Role Of Connexin43 In Promoting The Synaptic Structural Plasticity Of Cerebral Ischemia-Reperfusion Injury By Buyang Huanwu Decoction

ObjectiveCerebrovascular disease is one of the leading causes of death in China,ischemic cerebrovascular disease is the most common type of cerebrovascular disease,with high incidence,high recurrence rate,high disability rate and other characteristics.The prophylaxis and treatment of cerebrovascular disease has become a hot area in medicine today.Connexin43(Cx43)can transmit substances and information between cells and promote the repair after brain injury,which widely exists in astrocytes.Buyang huanwu decoction(BYHWD)is a representative prescription for the treatment of cerebral ischemia,which has the function of invigorating qi,activating blood circulation,removing stasis and clearing collaterals.BYHWD can maintain Basic Fibroblast growth factor(bFGF)and promote the expression of Cx43 during the recovery period of cerebral ischemia,Improving the morphology of neurons and promote the recovery of nerve function.The restoration of nerve function also depends on nerve remodeling,and synaptic remodeling is the strongest part of nerve remodeling.Synaptic plasticity is mainly regulated by synaptophysin(SYN)and growth associated protein-43(GAP-43).The changes of neuronal synaptic ultrastructure were observed by transmission electron microscopy,synaptic plasticity protein expressions were detected by Western blot and Immunofluorescence.To investigate the mechanism of connexin43 in BYHWD repairing synaptic structural plasticity after cerebral ischemia-reperfusion.MethodsMiddle cerebral artery occlusion and reperfusion model was established.1.The role of Connexin43 in impairing cerebral ischemia-reperfusion.SD rats were randomly divided into sham-operated group,model group,Gap26(Cx43 antagonist)group,GAP-134(Cx43 agonist)group.Rats in Gap26 group were intraperitoneally injected with Gap26 25 μg/kg once a day and GAP-134 group was treated with GAP-134 intragastrically 3 mg/kg twice a day both on the third day after surgery,while the MCAO and sham group were given the same dosage of saline in corresponding ways.Brain was taken out at 7 day.The changes of neuronal synaptic ultrastructure were observed by transmission electron microscopy.SYN and GAP-43 protein expressions were detected by Western blot.The target observation regions of immunofluorescence were hippocampal CA1,CA3 and DG areas.2.The role of Connexin43 in BYHWD impairing cerebral ischemia-reperfusion.SD rats were randomly divided into sham group,model group,BYHWD group and BYHWD+Gap26 group.BYHWD group was administrated with 16 g/kg intragastrically twice a day 2 h after the molding,Gap26 was intraperitoneally injected 25 μg/kg once a day on the third day after surgery,while the MCAO and sham group were given the same dosage of saline in corresponding ways.Brain was taken out at 7 day.The changes of neuronal synaptic ultrastructure were observed by transmission electron microscopy.SYN and GAP-43 protein expressions were detected by Western blot.The target observation regions of immunofluorescence were hippocampal CA1,CA3 and DG areas.3.The role of bFGF in Cx43 impairing cerebral ischemia-reperfusion.SD rats were randomly divided into sham-operated group,model group,bFGF group,bFGF neutralizing antibody group,BYHWD group,BYHWD+bFGF neutralizing antibody group,bFGF+Gap26 group.BYHWD group was administrated with 16 g/kg intragastrically twice a day 2 h after the molding,bFGF was intraperitoneally injected 100 μg/kg once a day,and Gap26 was intraperitoneally injected 25μg/kg once a day both on the third day after surgery,bFGF neutralizing antibody intragastrically 0.1 mg/kg once a day on the third day after surgery,while the MCAO and sham group were given the same dosage of saline in corresponding ways.Brain was taken out at 7 day.Synaptic plasticity protein expressions were detected by Western blot.The target observation regions of immunofluorescence were hippocampal CA1,CA3 and DG areas.Results1.Western blot revealed an increase of Cx43 at 3 d,7 d,and 14 d after MCAO(P<0.05).Furthermore,in the 7 d after MCAO,the protein expression levels was reduced compared to the 3 d(P<0.05),and in the 14 d after MCAO the protein expression levels was still reduced compared to the 7 d.The ultrastructure of the synapse in the sham group remained intact and clear.The structure was destroyed and the synaptic cleft was dim,fusion of synaptic space,loss of synaptic vesicles,incomplete synaptic structure,and swelling of the presynaptic terminal were observed in the MCAO and Gap26 group.However,these changes were obviously improved in GAP-134 group.Compared with the sham-operated group,model group up-regulated the expressions of SYN and GAP-43(P<0.05).Gap26 group decreased the expressions of SYN and GAP-43(P<0.05).However,GAP-134 up-regulated the expressions of SYN and GAP-43(P<0.05).2.The structure of synapses was integrated in the sham group.While in model group hippocampus,the structure was destroyed,Compared with the sham-operated group,model group up-regulated the expressions of SYN and GAP-43(P<0.05).In BYHWD group hippocampus,the structure was close to the normal,Furthermore,BYHWD up-regulated the expressions of SYN and GAP-43(P<0.05).However,in the co-administration with Cx43 inhibitor(Gap26)groups the damage of synaptic structural decreased,the effect of BYHWD on SYN and GAP-43 was inhibited(P<0.05).3.Only weak expressions of Cx43 can be observed in the sham group.In the MCAO group,the protein expression levels were dramatically increased.However,compared to the MCAO group,the expression levels of these proteins in the BYHWD group were distinctly upregulated.Furthermore,in the co-administration with bFGF neutralizing antibody group the effect of BYHWD on Cx43 was inhibited(P<0.05).Compared with the model group,bFGF group up-regulated the expressions of SYN and GAP-43(P<0.05).However,in the co-administration with Cx43 inhibitor(Gap26)groups the effect of bFGF on SYN and GAP-43 was inhibited(P<0.05).Conclus ions1.Cx43 could promote the intervention of SYN and GAP-43,which prove thehippocampal synaptic structural plasticity obviously after the CIRI.2.BYHWD could improve the hippocampal synaptic structural plasticity obviously after the CIRI.The mechanism may be related to increase the expression of Cx43 and promote the intervention of SYN and GAP-43.3.BYHWD could maintain Basic Fibroblast growth factor(bFGF)and promote the expression of Cx43,which promote the intervention of SYN and GAP-43 improve the hippocampal synaptic structural plasticity obviously after the CIRI.