Roles And Mechanisms Of Zedoary Guaiane-Type Sesquiterpene Compounds In Restenosis

Delayed vascular endothelial healing induced by drug-eluting stent(DES)is one of the main pathological factors in major adverse cardiovascular events(MACE),including stent thrombosis and restenosis.Therefore,simply inhibiting vascular smooth muscle cells(VSMC)proliferation,it is unsuitable to solve the problem of stenosis,what's more,it may increase the risk of MACE.More attention has paid on how to inhibit VSMC proliferation,meanwhile,promote vascular re-endothelialium in field of percutaneous coronary intervention(PCI).Stent implantation process,often accompanied endothelium collapse,produces waterfall-like reactive oxygen species(ROS),and permanent metal struts also constantly stimulate vascular tissues in state of oxidative stress.Therefore,we first assessed the effect of antioxidant on MACE in patients with coronary artery disease undergone PCI.The results showed that antioxidant(probucol)combined with conventional treatment was superior to conventional treatment in reducing the incidence of MACE in PCI patients.This beneficial effect was closely related to the reduction of revascularization and improvement of lipid profile.Of course,this research also threw light on selecting suitable experimental model in our study.Pharmacological research have demonstrated that main active ingredients of curcuma zedoaria have beneficial effect in antioxidant stress,anti-inflammation,and prevention of atherosclerotic plaque formation and development.Zedoary guaiane-type sesquiterpene compounds(ZGS)are important active ingredients incurcuma zedoaria.Our previous study showed that ZGS stents increased endothelial cells coverage,and reduced neointimal formation in a porcine coronary injured model.However,the mechanism is not yet clear.Therefore,this paper investigated the effect of ZGS on vascular healing,and the following studies were carried out:(1)Effect of ZGS on oxidative stress-induced endothelial cell injury;(2)Effect of ZGS on TNF-a-induced vascular smooth muscle cell phenotypic switch,proliferation and migration;(3)Effect of ZGS on vascular repair in a rat balloon injury model.Part 1 Effect of antioxidant on MACE in patients with coronary artery disease after percutaneous coronary intervention:a meta analysisObjective:To examine the effect of antioxidant(probucol)on MACE in patients with coronary artery disease undergone PCI.Methods:Electronic databases,including PubMed,EMBASE,ScienceDirect and the Cochrane Central Register of clinical trial,CNKI database,and Wan-Fang Database,were used.The search items of "antioxidant" or "probucol" or "vitamin C" or"vitamin E" or "N-acetylcysteine" and "angioplasty" or "stent" and"randomized" were selected.RevMan 5.3 providing by the Cochrane was used to perform this analysis.Results:In the enrolled 349 articles,there were 7 articles reported MACE events in patients with coronary heart disease undergone PCI.There were 72 events in antioxidant group(72/352,20.5%).111 events were occurred in control group(111/354,31.4%).Compared to control group,antioxidant(probucol)group significantly decreased the incidence of MACE(RR,0.65,95%CI,0.51 to 0.84,P =0.0008).Compared with control group(86/352,24.3%),there was a lower rate of repeat revascularization in antioxidant group(52/352,14.8;RR,0.61,95%CI,0.44 to 0.83,P = 0.002).No difference was observed in myocardial infarction and death between groups(P = 0.34,P=0.49).In addition,total cholesterol(TC)and low density lipoprotein-cholesterol(LDL-C)were significantly decreased in probucol group when compared with control group(standard mean reduction[SMD],-0.68,95%CI,-0.87 to-0.49;P<0.00001;SMD,-0.28,95%CI,-0.46 to-0.11;P = 0.001,respectively).Conclusion:Antioxidant probucol combined with conventional treatment significantly decreased the rate of MACE in patients with coronary artery disease undergone PCI.This benefit may be related to the improvement of repeat revascularization and lipid profile.Part 2 Effect of ZGS on oxidative stress-induced endothelial cell injuryObjective:The present study aimed to investigate the effect of ZGS on oxidative stress-induced endothelial injury and underlying mechanism.Methods:H2O2 was used to inhibit human umbilical vein endothelial cells(HUVECs)migration and viability.Treated HUVECs with ZGS,transwell-and wound-healing-assays were used to detected cell migration;Western blot,RT-PCR,small interference(si)technology and immunofluorescence were used to investigate the mechanisms,involving in Cav-1,eNOS,SDF-1?,MAPKs and Nrf2.Results:(1)Incubating HUVECs with H2O2(500ug/ml)for 24 h,significantly injured cell migration and viability when compared with control(both P<0.01).However,ZGS(5,10,20 and 40ug/ml)concentration-dependently reversed H2O2-impaired cell migration.(2)H2O2 increased Cav-1 protein expression,and decreased eNOS protein and phosphorylation(Ser1177)levels,while ZGS treatment down-regulated H2O2-induced Cav-1 protein expression,and up-regulated eNOS phosphorylation(Ser1177)in concentration-dependent manner,but eNOS protein level was unaltered(all P>0.05).In addition,eNOS inhibitor,L-NAME,reversed ZGS+H2O2-increased eNOS phosphorylation(Ser1177)levels(all P<0.01)and cell migration(both P<0.01).And deleting eNOS or Cav-1 by siRNA technology,ZGS did not alter H2O2-induced eNOS phosphorylation(Ser1177)level(P>0.05)and Cav-1 expression(P>0.05).(3)Compared with H2O2 group,ZGS increased H2O2-decreased SDF-la and P-P38 expression,but not P-JNK and P-Erkl/2 levels.When deleted Cav-1 protein expression,ZGS did not change H2O2-induced-SDF-1?and P-P38 expression(both P>0.05).(4)H2O2 inhibited Nrf2 release from Cav-1 and decreased its nuclear accumulation,while ZGS reversed Nrf2 nuclear accumulation via partially down-regulating Cav-1 protein expression.Conclusions:ZGS improved oxidative stress-induced HUVECs migration and increased the ability of antioxidant stress,which were involved in regulating Cav-1/P38/eNOS signaling pathway and Nrf2 nuclear accumulation.Part 3 Effect of ZGS on TNF-a-induced phenotype switch,proliferation and migration in vascular smooth muscle cellObjective:The present study assessed the effect of ZGS on tumor necrosis factor-a(TNF-a)-stimulated phenotype switch,proliferation and migration in SMC.Methods:TNF-a induced human coronary SMC(HCSMCs)from contractile to synthetic phenotype.Treated HCSMCs with ZGS,wound-healing assay and transwell assay were performed to examine cell motility;Flow cytometry observed cell cycle;Western blot and si RNA technology assessed the underlying mechanism.Results:(1)TNF-a(50 ng/ml)induced HCSMCs phenotype switch from contractile to synthetic status,the latter was increased cell proliferation(P<0.01).Compared with TNF-a group,TNF-a+ZGS groups were gradually restored to the "Hill and Valley" status.(2)ZGS(5,10,20 and 40 ug/ml)concentration-dependently inhibited HCSMCs proliferation at G0/G1 phase and suppressed HCSMCs migration in response to TNF-a.(3)Compared with TNF-a group,the expression of SMH-HMC and calponin was up-regulated and the S100A4 expression were down-regulated in TNF-a+ZGS groups,while SM-a-actin were not changed among groups.(4)ZGS decreased TNF-?-induced Cx43 protein,and increased Cx40 and Cx37 protein levels in a concentration dependent manner in response to TNF-a.(5)ZGS concentration-dependently increased P-Cav-1 level,and down-regulated P-stat3 and CyclinDl levels induced by TNF-a.Conclusions:ZGS increased contractile protein expression,and down-regulated synthetic protein expression in response to TNF-?,facilitating maintaining HCSMCs in a contractile phenotype,reducing proliferation and migration.The mechanism was associated with the Cxs(Cx43,Cx40 and Cx37)and Cav-1/Stat3/CyclinDl signaling pathway.Part 4 Effect of ZGS on vascular healing in a rat balloon injury modelObjective:The purpose of this study was to verify the effect of ZGS on vascular repair in a rat balloon injury model.Methods:A balloon-induced rat abdominal aortic injury model was established.SD rats were divided into five groups:sham group(n=10),model group(n=10),ZGS20 group(20mg/kg/d,n= 10),ZGS40 group(40mg/kg/d,n=10)and ZGS20+Cav-1 inhibitor group(M?CD,n=10).HE staining was used to observe the neointimal hyperplasia in each group;Immunohistochemistry assessed the expression of SMC phenotypic marker proteins;Cav-1 and connexins(Cxs)expression were observed by immunofluorescence.Results:(1)Compared with sham group,significantly increased neointima thickness was observed in model group(P<0.01),implying a successful balloon injury model.Compared with model group,ZGS20 and ZGS40 groups decreased neointima thickness(P<0.05,P<0.01,respectively);The inhibitory effect of ZGS on neointimal hyperplasia was reversed by Cav-1 inhibitor,M?CD(both P<0.05).(2)Compared with model group,the SMH-HMC were increased(both P<0.01)and S100A4 were decreased(both P<0.01)in ZGS20 and ZGS40 groups,while additional M?CD treatment changed the SMH-HMC(both P<0.01)and S100A4(both P<0.01)expression.(3)Compared with model group,the expression of P-Stat3 in ZGS20 and ZGS40 groups were reduced(both P<0.01).Additional M?CD reversed ZGS decreased P-Stat3 level(both P<0.01).(4)Compared with sham group,P-Cav-1,Cx40 and Cx3 7 expression were reduced,and Cx43 expression was increased in model group;while ZGS reversed P-Cav-1 and Cxs expression in response to balloon injury.(5)The expression of CD34,a marker of endothelial cells,were increased in ZGS20 and ZGS40 group(both P<0.01),when compared with model group.Conclusion:ZGS maintained SMC contractile phenotype and reduced neointimal thickening in a balloon injury model.The mechanism was associated with regulating of Cxs(Cx40,Cx37 and Cx43)and Cav-1/Stat3 signaling pathway.