The Effect Of Repulsive Guidance Molecule A On Cortex Neuron Autophagy In Rats After Cerebral Ischemia/Reperfusion Injury

Background and Objective:Cerebrovascular disease is one of the three main cause of death,and ischemic cerebrovascular disease is the main type of cerebrovascular disease.Brain tissue ischemia will lead to brain damage,and it often aggravate the damage after cerebral ischemia reperfusion.Autophagy is a highly conservative process in eukaryotic cells,it use lysosoms to degrade long-term protein,damaged protein and damaged organelles,this process provide nutrients and energy for cell growth,development and reconstruction,and maintaining cellular homeostasis.Numerous studies have found that the brain ischemia-reperfusion can induce cell autophagy,and mainly in neurons,it is concerned with hunger,lack of growth factors,infection and so on,and it is an important part of the brain ischemia reperfusion injury.This process is regulated by a series of complex signaling molecules,Beclin1 molecules is one of the most main molecular of autophagy,it combines with PIK3C3 and formed Beclin1-PIK3C3 complex,which widely involved in the formation and mature ofautophagosome.Many researchers have found that their moleculars affect autophagy process by acting on Beclin1-PIK3C3 complex,this process is called "Beclin1-dependent autophagy",but some researchers also have found that their moleculars is not through Beclin1 to affect autophagy process,this process is called "Beclin1-independent autophagy".In recent years,studies have found that it can cause neurons autophagy process after cerebral ischemia reperfusion,and Beclin1-independent autophagy related to promote apoptosis and inhibit the neurological function.Therefore,autophagy may be an important target in stroke treatment in the future.RGMa is a kind of repulsive guidance molecules,a large number of studies have found that its expression increased after cerebral ischemia reperfusion,it is because of these axon growth inhibitors,nerve regeneration is very difficult for the central nervous system(CNS)after injury.Our previous studies have found that it can significantly improve the growth conditions of damaged axons and promote neural functional recovery effectively when intervening the expression of RGMa after brain injury in rats.6FNIII peptide contain six structural domains like Fibronectin type ? in neogenin molecule which is the receptor of RGMa,and it will inhibit the biological function of RGMa in vivo when combine with it.This experiment inhibit the function of RGMa by 6FNIII peptide via RGMa/Neogenin pathways and intervene the expression of RGMa by recombinant adenovirus r Ad-sh RGMa via RNA interference in rats MCAO/Reperfusion model,assess whether RGMa affect autophagy process and neurological function,and we also explore whether RGMa affect the formation of Beclin1-PIK3C3 complex.Methods:1.The adult male Sprague-Dawley rats were randomly divided into five groups: sham operated group(sham),ischemia/reperfusion(I/R)group,I/R plus low concentration 6FN? group(I/R+L-6FN?),I/R plus medium concentration 6FN? group(I/R+M-6FN?),I/R plus high concentration6FN? group(I/R+H-6FN?),each group had 6 rats.Different concentration of 6FNIII were injected into the right lateral ventricles of these rats before undergoing I/R surgery.The expression of CRMP-2 in the brain tissue were detected by Western blotting to determine the optimal concentration of 6FNIII.2.The adult male Sprague-Dawley rats were randomly divided into six groups: sham operation group(sham),cerebral I/R group(I/R),I/R plus normal saline group(I/R+NS),I/R plus 6FNIII intervention group(I/R+6FNIII),I/R plus r Ad-HK group(I/R+r Ad-HK),I/R plus r Ad-sh RGMa intervention group(I/R+r Ad-sh RGMa),each group had 18 rats.NS and 6FNIII were injected into the right lateral ventricle of the rats before undergoing I/R surgery;I/R+r Ad-HK and r Ad-sh RGMa were injected into the right cortex of the rats before 3 days of I/R surgery.The blood was occluded for 120 min,followed by 24 h reperfusion.Theexpression of LC3II/I in the brain tissue were detected by Western blotting and immunohistochemical assay.Transmission electron microscope was used to observe the autophagosome formation.TTC staining was employed to observe the cerebral infarct volume,Neurological function was also evaluated.In addition,we explora whether RGMa affect the formation of Beclin1-PIK3C3 complex via combine with Beclin1 or PIK3C3 in rats MCAO/Reperfusion model.Results:1.RGMa Intervention decreased LC3II/I levels(P<0.05),attenuated autophagosome formation,lessen cerebral infarct volume(P<0.05),and improved neurological deficits(P<0.05)at 24 hours after cerebral I/R injury in rats.2.RGMa did not affect the formation of Beclin1-PIK3C3 complex or combine with Beclin1 or PIK3C3 after cerebral I/R injury in rats.Conclusions:Biological roles of RGMa could be blocked by exogenous 6FN?peptide after MCAO/Reperfusion in rats.RGMa may delay neural function recover through Beclin1-independent autophagy in acute focal cerebral ischemia.