The Effect Of Inhibit EphA4 Receptor On Aspirin Promoting Oligodendrocytes Development

With the development of population aging process,the incidence of white matter lesions(WML)in the elderly is increasing,resulting in cognitive function impairment in the elderly,which seriously affecting their quality of life and health.Chronic cerebral hypoperfusion is considered the most important factor leading to WML,but the pathogenesis and treatment are not clear.Previous studies have shown that the main pathological changes in WML are loss of oligodendrocytes(OLs),demyelination and axonal degeneration in the brain.Therefore,the important straregy for preventing WML is to supplement the lost and dead OLs,repair damaged myelin,and then ameliorate cognitive dysfunction.As the central nervous system myelin sheath cells,OLs derived from oligodendrocyte precursor cells(OPC)of the white matter situ and pluripotent neural stem cells of subventricular zone(SVZ).OPC can supplement the lost OLs by proliferation and differentiation in the process of repairing the white matter demyelination.Hence,the method of promoting OPC proliferation and differentiation is the key to supplement the lost OLs and treat WML.In the previous study,our group has found that aspirin(ASA)can promote OPC proliferation,differentiation and maturation,thereby repair damaged myelinand improve cognitive function in rats.Its possible mechanism involves ASA affects downstream ERK and RhoA activity by inhibiting EphA4 receptors on OPC and OLs,and thus promotes OPC proliferation,differentiation and maturation.In this study,we will focus on the expression of EphA4 receptor in OPC and OLs and the effect of ASA on it;and then block the EphA4 receptor activity by specific inhibitors,research the effect of OPC proliferation,differentiation and myelination in situ of WML model rats damage in vivo;as well as the effect of cultured OPC proliferation and differentiation in vitro;and finally to detect the change of downstream signal ERK/p-ERK,RhoA.In a word,provide experimental basis for the treatment of WML by ASA,and new targets for drug development in the treatment of WML.Experiment 1 The expression of EphA4 receptor in oligodendrocyte cell lines and the effect of ASA on it Objective:To study the expression and cellular localization of EphA4 receptor in the corpus callosum of rats and the cultured OPC,OLs in vitro,and the effect of ASA on it;Methods:1)normal rat brain tissue samples were obtained by frozen section,immunofluorescence staining was used to observe the co-expression of NG2/EphA4,CNPase/EphA4 in CC;2)OPC and OLs were obtained by cell primary culture,the co-expression of NG2/EphA4 in OPC and CNPase/EphA4 in OLs were observed by immunofluorescence staining;cultured OPCs and OLs were divided into ASA(1.0?mol/L)group and the same volume of ethanol,the expression of EphA4 receptor was examined by immunofluorescence staining and Western blot;Results:1)Immunofluorescence staining of rat brain tissue showed that OPC and OLs expressed EphA4 receptor in CC;2)Immunofluorescence staining of cell showed that cultured OPC and OLs expressed EphA4 receptor;compared with the control group,the number of NG2/EphA4 double-stained OPCs and CNPase/EphA4 double-stained OLs in ASA(1.0?mol/L)group were significantly decreased,the difference is statisticallysignificant(p<0.05);Western Blot showed that the EphA4 expression in ASA(1.0?mol/L)group was significantly decreased,as compared to the control group,the difference is statistically significant(p < 0.05);Conclusions:OPC and OLs could express EphA4 receptor,and its expression can be inhibited by ASA.Experiment 2 The effect of inhibit EphA4 receptor on ASA treating WML model rats Objective:To study the effect of EphA4 receptor inhibitor(EphA4-FC)on ASA treating WML model rats;Methods:CCAO method was use to make the WML model rats,then treated with 25mg/kg/d ASA,EphA4-FC(lateral cerebral ventricle injection250nM),25mg/kg/d ASA+EphA4-FC(lateral cerebral ventricle injection 250nM)and the same proportion of saline for 4 weeks,the expression of NG2 and CNPase positive cells in CC was observed by immunofluorescence staining,and the protein levels of PDGF?R and MBP were detected by Western blot;Moreover,the change of myelin structure in CC was examined by electron microscope;Results:1)Immunofluorescence staining showed that compared with the normal group,the number of NG2 positive cells was increased and the number of CNPase positive cells was decreased in the control group,however,the number of NG2 and CNPase positive cells was significantly increased by ASA,EphA4-FC,ASA+EphA4-FC treatment,and the ASA+EphA4-FC group increased more than others,the difference is statistically significant(p < 0.05),nevertheless,ASA group and EphA4-FC group have the same increased degree,the difference has no statistically significant(p>0.05);2)Western Blot showed that the expression of PDGF?R in control group was higher than normal group,and compared with the control group,the expression of PDGF?R in ASA,EphA4-FC,ASA+EphA4-FC group all incereased,and the ASA+EphA4-FC group increased more significantly,the difference is statistically significant(p<0.05);By contrast,the expression of MBP decreased after WML,while the effect was reversed by ASA,EphA4-FC,ASA+EphA4-FC treatment,and theASA+EphA4-FC group was more prominent,the difference is statistically significant(p<0.05);3)The electron microscope showed that compared with the normal group,demyelination,decrease of myelin thickness and increase of G-ratio were observed in WML model rats,while the effect was reversed by ASA,EphA4-FC,ASA+EphA4-FC treatment,and the ASA+EphA4-FC group was more prominent,the difference is statistically significant(p < 0.05);Conclusions:Inhibition of EphA4 receptors could promote OPC proliferation,differentiation and myelination in the CC of WML model rats,and enhance the therapeutic effect of ASA on WML model rats.Experiment 3 The effect of inhibit EphA4 receptor on ASA promoting OPC proliferation and differentiation Objective:To study the effect of EphA4 receptor inhibitor(EphA4-FC)on ASA promoting OPC proliferation and differentiation;Methods:Cultured OPCs and OLs were treated with ASA(1.0?mol/L),EphA4-FC(250nM),ASA(1.0?mol/L)+EphA4-FC(250nM)and the same volume of ethanol,the expression of NG2/Ki67 and CNPase positive cells was observed by immunofluorescence staining,and the protein levels of PDGF?R and MBP were detected by Western blot;Results:1)Immunofluorescence staining showed that compared with the control group,the number of NG2/Ki67 and CNPase positive cells was significantly increased in ASA,EphA4-FC,ASA+EphA4-FC treatment group,and the ASA+EphA4-FC group increased more than others,the difference is statistically significant(p < 0.05);2)Western Blot showed that PDGF?R and MBP expression was significantly increased in each treatment group,as compared to the control group,and the ASA+EphA4-FC group was more prominent,the difference is statistically significant(p<0.05);Conclusions:Inhibition of EphA4 receptors could promote OPC proliferation and differentiation in viro,and enhance the effect of ASA promoting OPC proliferation and differentiation.Experiment 4 The effect of inhibit EphA4 receptor on ERK and RhoA signaling pathways Objective:Recent study has shown that ASA can regulate ERK and RhoA signaling pathways,and the previous experiments have confirmed that ASA can inhibit EphA4.This study is to investigate the effect of EphA4 receptor inhibitor(EphA4-FC)on ERK and RhoA signaling pathway;Methods:the brain tissue and cell proteins were obtained according to the methods of experiment 2 and 3,the protein levels of ERK/p-ERK and RhoA in each group were detected by Western blot;Results:1)The Western blot results of tissue showed that compared with the normal group,the expression of p-ERK decreased in control group,however,the expression of p-ERK increased significantly in ASA,EphA4-FC,ASA+EphA4-FC treatment group,as compared to the control group,the difference is statistically significant(p<0.05),while the protein levels of total ERK had no change;By contrast the ERK,RhoA activity in control group was significantly higher than normal group,but decreased greatly after ASA,EphA4-FC,ASA+EphA4-FC treatment,the difference is statistically significant(p<0.05);2)The Western blot results of cell showed that compared with the control group,the expression of p-ERK increased significantly in each treatment group,the difference is statistically significant(p < 0.05),total ERK level was no difference among the groups;While the RhoA activity of each treatment group decreased compared with the control group,the difference is statistically significant(p<0.05);Conclusions:Inhibition of EphA4 receptor activity can activate ERK while inhibiting RhoA signaling pathway.