| Object: By establishing middle cerebral artery occlusion(MCAO)model in rats,we observed the effect of Wnt/?-catenin signaling pathway on isoflurane postconditioning to reduce cerebral ischemia-reperfusion injury.The specific inhibitor of Wnt/?-catenin signaling pathway and TGF-?1/Smad3 pathway-related inhibitors were applied to explore the role of TGF-?1/Smad3 pathway in the regulation of Wnt/?-catenin pathway in ischemic reperfusion injury in rats after isoflurane postconditioning.Methods: The model of focal cerebral ischemia-reperfusion injury in SD rats was established by middle cerebral artery occlusion(MCAO)ischemia for 90 min and reperfusion for 24 hours.Postconditioning by inhalation of ISO(1.5%)was performed for 60 min after ischemia at the onset of reperfusion.Each inhibitor group was injected with different inhibitors(Wnt pathway inhibitor DKK-1,TGF-?1 inhibitor LY2157299,Smad3 inhibitor SIS3)through the abdominal cavity or lateral ventricle 30 min before surgery.Various indicators of the rats were observed after 24 hours.Neurological deficit score and TTC staining were used to evaluate brain injury.TUNEL staining was used to observe the degree of apoptosis in hippocampal CA1;Nissl staining was used to observe the degree of damage of Nissl bodies;Real-time quantitative PCR technique(qRT-PCR)was applied to detect the mRNA levels of Wnt3 a,?-catenin and CyclinD1;immunofluorescence(IF)staining was used to observe the localization of ?-catenin and CyclinD1 in neuronal cells.The expression levels of ?-catenin both in the cytoplasm and nucleus,Wnt3 a,GSK-3?,CyclinD1,VEGF,Caspase3,TGF-?1,Smad3 and p-Smad3 of hippocampal CA1 region were detected by Western blot.Results:(1)Compared with the sham(S)group,the neurological deficit score,infarct volume,neuron apoptosis in the hippocampal CA1 area,and the damage degree of Nissl body were significantly increased in the ischemia-reperfusion injury(M)group(P<0.05),the expression levels of Wnt3 a,?-catenin and CyclinD1 significantly decreased(P<0.05),while the expression level of GSK-3? increased(P<0.05).In the isoflurane postconditioning(M+I)group,compared wtih the M group,the neurological deficit score,infarct volume,neuronal apoptosis and the damage degree of Nissl body significantly mitigated(P<0.05).The expression levels of Wnt3 a,?-catenin,VEGF and CyclinD1 significantly increased(P<0.05)while the expression level of GSK-3? was significantly decreased(P<0.05).(2)After the application of Wnt/?-catenin pathway inhibitor DKK-1,the neurological deficit score,infarct volume,neuronal apoptosis and the damage degree of Nissl body increased(P<0.05).The expression levels of Wnt3 a,?-catenin,VEGF and CyclinD1 significantly decreased(P<0.05),and the expression level of GSK-3? increased(P<0.05).(3)The expression levels of p-Smad3 and TGF-?1 protein decreased after pre-injection of the TGF-?1 inhibitor LY2157299(P<0.05).Compared with the M+I group,the expression level of ?-catenin protein in the cytoplasm and nucleus both obviously decreased(P<0.05).When Smad3 targeted inhibitor SIS3 was used,the expression level of p-Smad3 decreased(P<0.05),and the expression level of ?-catenin protein in cytoplasm and nucleus reduced than that of MI group(P<0.05).When DKK-1 was injected,the expression levels of TGF-?1 and p-Smad3 protein remained almost unchanged(P>0.05).In addition,the expression level of Smad3 protein in each group was almost no change(P>0.05).Compared with the MI group,the expression level of Caspase 3 significantly increased in all inhibitor groups(P<0.05).(4)In the MI group,the expression level of ?-catenin in the hippocampal CA1 region was higher than that of the other inhibitor groups(P<0.05).Conclusion:(1)Isoflurane post-conditioning can activate Wnt/?-catenin signaling pathway,inhibit neuronal apoptosis and play a protective role in the cerebral ischemia-reperfusion injury;(2)In the protective effect induced by isoflurane post-conditioning,TGF-?1/Smad3 signaling pathway has certain effect on the Wnt/?-catenin signaling pathway. |
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