects, includingantioxidant and antiradical action, anti-inflammtory, immune regulation,cardiovascular protection, antilipemic, reducing blood glucose, improvinginsulin resistance, anti-tumor and estrogen-like activity. Resveratrol canprevent AS through anti-oxidation, involved in inflanmmation, proliferation ofVSMCs.Nuclear factor-κB (NF-κB), which exists in mononuclrar cells, vascularsmooth muscle cells and vascular endothelial cells, is a transciption factor,play a key role in the initiation and progression of atherosclerosis. Bothmonocyte chemoattractant protein-1(MCP-1) and vascular cell adhesionmolecule-1(VCAM-1) are the downstream factors of NF-κB activation. Theactivation of NF-κB and the injuring stimulation induce the expression ofMCP-1and VCAM-1through NF-κB signal transuction pathway, promotingthe peripheral white blood cell and monocyte adhesion, chemotaxis andmigration to the endothelium, phagocytosis lipid into foam cells, VSMCsproliferation, involved in the development of AS. We design this experiment to observe the proliferation of rat aorticsmooth muscle cells and the secretion of MCP-1, VCAM-1and NF-κB incultured rat aortic smooth muscle cells with different density of glucose, andresveratrol and inhibitor LY294002, to observe the effect of resveratrol onproliferation of VSMCs and explore the possible mechanism of anti AS ofresveratrol, provide experimental basis for clinical application of resveratrol..Methods：1Vascular smooth muscle cell lines A7r5were purchased from CCTCC(China Center for Type Culture Collection). Put the cells in the37℃,5%CO2incubator. The cells of exponential phase of growth were inoculated in50mlculture bottles and on96pore culture boards for48hours. The above cellswere replaced by DMEM media with0%FBS for24hours to make the cellsin phase G0/G1and then divided into seven groups:①Normal glucose contorlgroup:5.5mmol/L glucose②High glucose group:25mmol/L glucose③Normal glucose+inhibitor group:5.5mmol/L glucose and10/20/30μmol/LLY294002④High glucose+inhibitor group:25mmol/L glucose and10/20/30μ mol/L LY294002⑤Normal glucose+Res-treated group:5.5mmol/Lglucose and25/50/100μmol/L Res⑥High glucose+Res-treated group:25mmol/L glucose and25/50/100μmol/L Res.2The 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.3The contents of MCP-1and VCAM-1in the supernatant were measuredwith the method of Enzyme linked immunosorbent assay (ELISA).4The effect of Res and inhibitor LY294002on NF-κB, p-Akt, Aktprotein expression in VSMCs incubated in high glucose was detected byWestern blot.Results:1Compared with the normal glucose group，the proliferation activity ofVSMCs induced by the high glucose was notably increased (P＜0.05), Res(25,50,100μmol/L) and inhibitor LY294002(10,20,30μmol/L) can inhibit the proliferation of VSMCs induced by high glucose in a dose-dependentmanner (P＜0.05).2The expression level of MCP-1and VCAM-1in HG group weresignificant higher than that in NG group (P＜0.05),The density of MCP-1andVCAM-1were significant inhibited by Res(25,50,100μmol/L) and inhibitorLY294002(10,20,30μmol/L) in a dose-dependent manner (P＜0.05).3Western blot detection results show⑴High glucose significantly up-regulated the expression of NF-κBprotein and p-Akt protein compared with normal glucose group (P＜0.05).Compared with normal glucose group, the expression of total Akt in highglucose group was no significant difference (P＞0.05).⑵Treated VSMCs with different concentrations of LY294002, thephosphorylation of Akt was inhibited and the expression of p-Akt proteindecreased (P＜0.05). Res (25,50,100μmol/L) significantly decreased theexpression of p-Akt protein in a dose-dependent manner (P＜0.05).⑶Treated VSMCs with different concentration of LY294002, theexpression of Akt no significant difference, compared with normal glucosegroup (P＞0.05). But the expression of Akt protein decreased by differentconcentrations of Res, but no dose dependence.⑷The expression of NF-κB protein decreased by different concentrationsof LY294002in a dose-dependent manner compared with high glucosegroup (P＜0.05). The expression of NF-κB protein decreased by differentconcentrations of Res in a dose-dependent manner compared with highglucose group (P＜0.05).Conclusions:1High glucose can increase the proliferation activity of VSMCs.2High glucose can promote VSMCs proliferation through activatingPI3K/Akt signaling pathway, and increasing NF-κB and MCP-1, VCAM-1expression.3Resveratrol can inhibit the proliferation of VSMCs. Resveratrol inhibitthe expression of NF-κB protein through inhibiting activation of PI3K/Akt signal pathway induced by high glucose, decreased the expression of MCP-1and VCAM-1to inhibit VSMCs proliferation.