| Background and objective:Ischemic cerebrovascular disease is a common clinical disease and frequently-occurring disease.Currently,the morbidity rate,disability rate,recurrence rate,and mortality rate are getting higher and higher,which brings a heavy economic burden to society and families.This disease mainly results of lack of the blood supply in brain,which causes necrosis or softening of brain tissue in the corresponding blood supply.Restoring blood supply and improving microcirculation are key strategies to reduce the infarct volume of brain.However,restoring blood supply cannot ameliorate the injury caused by cerebral ischemia sometimes and it maybe cause more serious or irreversible injury,which is called cerebral ischemia-reperfusion injury(CIRI).Considerable studies indicate that CIRI is an important factor of exacerbation of ischemic cerebrovascular disease.Thus,the treatment of ischemic cerebrovascular disease is not only to restore blood supply,but also to avoid CIRI.In recent years,many studies have shown that the destruction of blood-brain barrier(BBB)play a major role in CIRI.The BBB is mainly composed of brain microvascular endothelial cells,tight junctions(TJs),basement membranes,astrocytes,and pericytes.The tight junction between endothelial cells is an important structural component of the blood-brain barrier.The degradation of tight junction proteins and extracellular matrix is the pathological basis of blood-brain barrier damage.Changes in TJ-associated proteins will lead to changes in BBB function.Claudi-5 and Occludin are two important tight junction proteins.Gelatinase MMP-9 can directly degrade extracellular matrix,leading to destruction of blood-brain barrier,and is closely related to reperfusion injury.Zo-1 is the most important cytoplasmic protein associated with TJ function,linking the C-terminus of Claudi-5 and Occludin proteins to the actin cytoskeleton.Cerebral ischemia-reperfusion can lead to decreased expression of tight junction proteins in the vascular endothelial cells,degradation of the basement membrane,and increased permeability of the blood-brain barrier,thereby increasing the degree of brain injury.Therefore,the study on the molecular structure of BBB can further understand the possible mechanism of ischemia-reperfusion,which provides new strategies of prevention and treatment of ischemic stroke.Repulsive guidance molecule a(RGMa),a new axonal molecule,has attracted more and more researchers' attention.It could regulate the neuronal differentiation and proliferation.Studies have shown that RGMa causes collapse of growth cone through activation of the small GTPase Ras homologous A(Rho A),followed by activation of its downstream protein Rho kinase.Both Rho and cell division cycle protein 42(CDC42)belong to the Ras superfamily and have a wide range of biological behaviors and functions involved in cytoskeletal related activities such as cell migration,axon guidance,cell deformation,contraction and adhesion.CDC42 can bind to guanine trinucleotide(GTP)and play an important role in "molecular switch" in mammalian cell signal transduction systems,and it plays an important role in regulating the dynamic changes of cytoskeleton.The inhibition of CDC42 protein activity by RNAi technology successfully prevented the polarization localization of P21 protein-activated protein kinase(PAK),resulting in non-normal polarization of nematode embryonic cells,indicating that PAK is one of the downstream proteins of CDC42.Previous studies have shown that RGMa and its receptor Neogenin are expressed on cortical neurons and vascular endothelium after cerebral ischemic reperfusion in rats,and can affect the regeneration of cortical blood vessels around the infarcted lesions.Changes in the tight junction proteins between the vascular endothelium can cause changes in the blood-brain barrier structure.Thus we hypothesize that RGMa,as a upstream protein of Rho A/CDC42,can affect the BBB.6FNIII is a polypeptide that can interact with RGMa and interact with its full length.Studies have shown that the fragment of 6FNIII can block the inhibitory effect of RGMa protein on retinal axons in vitro.As one of the members of the Collapse Response Regulatory Protein family,CRMP-2 is abundantly expressed in the developing brain tissue and participates in the process of axonogenesis,development and regeneration.It is one of the downstream substrate molecules that RGMa exerts through Rho kinase.Our team has studied the expression of VEGF,Ang2 and Ang1 after 6FNIII blockade of RGMa/Neogenin signaling,indicating that RGMa's regulation of angiogenesis may be mediated by its receptor Neogenin and downstream molecule CRMP-2.In this study,we attempted to silence RGMa by adenovirus and CRMP2,a downstream protein of RGMa,by 6FNIII to observe the expression of tight junction proteins and to explore the possible mechanism of RGMa's influence on blood-brain barrier.PART I: THE EFFECT OF ADENOVIRUS MEDIATEDRGMA DOWNREGULATION ON BLOOD-BRAINBARRIER IN RATS WITH ISCHEMIA-REPERFUSION.Objective:To detect the effect of adenovirus mediated RGMa downregulation on blood-brain barrier in rats with ischemia-reperfusion.Methods:1.40 adult male Sprague-Dawley(SD)rats underwent injection of adenovirus(control)and RGMa adenovirus(sh-RGMa group)by stereotaxicapparatus.After one day and three days of injection,the distribution and intensity of the green fluorescent protein(GFP)were observed by laser confocal microscopy to determine whether the transfection was successful.Westting blot was used to detect the expression level of RGMa protein in the control group and the sh-RGMa group.2.154 rats were randomly divided into the sham group(sham),the ischemia reperfusion group(I/R group),the ischemia reperfusion rats treated with empty adenovirus group(I/R + sh-con group)and the ischemia reperfusion rats treated with RGMa adenovirus group(I/R+sh-RGMa group).After two days of adenovirus injection,middle cerebral artery occlusion(MCAO)was performed in rats.In sham group,the common carotid artery,external carotid artery and internal carotid artery were exposed and isolated without occlusion of the middle cerebral artery.3.After three days of MCAO,following studies were performed: a)neurological function score;b)detection of encephaledema;c)cerebral infarction volume detected by TTC;d)leakage rate of evans blue to detect the permeability of BBB;e)ultrastructure of BBB by electron microscope.4.The interaction between RGMa and claudin-5,MMP-9 and ZO-1 were observed by co-immunoprecipitation.The expression of Claudin-5,MMP-9 and ZO-1 were detected by Western blotting.5.The interaction between RGMa and CDC-42/PAK-1 were detected by co-immunoprecipitation,and the expression of CDC-42 and PAK-1were detected by Western blotting.Results:1.Fluorescence microscope showed that the virus was successfully infected with brain tissue in rats.The expression of GFP on the third day after injection of adenovirus was higher than the expression of GFP on the first day after injection of adenovirus.Western blotting indicated that expression of RGMa on the third day after injection of adenovirus was lower than the expression of RGMa on the first day after injection of adenovirus(p<0.05).2.a)The neurological function score indicated that I/R rats including I/R group,I/R+sh-con group and I/R+sh-RGMa group showed severe neurological deficits compared with sham rats.However,compared with I/R group and I/R+sh-con group,the neurological function of the I/R+sh-RGMa group was significantly improved(p<0.05).b)Detection of brain edema found that cerebral edema was more serious in I/R rats including I/R group,I/R+sh-con group and I/R+sh-RGMa group compared with sham group.However,compared with I/R group and I/R+sh-con group,brain edema in I/R+sh-RGMa group was significantly improved(P <0.05);c)Detection of cerebral infarction area indicated that higher infarction area in I/R rats including I/R group,I/R+sh-con group and I/R+sh-RGMa group compared with sham group.However,compared with I/R group and I/R+sh-con group,infarction area in I/R+sh-RGMa group was significantly improved(P <0.05);d)Detection of BBB permeability indicated that higher BBB permeability in I/R rats including I/R group,I/R+sh-con group and I/R+sh-RGMa group compared with sham group.However,compared with I/R group and I/R+sh-con group,BBB permeability in I/R+sh-RGMa group was significantly reduced(P <0.05);e)Transmission electron microscope found that the base membrane and tight junction in I/R rats including I/R group,I/R+sh-con group and I/R+sh-RGMa group was broken more seriously than that in sham group.However,compared with I/R group and I/R+sh-con group,the base membrane and tight junction in I/R+sh-RGMa group was more integrated.3.Co-immunoprecipitation showed interaction between RGMa and claudin-5,MMP-9 and ZO-1.The expression of MMP-9 was higher in the I/R and I/R+sh-con groups than sham group(p<0.05)and the expression of MMP-9 in I/R+sh-RGMa was lower than I/R and I/R+sh-con groups(p<0.05).The expression of claudin-5 and ZO-1 was higher in the I/R and I/R+sh-con groups than sham group(p<0.05)and the expression of claudin-5 and ZO-1 in I/R+sh-RGMa was lower than I/R and I/R+sh-con groups(p<0.05).4.Co-immunoprecipitation showed interaction between RGMa and CDC42 and PAK-1.The expression of CDC42 and PAK-1 was lower in the I/R and I/R+sh-con groups than sham group(p<0.05)and the expression of CDC42 and PAK-1 in I/R+sh-RGMa was higher than I/R and I/R+sh-con groups(p<0.05).Conclusion:The break of BBB is detected in the cerebral ischemia-reperfusion injury.However,down-regulation of RGMa activates the CDC42/PAK-1 signaling pathway to improve BBB,reduce infarct volume,and improve neurological function.PART II: THE EFFECT AND MECHANISM OF 6FNIII ONBLOOD-BRAIN BARRIER AFTER CEREBRALISCHEMIA-REPERFUSION INJURY.Objective:To detect the effect and mechanism of 6FNIII on blood-brain barrier after cerebral ischemia-reperfusion injury.Methods:1.40 healthy male SD rats were injected with 6FNIII(0.25 ?g/?L,0.5 ?g/?L,1 ?g/?L).After three days of injection,the expression of RGMa and its downstream CRMP-2 were detected.2.144 rats were randomly divided into sham group;ischemia reperfusion group(I/R group),ischemia-reperfusion treated with saline group(I/R +NS group)and ischemia-reperfusion treated with 6FNIII(I/R+6FNIII group).3.After three days of MCAO,following studies were performed: a)neurological function score;b)detection of encephaledema;c)cerebral infarction volume detected by TTC;d)leakage rate of evans blue to detect the permeability of BBB;e)ultrastructure of BBB by electron microscope.4.The expression of claudin-5,MMP-9,ZO-1,CDC-42 and PAK-1 were detected by western blotting.Results:1.The expression of CRMP-2 increased obviously with the increase of 6FNIII concentration.But there was no significant difference in the expression of CRMP-2 in the 0.5 ?g/?L and 1 ?g/?L group.Thus 0.5 ?g/?L 6FNIII was used in the future experiment.6FNIII had no effect on the expression of RGMa.2.a)The neurological function score indicated that I/R rats including I/R group,I/R+NS group and I/R+6FNIII group showed severe neurological deficits compared with sham rats.However,compared with I/R group and I/R+NS group,the neurological function of the I/R+6FNIII group was significantly improved(p<0.05).b)Detection of brain edema found that cerebral edema was more serious in I/R rats including I/R group,I/R+NS group and I/R+6FNIII group compared with sham group.However,compared with I/R group and I/R+NS group,brain edema in I/R+6FNIII group was significantly improved(P <0.05);c)Detection of cerebral infarction area indicated that higher infarction area in I/R rats including I/R group,I/R+NS group and I/R+6FNIII group compared with sham group.However,compared with I/R group and I/R+NS group,infarction area in I/R+6FNIII group was significantly improved(P <0.05);d)Detection of BBB permeability indicated that higher BBB permeability in I/R rats including I/R group,I/R+NS group and I/R+6FNIII group compared with sham group.However,compared with I/R group and I/R+NS group,BBB permeability in I/R+6FNIII group was significantly reduced(P <0.05);e)Transmission electron microscope found that the base membrane and tight junction in I/R rats including I/R group,I/R+NS group and I/R+6FNIII group was broken more seriously than that in sham group.However,compared with I/R group and I/R+NS group,the base membrane and tight junction in I/R+6FNIII group was more integrated.3.The expression of MMP-9 was higher in the I/R and I/R+NS groups than sham group(p<0.05)and the expression of MMP-9 in I/R+6FNIII was lower than I/R and I/R+NS groups(p<0.05).The expression of claudin-5 and ZO-1 was higher in the I/R and I/R+NS groups than sham group(p<0.05)and the expression of claudin-5 and ZO-1 in I/R+6FNIII was lower than I/R and I/R+NS groups(p<0.05).The expression of CDC42 and PAK-1 was lower in the I/R and I/R+NS groups than sham group(p<0.05)and the expression of CDC42 and PAK-1 in I/R+6FNIII group was higher than I/R and I/R+NS groups(p<0.05).Conclusion:The break of BBB is detected in the cerebral ischemia-reperfusion injury.However,down-regulation of CRMP-2 by 6FNIII activates the CDC42/PAK-1 signaling pathway to improve BBB,reduce infarct volume,and improve neurological function. |
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