The Role And Mechanism Of Ghrelin Obestatin In The Pathogenesis Of Atherosclerosis Disease

Ghrelin is the only endogenous ligand of growth hormone secretagogue receptor up to now,it play a important role in the increasing appetide,improving stomach intestine function and adjusting energy metabolism.Recent study suggest that apart from promoting growth hormone releasing,they are related with multiple pathophysiology process,for example:obesity,cardiolvascular disease,Diabetes mellitus and senescence.Recently researchers successfully isolated a length of ghrelin associated peptide named obestatin from the stomach of rat.study demonstrate that obestatin has opposite effect to ghrelin.The found of obestatin was expand the ghrelin associated study field.there some report demonstrate that the level of obestatin in the coronary disease increase,obestatin can accelerate the cardic ischemia reperfusion injury.But there are no report about the effect of Ghrelin and Obestatin in the regression of atherosclerosis plaque.Therefore,we explore ghrelin and obestatin's effect on the atherosclerosis from below aspect:â… -In vitro study the effect of ghrelin and Obestatin on the Angâ…¡induced human umbilicus endothelial cell NO release and cell oxidative stress,proliferation,migration and inflammatory factor production and the signal pathway involved in;â…¡-In vivo study the impact on the atherosclerosis plaque of apoE^-/-mice and inflammatory factor level and NFκBp65 expression of vascular wall. Chapterâ… :impact and mechanism of Ghrelin on the function of human umbilical endothelial cellObject:To observe the effect of Ghrelin on human umbilicus endothelial cell ROS production,NO release and proliferation,migration and the adhesion of moncyte to endothelial cell,TNFα,IL-8,MCP-1 level and gene express and signal path way involved in.Method:I use different concentration of Angâ…¡(10^-9-10^-6mol/L) incubate HUVEC-12 in vitro(0(?)24h),to aquire best observation,and observe(10^-9(?)10^-6mol/L)Ghrelin's effect on Angâ…¡induced injury.â…¡pretreated with the inhibitor of MAPK signal pathway PD98059 25μmol/L,GHS-Rla receptor block[Dys³]GHRP-6 25μmol/L,and NFκB inhibitor PDTC1μmol/Land then observe the role to the effect of Ghrelin on Angâ…¡induced injury,adopt nitrate reductase method measure the NO of supernatant.WST-8 method measure the proliferation of cell, transwell method measure the cell migration.Measuring endothelilal cell ROS with fluorescent compound 2',7'-dichlorofuorenscein(DCF) method.RT-PCRmethod measure the gene expression of TNFα,IL-8,MCP-1.ELISA method measure the level of TNFα,IL-8,MCP-1 in the supernatant.westernblot measure the expression of total Akt,ERK1/2,totalNFκBp65,p-Akt,p-ERK1/2,p-NFκBp65,eNOS.Result:I Angâ…¡inhibit HUVEC-12 releasing NO with dose and time dependent response.Ghrelin reverse Angâ…¡induced NO release decreased with dose-dependent response.Pretreated with PD98059 has no effect to Angâ…¡induced NO release decreased,but[Dys³]GHRP-6 pretreated block ghrelin's effect of increasing NO release.â…¡Angâ…¡promote endothelial cell proliferation and migration Ghrelin inhibit Angâ…¡induced endothelial cell proliferation and migration with dose and time dependent.Pretreated with PD98059 can inhibit Angâ…¡induced endothelial cell proliferation and migration,pretreated with [Dys³]GHRP-6 block the inhibited effect of Ghrelin on endothelial migration and proliferation.â…¢Angâ…¡increased endothelial cell ROS production with time and dose response.Ghrelin inhibit Angâ…¡induced endothelial proliferation and migration with dose dependent response.pretreated with[Dys³]GHRP-6 block the inhibited effect of ghrelin on ROS production.â…£Ghrelin attenuate Angâ…¡induced TNFα,IL-8,MCP-1 level with dose depent response and gene expression with time dependent response.Pretreated with[Dys³]GHRP-6 block the effect of ghrelin on TNFα,IL-8,MCP-1release and gene expression.â…¤ghreln can increase the endothelial cell expression of pAkt,eNOS,decrease p-NFκBp65 and p-ERK1/2 expression,and meanwhile decrease monocyte adhesion to endothelial cellConclusion:ghrelin inhibit Angâ…¡induced endothelial injury,inflammatory response and oxidative stress,the mechanism involved in PI3K/Akt signal pathway,affect ERK1/2 and NFκB signal pathway activation Chapterâ…¡:the effect and mechanism of obestatin on human umbilicus endothelial cell functionObjective:To observe the effect of obestatin on HUVEC-12 ROS production,NO release and proliferation,migration and the adhesion of moncyte to endothelial cell,TNFα,IL-8,MCP-1 level and gene expression and the signal pathway involved in.Method:Different concentration of Angâ…¡(0(?)24h)incubate HUVEC-12 in vitro to observe best point of time and concentration,and observe obestatin interfere's effect to the Angâ…¡induced injury.â…¡pretreated HUVEC-12 with inhibitor of MAPK signal pathway PD98059,NFκB signal pathway PDTC,and then incubate HUVEC-12 with obestatin and AngⅡ。Adopt the same method as chapterâ… .Result:â… Angâ…¡inhibit HUVEC-12 release NO the same as chapterâ… ,obestatin further accelerate Angâ…¡inhibit HUVEC-12 release NO with time and dose dependent response,obestatin inhibit eNOS expression,and may inhibit NO secretion through PI3K pathway.â…¡obestatin increase HUVEC-12 proliferation,the same dose of ghrelin can opposite obestatin's effect.â…¢obestatin itself has no effect on HUVEC-12 migration,but promote Angâ…¡induced HUVEC-12 migration.PD98059 can block the effect of obestatin+Angâ…¡induced HUVEC-12 migration.â…£obestatin increase Angâ…¡induced HUVEC-12 ROS production with dose and time dependent response.â…¤obestatin increase Angâ…¡induced HUVEC-12's TNFα,IL-8,MCP-1 level and gene expression.PD98059 and PDTC block obestatin's effect.â…¥obestatin increase p-ERK1/2,p-NFκBp65protein expression,decrease p-AKt, eNOS,and promote monocyte adhesion to endothelial cell,and accelerate Angâ…¡'s effect.Conclusion:obestatin promote Angâ…¡induced HUVEC-12 injury,inflammatory response,oxidative stress,the mechanism may be related to inhibit PI3K/Akt,activate ERKl/2,NFκB signal pathway.Chapterâ…¢the effect of ghrelin on the regression of atherosclerosis plaque in ApoE^-/-mice aortaObjective:To observe the effect of ghrelin on reducing the apoE^-/- mice plasma IL-8,MCP-1,TNFαlevel and the NFκBp65 expression in vascular wall and the regression of atherosclerotic plaque.Method:8 week ApoE^-/-mice feed with western style meals,and the same age mice C57BL/6J feed with same meals as control.In the eighth week,ApoE^-/-mice was assign to ghrelin intraperitoneal injection and saline injection group randomly in the twelfth week.All of the groupswere drawed blood from eye sockets,isolate plasma to measure IL-8,MCP-1,TNFαby ELISA.Mice were killed to be observed undered stereomicroscope and paraffin imbedding for HE and immunohistochemistry,and frozen section for red oil stain.Result:â… the result of stereomicroscope,HE,oil red stain and image analysis equipment measurement demonstrate that no plaque at C57BL/6J mice vessels,and both apoE^-/-group and ApoE^-/-+ghrelin group has atherosclerosis plaque at vessels.â…¡contrast to C57BL/6J mice, apoE^-/-mice has higher plasma TNFα,IL-8,MCP-1 level,but apoE^-/+ghrelin mice has lower TNFα,IL-8,MCP-1 level than ApoE^-/mice.â…¢Contrast to C57BL/6J mice,apoE^-/-mice NFκBp65 immunohistochemistry positive cell integral calculus value are increase obviously,ghrelin can decrease the expression of NFκBp65 in apoE^-/-mice aorta.Conclusion:Ghrelin can inhibit inflammatory response to decrease ApoE^-/-mice atherosclerosis plaque formation.