| Objective: To explore the possible mechanism of hyperglycemia exacerbating cerebral ischemia reperfusion injury.To investigate the effects of lycium barbarum polysaccharide on ameliorating hyperglycemia-exacerbated cerebral ischemia reperfusion injury and the changes of Wnt/?-catenin signaling pathway.Methods: The experimental animals were randomly divided into normoglycemia group(NG),hyperglycemia group(HG),Lycium barbarum polysaccharide intervention group(LBP).According to the different treatment methods and time points of ischemia reperfusion,each group was divided into four subgroups: sham group,ischemia reperfusion 1-,3-,7-d group.The model of hyperglycemia was established by intraperitoneal injection of streptozotocin(STZ).After the hyperglycemia model was established,rats were given intraperitoneal injection of Lycium barbarum polysaccharide(LBP)for 4 weeks,during which body weight and blood glucose were monitored.Then the middle cerebral artery occlusion(MCAO)reperfusion model was established by thread embolization.At different reperfusion time points,the neurological deficits,survival rate,infarct volume,blood-brain barrier permeability,histological changes,apoptosis and neuronal ultrastructure were measured.The constructed components of neurovascular unit in cerebral ischemic penumbra cortex: neuron nuclear antigen(NeuN),glial fibrillary acidic protein(GFAP),type IV collagen(Collagen IV),vascular endothelial growth factor(VEGF)and blood-brain barrier related proteins: ZO-1,Occludin and Claudin-5 were detected by immunohistochemistry and Western blot.Microvessel density counts were measured by immunofluorescence.The whole structure of neurovascular unit was observed by double immunofluorescence labeling and triple immunofluorescence labeling techniques.The ultrastructure of microvascular was observed by transmission electron microscopy.The expression of key factors of Wnt/?-catenin signaling pathway: Wnt3 a,?-catenin,p-GSK-3? and GSK-3? in cerebral ischemic penumbra cortex was detected by Western blot and immunofluorescence.HT22 cells were cultured in hyperglycemia oxygen deprivation and reoxygenation state to simulate hyperglycemic cerebral ischemia reperfusion state in vivo.The experimental cells were divided into normal glucose group(NG),hyperosmotic group,normal glucose oxygen deprivation group(NG+OD),high glucose oxygen deprivation group(HG+OD),high glucose oxygen deprivation Lycium barbarum polysaccharide intervention group(HG+OD+LBP),high glucose oxygen deprivation LiCl intervention group(HG+OD+LiCl),high glucose oxygen deprivation LiCl and LBP joint intervention group(HG+OD+LiCl+LBP).MTT assay was used to detect cell activity.DHE assay was used to detect the production of reactive oxygen species(ROS).Western blot and immunofluorescence were used to detect the expression of key factors of Wnt/?-catenin signaling pathway: ?-catenin,p-GSK-3? and GSK-3?.Results: The body weight of HG group was lower than that of NG group at 3 day,1 week,2 week,3 week and 4 week after STZ administration(P<0.05).There was no significant difference between LBP group and HG group.Glucose levels in HG group were higher than those in NG group at 3 day,1 week,2 week,3 week and 4 week after STZ administration(P<0.05),while those in LBP group were lower than those in HG group at 3 week and 4 week after STZ administration(P<0.05).The neurological deficit score in HG group was higher than that in NG group(P<0.05),while that in LBP group was lower than that in HG group(P<0.05),but still higher than that in NG group(P<0.05).The survival rate was lower in HG group than in NG group(P<0.05),and higher in LBP group than in HG group(P<0.05)at 3 d and 7 d of ischemia reperfusion.The infarct volume of HG group was significantly larger than that of NG group(P<0.01),and that of LBP group was significantly smaller than that of HG group(P<0.01).The amount of IgG leakage from blood-brain barrier in HG group was significantly higher than that in NG group(P<0.01),and that in LBP group was significantly less than that in HG group(P<0.01).Number of nuclear pyknotic neurons in HG group was higher than that in NG group(P<0.05),while that in LBP group was lower than that in HG group(P<0.05).The percentage of apoptotic cells in HG group was significantly higher than that in NG group(P<0.01),while that in LBP group was significantly lower than that in HG group(P<0.01).HE staining,Nissl staining and transmission electron microscopy showed that HG group had more serious morphological changes than NG group,and LBP group had less morphological changes than HG group.The expression of neuron nuclear antigen(NeuN)was lower in HG group than that in NG group(P<0.05 or P<0.01)and higher in LBP group than that in HG group(P<0.01 or P<0.05)at 1 d,3 d and 7 d after ischemia reperfusion;NeuN expression increased with the prolongation of reperfusion time in NG group(P<0.05);HG group and LBP group showed a downward trend with the prolongation of reperfusion time(P < 0.05).The expression of glial fibrillary acidic protein(GFAP)in HG group was lower than that in NG group at 1 d and 3 d after ischemia reperfusion(P<0.05,P<0.01),higher in LBP group than that in HG group at 3d(P<0.05),higher in HG group than that in NG group at 7 d(P<0.01),and lower in LBP group than that in HG group at 7 d(P<0.05);with the prolongation of reperfusion time,the expression of GFAP in NG group increased first and then decreased(P<0.01),while that in HG group and LBP group increased(P<0.05 or P<0.01).The expression of Collagen IV was lower in HG group than in NG group(P<0.01 or P<0.05)and higher in LBP group than in HG group(P<0.05)at 1,3 and 7 d of ischemia reperfusion;with the prolongation of reperfusion time,the expression of Collagen IV increased in NG group,HG group and LBP group(P<0.05 or P<0.01).The expression of vascular endothelial growth factor(VEGF)in HG group was higher than that in NG group at 1 d of ischemia reperfusion(P<0.01),HG group was lower than that in NG group at 3 and 7 d(P<0.05,P<0.01),and LBP group was higher than that in HG group at 3d(P<0.05);with the prolongation of reperfusion time,the expression of vascular endothelial growth factor increased in NG group(P<0.05 or P<0.01),and decreased in HG group and LBP group(P<0.05).The number of microvessels in HG group was significantly less than that in NG group(P<0.01),and that in LBP group was more than that in HG group(P<0.05).Vascular dissociation index in HG group was significantly higher than that in NG group(P<0.01),and that in LBP group was significantly lower than that in HG group(P<0.01).The expression of Occludin and Claudin-5 in HG group was lower than that in NG group(P<0.05).The expression of ZO-1,Occludin and Claudin-5 in LBP group were higher than those in HG group(P<0.01 or P<0.05).The results of triple immunofluorescence staining showed that the degree of structural disorder of neurovascular unit in HG group was higher than that in NG group,and that in LBP group was lower than that in HG group.Transmission electron microscopy showed that the degree of ultrastructural disorder of microvascular in HG group was higher than that in NG group,and that in LBP group was lower than that in HG group.The expression of Wnt3 a in HG group was lower than that in NG group(P<0.05 or P<0.01)and higher in LBP group than that in HG group(P<0.01)at 1,3 and 7 d of ischemia reperfusion.The expression of ?-catenin in HG group was lower than that in NG group at 1 d and 3 d of ischemia reperfusion(P<0.05);the expression of ?-catenin in LBP group was higher than that in HG group(P<0.05 or P<0.01)at 1,3 and 7 d of ischemia reperfusion.The expression of p-GSK-3? in HG group was lower than that in NG group(P<0.01)and in LBP group was higher than that in HG group(P<0.01)at 1 d of ischemia reperfusion;HG group was higher than that in NG group(P<0.01)and LBP group was lower than that in HG group(P<0.05)at 3 d of ischemia reperfusion;HG group was higher than that in NG group(P<0.05)and LBP group was higher than that in HG group(P<0.05)at 7 d of of ischemia reperfusion.The expression of GSK-3? in LBP group was higher than that in HG group(P<0.05)at 1 d of ischemia reperfusion;HG group was lower than that in NG group(P<0.05),LBP group was higher than that in HG group(P<0.01)at 3 d of ischemia reperfusion;HG group was higher than that in NG group(P<0.05)and LBP group was lower than that in HG group(P<0.05)at 7 d of ischemia reperfusion.?-catenin was mainly expressed in neurons.Cell viability in HG+OD group was lower than that in NG+OD group(P<0.01),and that in HG+OD+LBP group was higher than that in HG+OD group(P<0.01 or P<0.05).The production of reactive oxygen species(ROS)in HG+OD group was higher than that in NG+OD group(P<0.05),while that in HG+OD+LBP group was lower than that in HG+OD group(P<0.05).The expression of ?-catenin in HG+OD group was lower than that in NG+OD group(P<0.05),while that in HG+OD+LiCl group,HG+OD+LBP group and HG+OD+LiCl+LBP group was higher than that in HG+OD group(P<0.05).The expression of p-GSK-3? in HG+OD group was lower than that in NG+OD group(P<0.05),while that in HG+OD+LBP group was significantly higher than that in HG+OD group(P<0.01).The expression of GSK-3? in HG+OD+LBP group was significantly lower than that in HG+OD group(P<0.01).Conclusion: Hyperglycemia exacerbates cerebral ischemia reperfusion injury,and hyperglycemia exacerbates cerebral ischemia reperfusion injury by damaging neurovascular units.Hyperglycemia exacerbates the neurovascular units injury of cerebral ischemia reperfusion involves the inhibition of Wnt signaling pathway.LBP can alleviate the hyperglycemia-exacerbated cerebral ischemia reperfusion injury by protecting neurovascular units,and this protective effect may be related to the activation of Wnt/?-catenin signaling pathway. |
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