Role Of Connexin43 In Promoting The Angiogenesis Of Cerebral Ischemia-reperfusion Injury By Buyang Huanwu Decoction

ObjectiveConnexin43(Cx43)is the most major gap junction(GJ)protein on astrocytes,which can transport toxic metabolites and nutrients among cells,and have a bidirectional regulation effect on the diffusion and restoration.Buyang Huanwu decoction(BYHWD)influences promoting qi flow and blood circulation.In this study,behavioral experiments,golgi-staining and immunofluorescence staining were used to examinate the neurological function recovery,neurogenesis and angiogenesis indicators,so as to explore the role of connexin43 in BYHWD impairing cerebral ischemia-reperfusion injury(CIRI)in rats.The formation of blood vessels is related to vascular endothelial growth factor(VEGF)and angiopoietin-1(Ang-1).This experiment for the concerned mechanism has carried on a preliminary exploration,which provided more scientific evidences for the study of new targets and ways of BYHWD restoring.MethodsThe middle cerebral artery occlusion(MCAO)model was established by Longa method.The rats after surgery up to standard were randomly divided into model group,GAP-134(Cx34 agonist)group,Gap26(Cx34 antagonist)group,BYHWD group and BYHWD+Gap26 group,and a sham-operated group was set up as control as well.Every group has 12 rats.Rats in GAP-134 group was treated with GAP-134 intragastrically 3mg/kg twice a day and Gap26 group were intraperitoneally injected with Gap26 25μg/kg once a day both since the 3rd day after surgery;BYHWD group was administrated with 16g/kg intragastrically twice a day 2 h after the molding,while the MCAO and sham group were given the same dosage of saline in corresponding ways.1.The role of Connexin43 in BYHWD impairing cerebral ischemia-reperfusion injury.At the 1st,3rd,and 7th day after the operation,beam-walking test(BWT)was performed to observe the motor integration ability and balancing ability;The Spontaneous alternation behavior test(SAB test)was to determinate the spatial memory ability.At the 7th day,the rats were sacrificed for golgi-staining and immunofluorescence.We observed neurons involving dendritic length,number of nodes,number of endings and density of dendritic spines,which reflected the complexity of neuron dendrites and the plasticity of dendritic spines.2.To investigate the mechanism of Connexin43 in BYHWD angiogenesis.At the 7th day after operation,quantifiing CD31 expression as microvessel density(MVD)was marked angiogenesis,and the expression of vascular endothelial growth factor(VEGF)and angiogenin factor 1(Ang-1)was detected by immunofluorescence.The target observation regions of golgi-staining and immunofluorescence were hippocampal CA1,CA3,and DG areas.The whole experiment resorted to a Laser scanning confocal microscope and an Image Pro Plus 6.0.Results1.Compared with MCAO group,the rats of GAP-134 group had lower scores of the beam-walking test at the 7th day(P<0.05)and higher percentage of relative alternation of SAB test(P<0.01);the dendritic length,numbers of nodes and endings,and the dendritic spines density were also increased.However,the scores of the beam-walking test at the 7th day in the Gap26 group were higher(P<0.05);the percent of relative alternation were lower(P<0.05);the dendritic length,numbers of nodes and endings,dendritic spines density decreased generally.The rats of BYHWD group had lower scores of the beam-walking test(P<0.05)and higher percentage of relative alternation of SAB test(P<0.05);the dendritic length,numbers of nodes and endings,and the dendritic spines density were also increased.Compared with BYHWD group,the scores of the beam-walking test at the 7th day in the BYHWD+Gap26 group were higher(P<0.05);the percent of relative alternation were lower(P<0.05);the dendritic length,numbers of nodes and endings,dendritic spines density decreased generally.2.Compared with MCAO group,the expressions of MVD in the hippocampal CAI.CA3 and DG areas(P<0.01)and VEGF in CA1/CA3 areas(P(0.05)and Ang-1 in the CA3 area(P<0.05)of GAP-134 group increased,However,the MVD decreased in CA1 and DG areas(P<0.05)in the Gap26 group(P<0.05),and the expression of VEGF decreased in the DG area(P<0.05),Ang-1 was significantly down-regulated in CA1 and DG regions(P<0.01);BYHWD group increased MVD in the hippocampus(P<0.05)and VEGF expressions in CA1 and CA3 areas of(P<0.05),and significantly up-regulated Ang-1 expression in three regions(P<0.01).Compared with BYHWD group,MVD expression levels decreased markedly(P<0.01)in hippocampus;VEGF expression in CA1 and DG regions(P<0.01)and Ang-1 expression in CA1 and CA3 regions(P<0.05)decreased significantly.Conclusions1.After cerebral ischemia-reperfusion,Buyang Huanwu Decoction can activate neurogenesis and angiogenesis and promote the rehabilitation of neurological function through astrocyte Cx43 to promote the repair of brain injury.2.The mechanism of Buyang Huanwu Decoction in inducing angiogenesis through astrocyte Cx43 is related to VEGF and Ang-1.