| BackgroundIschemic stroke is one of the most common causes of death and disability in the world.It has been confirmed that stroke can lead to cognitive dysfunction.However,due to severe physical disability,cognitive impairment after stroke is likely to be ignored.It is helpful to improve cognitive impairment and cognitive impairment after stroke.Some studies have shown that bone marrow mesenchymal stem cells(BMSCs)can repair endogenous brain injury after stroke by secreting exosomes to mediate intercellular communication.However,there are still many deficiencies in the targeting ability and in vivo efficacy of exosomes in clinical application.TCM believes that cognitive impairment after stroke belongs to the stage of stay and recovery and sequelae.The recovery and sequelae of stroke are mostly characterized by blood stasis,blood stasis,disturbance of brain collaterals,abnormal operation of blood and blood,and the use of blood loss fluid in the brain pulp,and then a series of symptoms change.Xionggui formula,which is composed of Angelica sinensis and chuanxiong,is a commonly used prescription for cerebrovascular diseases.In the early stage,the team developed shunaoxin dropping pill(SNX),a listed Chinese patent medicine developed by Xionggui formula,in collaboration with BMSCs exosomes,to intervene the rat model of transient middle cerebral artery occlusion(tMCAO),and found that it can not only promote the recovery of cognitive function of the model rats,but also promote the neurogenesis of the rats Synaptic remodeling and angiogenesis.However,how SNX cooperates with BMSCs exosomes to regulate synaptic remodeling in rats with ischemic stroke and its possible mechanism need to be further discussed.Objective:To observe the effect of SNX combined with BMSCs exosomes on neurological and cognitive function of tMCAO rats during recovery period,and to explore whether SNX combined with BMSCs exosomes can regulate synaptic plasticity through sema3 g / Nrp2 /plexina4 signaling pathway and improve cognitive function of tMCAO rats.Methods:1.BMSCs exosomes were prepared and identified by Western blot.2.The transient middle cerebral artery occlusion(tMCAO)model of SD rats was established by thread occlusion method,and the unqualified rats were eliminated by longa score.The animals were randomly divided into five groups: sham operation group(sham),model group(tMCAO),xiongguifang group(tMCAO + SNX),exosome group(tMCAO + BMSCsexos)and synergistic treatment group(tMCAO + BMSCs-exos + SNX).3.On the 1st,3rd,7th,14 th and 21 st day after modeling,the rats were scored with m NSS.On the 7th and 21 st day,the spatial memory function was evaluated with Y-maze.On the 21 st day,the anxiety state and short-term memory function were evaluated with elevated cross maze.Twenty one days after the model was established,the brain was perfused and the morphology of hippocampus and neuron damage were observed by HE staining and Nissl staining.4.The expression of synapse related protein SYN and PSD-95 in the infarcted hippocampus was detected by immunofluorescence staining.The morphology and density of dendritic spines in the infarcted hippocampus were detected by Golgi Cox staining.5.Western blot was used to detect the expression of Sema3 G,Nrp2,plexin A4 and Rac1 in Sema3G / Nrp2 / plexin A4 pathway,and to detect the synapse associated protein syn and PSD-95.Result:1.The results of neurological deficit score of rats in each group showed that the MNSs scores of exosomes group and synergetic group decreased compared with the model group 3days after cerebral ischemia-reperfusion(P < 0.05,P < 0.05).The MNSs scores of xiongguifang group decreased compared with the model group,but there was no significant difference(P >0.05).On the 14 th day after ischemia,the MNSs score of xiongguifang group was lower than that of model group(P < 0.05).On the 21 st day after ischemia,the MNSs score of the three groups decreased compared with the model group(P < 0.05).2.The results of Y-maze test showed that compared with sham operation group,the spontaneous alternative reaction rate of model group decreased significantly on the 21 st day after cerebral ischemia-reperfusion(P < 0.01);compared with model group,the spontaneous alternative reaction rate of xiongguifang group increased,but the difference was not statistically significant(P > 0.05);compared with model group,the spontaneous alternative reaction rate of exosome group and synergy group increased significantly(P<0.001,P<0.001)。3.The results of elevated cross maze test showed that on the 21 st day after ischemiareperfusion,the total number of entering the open arm and closed arm(OE + CE)in the model group was lower than that in the sham operation group(P < 0.05),the total number of entering the arm in the exosome group and the synergy group were higher than that in the model group(P < 0.05),There was no significant difference between Xionggui formula group and model group(P > 0.05).Compared with the sham operation group,the OT% of the model group was significantly increased(P < 0.01).Compared with the model group,the anxiety state of the three treatment groups was improved,but there was no statistical difference(P > 0.05).There was no significant difference in TLT before and after ischemia-reperfusion in each group.After ischemia-reperfusion,TLT in model group was significantly lower than that in sham operation group(P < 0.01).TLT in treatment group decreased compared with that in model group,but the difference was not statistically significant(P > 0.05).4.The results of HE staining showed that compared with the model group,the morphology of hippocampal neurons in the three treatment groups was relatively complete,the degree of damage was lighter,the cell necrosis was reduced,and the hippocampal formation was clear;Nissl staining showed that compared with the model group,the number of neurons in Xionggui formula group,exosome group and synergetic group increased significantly(P < 0.01).5.The results of immunofluorescence staining of syn protein in hippocampus of rats in each group showed that the number of syn positive cells in CA1 area of exocrine group and co group was significantly increased than that in model group(P < 0.05),and the number of syn positive cells in CA3 area of the co group was significantly increased than that in the model group(P < 0.01),and the ratio of syn to exosecretion increased(P < 0.05).The results of immunofluorescence staining of PSD-95 protein in hippocampus showed that the number of PSD-95 positive cells in the three groups was higher in CA1 area than in the model group(P <0.05).In CA3 area of hippocampus,the number of PSD-95 positive cells in three treatment groups was significantly increased compared with that of model group(P < 0.01),and the number of positive cells in ligusticgui group was higher than that in the exoderm group(P <0.05).6.Golgi Cox staining results showed that compared with the model group,the morphology of dendritic spines in the synergetic group was more uniform and the number increased;compared with xiongguifang group and exosome group,the number of dendritic spines in the synergetic group increased.In hippocampal CA3 area,compared with the model group,the morphology of dendritic spines in exosomes group and synergetic group was more complete and the number was significantly increased.In DG area of hippocampus,compared with the model group,the morphology of dendritic spines in SNX group was more complete and the number of dendritic spines was more,but the number of dendritic spines in exosomes group and synergy group was less than that in the model group.In addition,compared with the model group,the density of dendritic spines in xiongguifang group,synergetic group increased significantly(P < 0.05,P < 0.01),and the density of dendritic spines in synergetic group increased significantly compared with exosome group(P < 0.01).7.Western blot showed that the expression levels of sema3 g,Nrp2,plexina4,Rac1,syn and PSD-95 protein in the model group were lower than those in the sham operation group(P< 0.05);Compared with the model group,the expression levels of plexina4,Rac1,syn and PSD-95 in exosome group were increased(P < 0.05),and the expression levels of sema3 g and Nrp2 in exosome group were increased,but there was no significant difference(P > 0.05);Compared with the model group,the expression levels of sema3 g,Nrp2,plexina4,Rac1,syn and PSD-95 in xiongguifang group and synergetic group were significantly higher(P < 0.05).Conclusion:1.BMSCs-exos + SNX can improve the damage of neurons in the hippocampus of tMCAO rats,and promote the recovery of neurological,motor and cognitive functions.2.BMSCs-exos + SNX can promote synaptic density and dendritic spine growth in hippocampus of tMCAO rats,and promote synaptic remodeling in hippocampus of tMCAO rats.3.BMSCs-exos + SNX regulates synaptic remodeling in the hippocampus of tMCAO rats through Sema3 G / Nrp2 / plexin A4 signaling pathway,thus improving their cognitive function. |
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