The Effect And Mechanism Of Resveratrol On Lipopolysaccharide-induced Inflammatory And Osteogenic Responses In Human Vascular Smooth Muscle Cells

BackgroundVascular calcification(VC)is a common pathological changes of coronary atherosclerotic heart disease,hypertension,diabetes,and chronic renal disease.Studies have found that the overall risk in patients with artery calcification of which coronary artery calcification(CAC)is more common and the most serious have four times more likely for cardiovascular events(CVD)compared with healthy person.Microcalcification in the coronary arteries may destroy the fibrous cap,leading to the rupture of the plaque and exposure of the collagen fibers and thrombosis,resulting in the occurrence of acute myocardial infarction(AMI).While moderate to severe calcification increases intraoperative complication of percutaneous transluminal coronary angioplasty(PTCA)and percutaneous coronary intervention(PCI),and even coronary artery bypass grafting(CABG).Despite of substantial medical costs and extensive research work,there is still difficult to come up with effective strategies.Resveratrol(RSV)is a natural polyphenol compounds,which was first discovered in 1939.Several biologic cardiovascular protection effects of resveratrol and red wine have been reported so far,including anti-inflammation,antioxidant,antiaging,inhibition of platelet aggregation and thrombosis and so on.In addition,recent studies have found that RSV also play a regulatory role on mesenchymal stem cells in the process of bone formation in the skeletal system,not only regulating osteoblast differentiation,but also inhibiting osteoclast formation.Furthermore,the researchers also found that RSV could inhibit proliferation of smooth muscle cells.Therefore,considering the similar process in calcification of vascular smooth muscle cells and osteoblast differentiation,there is still no definite conclusion whether RSV also affect the osteoblast differentiation of human vascular smooth muscle cells.The first part The effect study of TLR4/NF-kB signaling pathway in lipopolysaccharide-induced vascular smooth muscle cell inflammatory and osteogenic responsesObjective:To investigate the effect of TLR4/NF-kB signaling pathway on the Imflammatory response and osteogenic differentiation of Human vascular smooth muscle cells induced by lipopolysaccharide(LPS).Methods:Human vascular smooth muscle cells were cultured in Dulbecco's Modification of Eagle's Medium(DMEM)containing 10%fetal bovine serum(FBS)in 6-well plates and treated with TLR4 agonist LPS respectively for 0.5-24h.The expression levels of inflammatory cytokines included intercellular adhesion molecule 1(ICAM-1),monocyte chemotactic protein(MCP-1),Tumor necrosis factor a(TNF-?)and osteogenic factors of bone morphogenic protein 2(BMP-2),Alkaline phosphatase(ALP)and also activation level of Nuclear factor kB(NF-kB)were analyzed with immunoblotting.The release of interleukin 6(IL-6),interleukin(IL-8),TNF-? in the cell culture medium were examined with Enzyme-linked immunosorbent assay(ELISA).The calcium salt deposition was observed with alizarin red staining.NF-kB inhibitor BAY11-7082 was applied to inhibit the activation of NF-kB and then the LPS stimulated 24h,The expression levels of BMP-2,ALP,ICAM-1,MCP-1,TNF-? and the release of IL-6,IL-8,TNF-? mediated by TLR4 were analyzed,as well as the calcium salt deposition with alizarin red staining.Results:LPS induced the protein expression of ICAM-1,MCP-1,TNF-alpha,BMP-2,ALP and release of IL-6,IL-8,TNF-? in human vascular smooth muscle cells,as well as the activation of NF-kB signaling pathway.Furthermore,Bay11-7082,a kind of NF-kB inhibitor,attenuated TLR4-mediated expression of ICAM-1,MCP-1,TNF-?,BMP-2,ALP and release of IL-6,IL-8,as well as the extent of calcification,detected by alizarin red staining.Conclusion:LPS induces the inflammatory and osteogenic responses and activation of TLR4/NF-kB signaling pathway in human vascular smooth muscle cells.NF-kB activation regulates TLR4-mediated inflammatory and osteogenic responses,the activation of the TLR4/NF-kB signaling pathway might be one of an important mechanisms for vascular calcification.The second part The study of effect and mechanism of Resveratrol on LPS-induced imflammatory response and osteogenic differentiation in Human vascular smooth muscle cellsObjective:To investigate the regulation and mechanism of Resveratrol(RSV)on the modulation of LPS-induced inflammatory response and osteogenic responses in Human vascular smooth muscle cells.Methods:Human vascular smooth muscle cells were bought from ATCC company of American and treated with RSV for 1h before treating with TLR4 agonist LPS for 0.5-24h.The expression Levels of ICAM-1,TNF-?,MCP-1 and BMP-2,ALP protein were analyzed with immunoblotting,as well as the activation level of NF-kB.ELISA method was used to analyze the release of IL-6,IL-8 and TNF-? in the medium.calcium nodules were detected with Alizarin red staining.Results:Inflammatory cytokines expression levels included ICAM-1,MCP-1,TNF-?,IL-6,IL-8 decreased in cultured human vascular smooth muscle cells pretreated with RSV.the expression levels of BMP-2,ALP also significantly reduced as well as the calcium nodules,detected by alizarin red staining.In addition,Pretreatment of RSV inhibited activation of NK-?B in human vascular smooth muscle cells treated with LPS after 4 and 8 hours.Conclusion:Resveratrol regulates TLR4-mediated inflammatory response and osteogenic responses in human vascular smooth muscle cells through inhibition of TLR4/NF-kB axis.Thus,Resveratrol might have therapeutic potential for the progression of vascular calcification.