The Role Of CD200/CD200R Signaling Pathway In Microglia Activation After Stroke

Objective: To observe the difference between CD200/CD200 R protein and inflammatory response in ischemic stroke and CD200-CD200 R signal enhancement,and to explore the role of CD200/CD200 R signaling pathway in microglial activation after stroke。Methods: Experiment 1,The mice were randomly divided into sham-operated group and ischemia reperfusion group,ischemia reperfusion group was divided into 3 subgroups by 1 d、3 d and 7 d.Middle cerebral artery occlusion(MCAO)reperfusion models were produced by suture emboli technique in C57BL/6J mice.Neurological deficit scores were evaluated before operation in each group.At 1d,3d and 7d after ischemia reperfusion,the neurological deficit score was performed in each group.TTC(2,3,5-Triphenyltetrazolium chloride)was applied to measure the volume of cerebral infarction.Then enzyme-linked immunosorbent assay(ELISA)was used to detect the serum cytokine interleukin 4(IL-4),interleukin 10(IL-10),tumor necrosis factor-α(TNF-α)and immunoblotting(Werstern blot)was employed to detection the four proteins expression of inducible nitric oxide syntheses(i NOS)and CD200,CD200 R,Arg-1.Immunofluorescence technique was used to label CD200/CD200 R,and then co localized with Neu N(with CD200),CD3e(with CD200R),DAPI.The positive cells of CD200+/Neu N+and CD200R+/ CD3e+ was detected by laser confocal microscope.Experiment 2,The mice were randomly divided into sham-operated group,ischemia reperfusion group,Ig G2 a group and CD200 fusionprotein agonist(CD200Fc)group.At 3d after ischemia reperfusion,the difference of neurological deficits and infarct volume,serum IL-4,IL-10 and TNF-α and CD200,CD200 R,i NOS,arg1 protein were detected in each group.Results: Experiment 1,The sham-operated group had no change of nerve function defect.In the ischemia reperfusion groups,the highest neurological deficit score was in the 3d group,and there was no significant difference between the 1d group and the 3d group(P>0.05),Compared with group 1d and group 3d,there was significant difference in 7d group(P<0.001).The sham-operated group had no infarction.In the ischemia reperfusion groups,the maximum infarct volume was in the 3d group,there were significant differences between the 1d group,3d group and 7d group(P<0.001).In the 1d,3d,7d three groups,serum IL-4,IL-10,TNF-α were significantly higher than the sham-operated group and there was significant difference(P<0.001)The CD200 protein in 3d group was significantly lower than sham-operated group(P < 0.05),while there was no difference between sham-operated group vs 1d group and 7d group(P>0.05).The CD200 R and i NOS protein in 1d group,3d group and 7d group were significantly higher than those in sham-operated group.The difference have statistical significance(P<0.05).The Arg1 protein were began to rise at 1d,reached the peak at 3d,and has decreased at 7d and there was significantly statistical difference compared with the sham-operated group(P<0.05).The positive cells of CD200+/Neu N+ in 1d group,3d group and 7d group were lower than that of the control.The cells of CD200R+/ CD3e+ in 1d group,3d group and 7d group were significantly higher than those in sham-operated group.The difference have statistical significance(P<0.05).Experiment 2,The sham-operated group had no change of nerve function defect.CD200 Fc group has lower nerve function defect than the stroke group and Ig G2 a group,with no statistical significance(P>0.05).The sham-operated group had no infarction.The infarct volume of CD200 Fc group was lower than that of ischemia reperfusion group and Ig G2 a group(P<0.05).In the CD200 Fc group,serum TNF-α and i NOS protein was decreased,when compared with ischemia reperfusion group and Ig G2 a group,with statistical significance(P<0.05).Compared with the other three groups,CD200 protein,CD200 R protein,Arg1 protein and serum IL-4,IL-10 were higher in the CD200 Fc group(P<0.05).There was no difference between the stroke group and the Ig G2 a group(P>0.05).Conclusion:(1)Ischemic stroke blocks the CD200-CD200 R signaling pathway and promotes microglial activation.(2)CD200Fc promotes the expression of Arg1,IL-4 and IL-10,inhibits the secretion of i NOS and TNF-,the CD200-CD200 R signaling pathway is involved in the inflammatory response of ischemic stroke.