| BackgroundIschemic stroke can cause a lot of neuron loss and nerve tissue damage,and the treatment for nerve regeneration and remodeling is still not satisfactory.Bone marrow mesenchymal stem cells(BMSCs)derived exosomes(BMSCs-derived exosomes,BMSCs-exos)are the current research hotspots in this field,which can regulate endogenous repair of damaged brain tissue by mediating intercellular signal communication.However,due to the difference between exosomes prepared in vitro and exosomes secreted by BMSCs in vivo,there is still a certain lack of efficacy in the treatment of ischemic brain injury;Xionggui prescription consists of Chuanxiong,Danggui is commonly used for the treatment of ischemic stroke,our team’s early use of the Chinese patent medicine Shunaoxin Pills SNX)developed by it to intervene in t MCAO model rats found that SNX can promote cell proliferation and neural function recovery.Proteomics and bioinformatics analysis showed that SNX can promote the upregulation of protein expression in the cholinergic synapse and dopaminergic synapse pathways of 72h after t MCAO injury rats’cortical,suggesting that SNX may be has a certain effect on nerve repair and synaptic reconstruction after injury.Whether Xionggui prescription can synergize with BMSCs-exos to improve the microenvironment in the brain,enhance nerve regeneration and nerve function repair,and its specific role is worth further discussion.ObjectiveXionggui prescription(SNX)joint with BMSCs-exos were used to intervene in t MCAO rat model,to evaluate its effect on neurological function recovery in rats with ischemic brain injury,and to explore its effect on nerve regeneration、remodeling and intervention link.Methods1.Male SD rats were divided into 5 groups,including sham group(Sham),model group(t MCAO),Xiongguifang group(t MCAO+SNX),BMSCs-exos group(t MCAO+exos),Xiongguifang synergistic BMSCs-exos group(t MCAO+SNX+exos).The t MCAO model was established by thread embolization method.Rats with successful modeling were randomly divided into(1)t MCAO group,500μl PBS by tail vein injection,1/2d,4 times in total;(2)t MCAO+SNX group,SNX 45mg/kg/d intragastric administration for 7 consecutive days;(3)t MCAO+exos group,BMSCs-exos 400μg/kg by tail vein injection,1/2d,a total of 4times;(4)t MCAO+SNX+exos group,The dosing regimen is the same as t MCAO+SNX group and t MCAO+exos group.In the Sham group,except for the insertion of the thread plug,the rest of the operation and administration methods are the same as t MCAO.In each group n=10,and was administered 24h after modeling.2.The weight、m NSS、beam-walking test、foot-fault test,wiring test and corner test were evaluated in each group on the 1d,3d,7d,14d,and 21d after the model was made,the rotarod test and open field test were evaluated in each group on the 7d,14d,and 21d,From the 15th day onwards,the rats in each group were evaluated for the Morris water maze test.The above tests evaluated motor balance and coordination,limb dysfunction,sensory dysfunction,anxiety and depression,learning and memory ability in rats with ischemic brain injury;weight is to assess the overall living conditions;m NSS is to assess the neurological deficit condition.3.Immunofluorescence staining was used to detect the expression of Neu N~+/Brd U~+,DCX~+/Brd U~+,nestin~+/Brd U~+positive cells in the infarcted side of striatum;the expression of NF-200 protein in neurons axons in the hippocampus of the infarcted side and olig2~+/Brd U~+;The expression of presynaptic vesicle synaptophysin SYN protein and post-synaptic membrane compact PSD-95 protein in the infarcted hippocampus.4.Golgi-Cox staining was used to detect the dendritic morphology,branching,length and its spines density of pyramidal cell neurons in the motion representative area and medial prefrontal cotex(m PFC)to assess the plasticity of nerves and the activity of nerve functions.Result1.Behavioral assessment(1)Evaluation of motion balance and coordination abilityRotarod test:the residence time of rats in t MCAO group was significantly reduced on the14d and 21d compared with Sham group,the difference was statistically significant(P<0.01);compared with t MCAO+SNX+exos group,the difference was statistically significant(P<0.01),compared with t MCAO+SNX group the difference was statistically significant on the21d day(P<0.05);in the 21d,the t MCAO+SNX+exos group has a statistically significant difference compared with the t MCAO+exos group(P<0.05);t MCAO+SNX+exos group was not statistically significant compared with the other two treatment groups(P>0.05);the beam-walking test:Sham group scored 0 points;t MCAO group’s score is significantly increased compared with t MCAO+SNX+exos group on the 3d,7d,14d,21d,the difference was statistically significant(P<0.01,P<0.05),on 3d,14d,is significantly increased compared with t MCAO+exos group,the difference was statistically significant(P<0.01,P<0.05)and was significantly higher than t MCAO+SNX group on 3d,the difference was statistically significant(P<0.01);t MCAO+SNX+exos group score decreased significantly compared with the other two treatment groups on the 21d the difference is statistically significant(P<0.05).(2)Evaluation of limb dysfunctionFoot-fault test:the Sham group scored 0 points;the t MCAO group scored obviously high on the 14d and 21d compared with the t MCAO+SNX group,t MCAO+exos group,the difference was statistically significant(P<0.01,P<0.05),on the 7d,14d and 21d compared with t MCAO+SNX+exos group,the difference was statistically significant(P<0.01,P<0.05);compared with other two treatment groups at each time the t MCAO+SNX+exos group’s difference was not statistically significant(P>0.05);wiring test:Sham group score was significantly lower than other groups,on1d,3d,7d,14d,21d the score is significantly reduced compared with t MCAO group,the difference is statistical significance(P<0.01,P<0.05);t MCAO group was not obviously differ from the 3 treatment groups(P>0.05);t MCAO+SNX+exos group had no difference compared with t MCAO+SNX,t MCAO+exos group(P>0.05).(3)Evaluation of sensory dysfunctionThe percentage of right-turn in the t MCAO group was significantly higher on the 3d,14d,and 21d compared with the Sham group,the difference was statistically significant(P<0.01,P<0.05);on the 14th,21d it was significantly higher compared with the t MCAO+SNX+exos group,the difference was statistically significant(P<0.01,P<0.05);there was no significant difference between the treatment groups(P>0.05).(4)Evaluation of anxiety and depressionFrom the total distance of motion,there was no statistical difference between the groups(P>0.05);the central stay time of the t MCAO group was significantly longer than that of the Sham group on the 21st day,and the difference was statistically significant(P<0.05);Compared with t MCAO+SNX group and t MCAO+SNX+exos group,the difference was statistically significant(P<0.01),compared with t MCAO+SNX+exos group on the 14th day,the difference was statistically significant(P<0.01),on the 21d,the t MCAO+SNX+exos group was significantly reduced compared with the t MCAO+SNX group,the difference was statistically significant(P<0.05);the standing time in the t MCAO group was significantly reduced compared with the Sham group on the 21st day,and the difference was statistically significant(P<0.01),significantly reduced compared with t MCAO+exos group(P<0.05);t MCAO+SNX+exos group showed no significant difference compared with the other two treatment groups at each time point(P>0.05).(5)Assessment of learning and memoryIn the navigation test,the latency period of the t MCAO group was significantly longer than the Sham group at the 3d,4d,5d,and the difference was statistically significant(P<0.01,P<0.05);at the 3d,5d compared with t MCAO+SNX+exos group the difference was statistically significant(P<0.01,P<0.05);at 3d and 5d,t MCAO+SNX+exos was significantly shortened compared with t MCAO+SNX group,the difference was statistically significant(P<0.05);in the probe trial,t MCAO group stayed in the target quadrant is significantly shorten compared with at 3d and 5d,t MCAO+SNX+exos was significantly shortened compared with Sham group,the difference significantly(P<0.05);there is no statistically significant difference between the remaining groups(P>0.05).2.Nerve regeneration and remodeling(1)Neuron regenerationCompared with the t MCAO group,the expression of Neu N~+/Brd U~+and DCX~+/Brd U~+cells in the 3 treatment groups increased significantly,and the difference was statistically significant(P<0.01);t MCAO+SNX+exos group’s Neu N~+/Brd U~+,DCX~+/Brd U~+is significantly increased compared with two other treatment groups,the difference was statistically significant(P<0.01,P<0.05);compared with t MCAO group,the expression of nestin~+/Brd U~+cells in the t MCAO+SNX+exos group was significantly increased,and the difference was statistically significant(P<0.01);there was no significant difference in the t MCAO+SNX+exos groups between the two other groups of nestin~+/Brd U~+(P>0.05).(2)Growth of neuronal axon、dendrite and remodeling of synapsesCompared with the t MCAO group,the expressions of NF-200,SYN,Olig2~+/Brd U~+in the3 treatment groups were significantly increased,the distribution was dense,the morphology was regular,and the difference was statistically significant(P<0.01,P<0.05);the number of Olig2~+/Brd U~+and PSD-95 positive cells in the t MCAO+SNX+exos group increased significantly compared with the other two groups,the difference was statistically significant(P<0.01,P<0.05).Compared with the t MCAO+exos group,SYN positive cells in the t MCAO+SNX+exos group increased significantly,the difference was statistically significant(P<0.05),there was no significant statistical difference in the expression of NF-200(P>0.05).Golgi-Cox staining results showed that in the m PFC area,the dendritic length and dendritic spine density of pyramidal cells in the t MCAO+SNX+exos group were significantly higher than those in the t MCAO+SNX and t MCAO+exos group(P<0.01,P<0.05).Although there is an increasing trend in the motion representative area,there is no statistical significance(P>0.05).Conclusion1.SNX+exos can promote the recovery of nerve function and strengthen the reconstruction of brain function.2.SNX+exos can improve the balance and coordination ability of cerebral ischemia injury rats,ameliorate limb dysfunction,relieve anxiety and depression,regulate emotional disorders,and enhance learning and memory.3.SNX+exos can promote neuron regeneration,regulate neurite outgrowth and synapse remodeling. |
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