| Background:Cardiovascular disease(CVD)which is caused by atherosclerosis(AS),is the leading cause of death in the aged and patients with metabolic diseases,including diabetes.Inflammation is the main pathological change of AS,oxidized low density lipoprotein(ox-LDL)is a risk factor for atherosclerosis,and aging is a directly related risk factor to promote the progression of AS.The activated MMPs can degrade collagen,elastin and other extracellular molecules,which may lead to aging of arterial walls,hypertension and AS.However,the mechanism by which platelets release MMPs is unclear.Resveratrol is a natural polyphenol product with the effects of anti-inflammatory,antioxidant and the inhibition of platelet function.The specific molecular mechanism of anti-AS is still to be further studied.Objectives:1.To investigate the effects of resveratrol on toll-like receptor 4(TLR4),MMP3 and MMP9 activated by ox-LDL and its potential molecular mechanisms.2.To explore the effect of resveratrol on tyrosine kinase Syk and the activation of NLRP3 inflammasome in ox-LDL-stimulated platelet.3.To investigate the effects of resveratrol on the changes of plasma MMP3 and MMP9 levels,the expression of TLR4,MMP3,MMP9 and vascular structure of aortic in mice.To provide a theoretical basis for the prevention and treatment research and clinical application of resveratrol to improve the aging and high-fat diet,which are two risk factors promote the progress of AS.Methods:1.Preparation and treatment of platelets:Platelets were obtained by venous blood washing and purification in healthy people,and preincubated with resveratrol(1,10,and 100?M),CLI-095(0.1,0.3,and 1 ?M),R788(10,30,and 100 nM),or MCC950(1,10,and 100 nM)for 10 min,then ox-LDL(0.1 mg/ml)or LPS(5 ?g/ml)was added for 10 min.2.Animal model:The male C57/BL mice mice(8 and 52 weeks old)were randomly divided into three groups.The control group was fed a normal diet,and given physiological saline per day.High-fat chow group was fed a high-fat diet,which was contained 21%fat supplemented with 1.25%cholesterol,and given physiological saline per day.Resveratrol(22.4 mg/kg/day)was administered intragastrically in the high-fat chow+RSV group.The treatment time of each group was 12 weeks.3.Western blot analysis:Detection of protein expression of TLR4,MMP3 and MMP9,phosphorylation of Spleen tyrosine kinase(Syk),nucleotide binding domain leucine rich repeat containing protein 3(NLRP3),cysteine-requiring Aspartate protease-1(caspase-1),interleukin-1?(IL-1?),p53 and p21.4.Immunofluorescence:The morphological changes and caspase-1 expression and distribution of resveratrol on ox-LDL-induced platelet activation were detected.5.Flow cytometry:Detection of resveratrol on ox-LDL-induced platelet TLR4 expression.6.Enzyme linked immunosorbent assay(ELISA):detect MMP3,MMP9 and IL-1? platelet secretion and mouse plasma content.7.Biochemical methods:Detection of four items of blood lipids in mice,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),total cholesterol(total Cholesterol(TC)and triglyceride(TG)are detected by fully automatic biochemical analyzer.8.Immunohistochemical staining:detect the expression of MMP3 and MMP9 in aortic vascular tissue.9.Senescence-associated ?-galactosidase(SA-?-gal)staining:detect senescent cells in aortic vascular tissue.10.Sirius red staining:detection of collagen in blood vessels.Results:1.Resveratrol(100?M)inhibited TLR4 expression in ox-LDL-activated platelets,and reduced ox-LDL-stimulated platelet adhesion to fibrinogen and pseudopod extension Western blot and flow cytometry analysis of TLR4 expression showed that resveratrol pretreatment inhibited the expression of TLR4 stimulated by ox-LDL.Immunofluorescence showed that ox-LDL treatment caused platelets to have a pseudopod appearance and spread.Less spreading was observed in resveratrol-treated platelets.2.The inhibition of MMP3 and MMP9 expression by resveratrol is associated with the inhibition of TLR4 activationResveratrol(100 ?M)inhibited the expression and secretion of MMP3 and MMP9 in ox-ldl-stimulated platelets.TLR4 inhibitor CLI-095(1 ?M)inhibited the expression and secretion of platelets MMP3 and MMP9 stimulated by ox-LDL or TLR4-specific activator LPS.Lower concentrations of resveratrol and CLI-095 synergically inhibited the expression of MMP3 and MMP9 in ox-LDL and LPS-activated platelets.3.Resveratrol reduces the expression and secretion of MMP3/MMP9 by inhibiting the activation of Syk/NLRP3Resveratrol treatment inhibited ox-LDL and LPS stimulated Syk phosphorylation and NLRP3,caspase-1 and IL-1? expression.Syk inhibitor R788 inhibited the expression of MMP3,MMP9,NLRP3,caspase-1 and IL-1? in ox-LDL-stimulated platelets.Lower concentrations of resveratrol and R788 synergistic inhibited the expression of MMP3,MMP9,NLRP3,caspase-1 and IL-1? in ox-LDL and LPS-activated platelets.NLRP3 inhibitor MCC950 also inhibited the expression of MMP3,MMP9,caspase-1 and IL-1? in ox-LDL-stimulated platelets,while lower concentrations of resveratrol and MCC950 synergistic inhibited the expression of MMP3,MMP9,caspase-1 and IL-1? in ox-LDL-activated platelets,which suggested an interaction between the Syk and NLRP3 molecules.The results of co-immunoprecipitation showed that when ox-LDL stimulated platelet activation,Syk and NLRP3 showed a binding state,which was reversed by resveratrol treatment.4.Protective effect of resveratrol on blood vessels in aged high-fat miceResveratrol reduced levels of LDL-C,TG,and TC in blood lipids,and decreased levels of MMP3,MMP9,and IL-1? in plasma in mice on the high-fat diet.Analysis of vascular inflammatory protein levels showed that resveratrol intervention reduced the expression of TLR4,MMP3 and MMP9 in vascular tissues.SA-?-Gal staining showed that the resveratrol intervention reduced the number of vascular senescent cells in the 52-week-old high-fat diet mice compared with the high-fat diet group.The Sirius staining showed that the resveratrol intervention reduced collagen degradation in vascular tissue of 52-week-old high-fat diet mice.Conclusions:1.Resveratrol inhibited the activation of TLR4 and the expression and secretion of MMP3 and MMP9 in platelets stimulated by ox-ldl,and the molecular mechanism is related to the inhibition of the activation of the TLR4/Syk/NLRP3 pathway.2.Resveratrol inhibited the activation of ox-LDL stimulating platelet NLRP3 inflammasome and reduced the extracellular release of IL-1?.3.The intervention of resveratrol improved the vascular collagen structure disorder and elastic fiber rupture in the aging mice fed with high-fat diet,reduced the expression of TLR4,MMP3 and MMP9 in the aorta of the mice,reduced the expression of senescent cells in the vascular wall,and had a protective effect on the vascular injury caused by the aging high-fat diet... |
PDF Full Text Request