| Objective: To determine the effect of blockage of P2X7 receptor(P2X7R)against cerebral ischemia/reperfusion injury in rats and its protential mechanism.Methods: 1.SD rats were divided into five goups: sham group,ischemia/reperfusion(I/R)group,I/R+BBG group,I/R+PD98059 group,I/R+BBG+PD98059 group.A thread occlusion method was used to make a middle cerebral artery occlusion(MCAO)model in the rats for 2 h followed by reperfusion for 24 h.Neurobehavioral scores was detected by Longa’5.TTC staining was used to measure the cerebral infarction volume and Western blot was performed to detect the expressions of P2X7 R,Cx43,ERK,p-ERK,Bax and Bcl-2 in the brain of the rats.PD98059 0.3mg·kg-1 was injected 20 min before ischemia by tail vein,BBG 30 mg·kg-1 was injected 30 min after ischemia intraperitoneally.To ensure that each group of models were prepared successfully up to 10 rats.2.Astrocytes of the study were divided into control group,hypoxia/reoxygenation(H/R)group,H/R+BBG group,H/R+PD98059 group and H/R+BBG+PD98059group.Astrocytes viability and the cell apoptosis were detected by Methyl thiazolyltetrazolium(MTT)assay and Hochest 33258.The expressions of Bax,Bcl-2,ERK,p-ERK,Cx43 were detected by Western blot.The function of gap junction was evaluated by fluorescent tracer.Results:1.The neurobehavioral scores were assessed among the different groups.No neurological deficits were observed in the sham group.After 24 h of reperfusion,compared with the MCAO group,the neurobehavioral scores were greatly decreased when rats administrated with BBG or PD98059.Moreover,the neurobehavioral score of rats administrated with BBG+PD98059 was much lower than that rats administrated with BBG or PD98059.The infarct volume of the brain tissue in rats from the different experimental groups was examined by the TTC staining.No infarcted area was observed in the sham group,whereas a significant infarction was shown in the MCAO group.However,the infarct volume was significantly reduced when rats administrated with BBG or PD98059 compared with MCAO group.Notably,rats treated with BBG and PD98059 showed a smaller infarct volume than rats treated with BBG or PD98059.Together,these results indicated that the combination of BBG and PD98059 treatment was superior to BBG or PD98059 treatment in attenuation of the brain injury in MCAO.2.Compared with the sham group,the expression levels of P2X7 R,p-ERK,Cx43 were significantly increased in MCAO group.BBG treatment obviously decreased p-ERK,Cx43 expression levels compared to MCAO group.Compared with BBG or PD98059 treatment,the expression levels of p-ERK,Cx43 were significantly decreased when rats administrated with BBG and PD98059.To further investigate the effects of BBG on the apoptosis of the brain tissue in rats of MCAO,the levels of Bax/Bcl-2 was measured by Western blot.Compared to the sham group,the ratio of Bax/Bcl-2 was significantly increased in MCAO group.Compared to MCAO group,the ratio of Bax/Bcl-2 was significantly reduced when rats treated with BBG or PD98059.Furthermore,the ratio of Bax/Bcl-2 was greatly decreased when rats treated with BBG and PD98059 compared with the BBG or PD98059 treatment.3.In order to observe the protective effect of blockage of P2X7 R on astrocytes hpoxia/reoxygenation injury,MTT assay was detected after hpoxia/reoxygenation,the astrocytes viability was reduced significantly.Administration of BBG or PD98059 could increase the astrocytes viability greatly after hpoxia/reoxygenation.BBG combined with ERK kinase inhibitor PD98059 significantly reduced the astrocytes viability.4.To further investigate the effects of BBG on the apoptosis of the brain tissue in rats of MCAO,the levels of Bax,Bcl-2 were measured by Western blot.Compared with the sham group,the ratio of Bax/Bcl-2 was significantly increased in MCAO group.Compared to MCAO group,the ratio of Bax/Bcl-2 was significantly reduced when rats treated with BBG or PD98059.Furthermore,the ratio of Bax/Bcl-2 was greatly decreased when rats treated with BBG+PD98059 compared with the BBG or PD98059 treatment.5.The results show that after hpoxia/reoxygenation the expression of Cx43,p-ERK and Bax/Bcl-2 ratio were both increased.Cx43,p-ERK protein expression and reduce the Bax/Bcl-2 ratio after administration of BBG or PD98059.Administration of BBG combined with PD98059,reduced the expression level of Cx43,p-ERK,both of the Bax/Bcl-2 ratio.6.The function of gap junction was detected by fluorescent tracer.The results show that compared with control group,the function of gap junction increased significantly in hpoxia/reoxygenation group.Compared with hpoxia/reoxygenation group,administration of BBG or PD98059 could increase the function of gap junction.Administration of BBG combined with PD98059,reduced the function of gap junction greatly.Conclusions: 1.Blockage of P2X7 R reduced cerebral I/R injury was associated with inhibition of ERK kinase.2.Blockage of P2X7 R reduced cerebral I/R injury was accompanied with inhibition of gap junction and the expression of Cx43. |
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