| Objective:Stroke is the second leading cause of death and the third leading cause of disability in the world,characterized by high morbidity,mortality and disability.Ischemic stroke is one of the major types of stroke,accounting for 60%-80% of all strokes.After ischemic stroke,a series of oxidative stress and inflammatory reactions occur in the brain tissue due to ischemia and hypoxia,resulting in severe neurological deficits,such as limb motor dysfunction and cognitive dysfunction,which not only affect the prognosis and life of patients,but also impose a heavy economic and social burden on both patients and families.Therefore,it is very important to find a safe,effective,simple and inexpensive rehabilitation treatment strategy.Many studies have shown that the opposing needling method can improve post-ischemic stroke limb motor dysfunction,but the mechanism of action is not clear.In the present study,we investigated the potential mechanism of the treatment from the perspective of the remodeling of the healthy side of the brain to improve the motor dysfunction of the affected limb in the Middle Cerebral Artery Occlusion(MCAO)model rats on the basis of the efficacy of the opposing needling method,and provided a reference for the clinical application of the method.Methods:Experiment 1 Efficacy of opposing needling in the treatment of motor dysfunction in MCAO model ratsForty-eight healthy male Sprague Dawley(SD)rats were randomly divided into shamoperated group(Sham group,S group),model group(Model group,M group),opposing needling group(Model + Contralateral Acupuncture,MCA group),and affected-sided acupuncture group(Model + Ipsilateral Acupuncture,MIA group).The MCAO rat model was established according to Longa’s modified method,and the four acupuncture points of Hegu and Neiguan on the healthy side of the MCA group and those on the affected side of the MIA group were selected for electroacupuncture intervention for 30 min each time,once a day for 7days.At 1d,4d and 7d after model preparation,the comprehensive motor function score(suspended tail lift score + balance beam walking score + rotating stick walking score + mesh screen experiment score)with modified rat neurological deficit score was performed for each group of rats,and the changes of body weight and mortality were recorded at 7d,and TTC staining was performed to evaluate the brain infarct volume of each group of rats at the 7th day.Experiment 2: The role of SHP-1/BDNF/Trk B signaling pathway in improving motor dysfunction in MCAO model rats by opposing needlingTwenty-four healthy male SD rats were divided into sham-operated group,model group,opposing needling,and affected side acupuncture group,and the procedure of modeling,acupuncture intervention and motor function observation was the same as that of Experiment 1.7 d after modeling,the healthy side cortex of rats in each group was taken,and the SH2 The Src homology 2 domain-containing protein tyrosine phosphatase-1(SHP-1),brain-derived neurotrophic factor(BDNF),and tyrosine protein kinase receptor(TPGR)were detected by RTq PCR.Detection of total protein of SHP-1,BDNF and Trk B by protein blotting(Western Blot,WB)and p-Trk B phosphorylated protein levels.The protein content of axonal growth marker GAP-43 in the healthy cortex,C4-C7 denervated lateral(left)spinal cord,and C4-C7 healthy lateral(right)spinal cord was measured by WB method.Experiment 3: Molecular mechanism of improving motor dysfunction in MCAO model rats based on gene sequencing screening opposing needling methodTwenty-four healthy male SD rats were divided into sham-operated group,model group,opposing needling,and affected side acupuncture group,and the process of modeling,acupuncture intervention and motor function observation were the same as Experiment 1.7 d after modeling,the total RNA of the healthy side cortex of each group was extracted for highthroughput gene sequencing,and the gene content of each sample was analyzed statistically between groups,and the genes with statistical difference P<0.05 between groups were localized as differential genes.The key pathways and key genes associated with this model and the effect of acupuncture were screened using GO and KEGG analysis.Results:1.Opposing needling improves motor dysfunction in MCAO model rats(1)The results of the comprehensive score of motor function showed that the comprehensive score of motor function was 0 in all groups before the model preparation,the neuromotor function was normal.At 1d,4d,and 7d after the model preparation,the motor function composite score was 0 in the sham-operated group;at 1d after the model preparation,the rats in the model group,the opposing needling group,and the affected needling group all showed significant motor dysfunction,and the motor function composite score was significantly higher than that in the sham-operated group(P < 0.05),and there was no significant difference between the groups;at 4d after the model preparation,the motor function composite score was lower in the opposing needling group and the affected needling group than that in the model group(P < 0.001).At 7d after modeling,the overall motor function scores of rats in both the opposing needling group and the affected needling group were significantly lower than those in the model group(P < 0.001),and the scores in the affected needling group were lower than those in the opposing needling group(P < 0.01),suggesting that acupuncture on the affected side at 7d in this experimental acupuncture protocol was slightly better than opposing needling in improving motor dysfunction.The 7d acupuncture on the affected side was slightly better than the opposing needling in improving motor dysfunction.(2)The results of m NSS scores showed that the m NSS scores of rats in all groups before the model preparation were 0,the neuromotor function was normal.At 1d,4d and 7d after model preparation,the rats in the sham-operated group scored 0.At 1d after modeling,the rats in the model group,the opposing needling group and the affected needling group all showed significant signs of neurological deficits,and the m NSS scores were significantly higher than those in the sham-operated group(P < 0.05),and there was no significant difference between the three groups;at 4d after modeling,the m NSS scores in the two acupuncture groups were not significantly different compared with those in the model group,and there was no significant difference between the acupuncture groups.The m NSS scores were significantly lower in each acupuncture group compared with the model group at 7 d after modeling(P < 0.01),and there was no difference in m NSS scores between the opposing needling group andthe affected needling group.It is suggested that all needling in this experimental needling protocol for 7d can effectively improve the neurological deficits in MCAO model rats.(3)At 7d after modeling,the body weight of the rats in the model group,the opposing needling group and the affected needling group all continued to decrease,and there was no statistical difference in body weight between the three groups.(4)Mortality within 7d in each group: 0% in the sham-operated group,25% in the model group,18.75% in the opposing needling group,and 12.5% in the affected needling group.(5)TTC staining results: At 7 d after modeling,the cerebral infarct volume was 0±0 in the sham-operated group,0.24±0.01 in the model group,0.18±0.01 in the opposing needling group,and 0.19±0.02 in the affected needling group,and the cerebral infarct volume was significantly lower in each acupuncture group compared with the model group(P<0.001),and there was no difference between the opposing needling group and the affected needling group.2.Opposing needling modulates Trk B-related signaling pathways in healthy cortex(1)Changes in gene expression of Trk B and other related factors in the healthy cortex:7d after modeling,compared with the model group,the Trk B gene content was significantly higher in the opposing needling group and the affected needling group(P<0.05,P<0.01);compared with the model group,the BDNF gene content in each acupuncture group showed an increasing trend,but there was no statistical difference(P>0.05),and there was no statistical difference between the acupuncture groups(P>0.05).The SHP-1 gene content in each acupuncture group showed an increasing trend compared with the model group without statistical difference(P>0.05),and there was no statistical difference between the acupuncture groups(P>0.05).(2)Changes in the amount of Trk B,p-Trk B,SHP-1 and BDNF protein expression in the healthy cortex: At 7d after modeling,the amount of Trk B protein expression was significantly increased in the model group compared with the sham-operated group(P < 0.05);compared with the model group,the amount of Trk B protein expression was on the rise in the opposing needling group and the affected needling group,but there was no statistical difference(P > 0.05);compared with the sham-operated group,the amount of p-Trk B protein expression was reduced in all model preparation groups(P < 0.001);the amount of the p-Trk B protein expression was significantly increased in the opposing needling group compared with the model group(P < 0.05).Compared with the sham-operated group,there was an increasing trend of the amount of SHP-1 protein expression in the model group,opposing needling group,and affected needling group,but there was no statistical difference;compared with the model,there was an increasing trend of the amount of SHP-1 protein expression in the opposing needling group,but there was no statistical difference.There was no significant change in the amount of BDNF protein expression in each group.(3)Changes in the amount of GAP-43 protein expression in the healthy cortex: At 7d after modeling,there was no significant change in the amount of GAP-43 protein expression in the sham-operated group,the model group,the opposing needling group,and the affected needling group.Changes in the amount of GAP-43 protein expression in the C4-C7 denervated lateral(left)spinal cord: compared with the sham-operated group,there was a decreasing trend in the amount of GAP-43 protein expression in the model group,the opposing needling group,and the affected needling group,but there was no GAP-43 protein expression in the C4-C7 sound side(right)spinal cord: At 7d after modeling,compared with the sham-operated group,the amount of GAP-43 protein expression in the model group decreased significantly(P < 0.05),while that in the opposing needling group and the affected needling group decreased significantly(P < 0.05),there was a decreasing trend of GAP-43 protein expression in the opposing needling group and the affected needling group,but there was no statistical difference(P>0.05);compared with the model group,there was an increasing trend of the amount of GAP-43 protein expression in the opposing needling group and the affected needling group,but there was no statistical difference(P>0.05);there was no difference of GAP-43 protein expression between the opposing needling group and the affected needling group.3.Opposing needling can regulate several signaling pathways in the healthy cortex of MCAO model rats,which is presumed to be involved in the recovery of motor dysfunction in model rats(1)A total of 103 differential genes were obtained in the model group and shamoperated group,81 and 62 differential genes were obtained in the opposing needling group and the affected needling group compared with the model group,respectively.The sequencing data were further analyzed,and the differential genes obtained from two comparisons between the model group,sham-operated group and opposing needling group were taken as an intersection to obtain 18 differential genes,and similarly the differential genes obtained from two comparisons between the model group,sham-operated group and affected needling group were taken as an intersection to obtain 20 differential genes,and finally the differential genes obtained from four groups of two comparisons between the model group,sham-operated group,opposing needling group and affected needling group were taken as an intersection to obtain 9differential genes.(2)The enrichment results of KEGG pathway showed that the regulatory effects of opposing needling in the healthy cortex included multiple pathways such as cytokine-cytokine receptor and B-cell receptor signaling pathway,and the gene changes of Atp1b4,Lilrb2,Hbaa3 and Krt42.The regulatory effects of affected needling in the healthy cortex included TGFβsignaling pathway and other pathways,and the genes such as Plk1,Enpp6,Clca4 l,Bambi,Krt42,Plk1,Ctse,Drd5,and Olr1374.The modulatory effects of opposing needling and affected needling in the healthy cortex included olfactory transduction signaling pathway and the genes such as Krt42,Ctse,Olr1374.Conclusion.(1)Both opposing needling and affected needling for 7d were effective in improving the motor function of the affected limb,reducing the infarct volume and improving the survival rate in the MCAO model rats,but there was no difference in the effect of opposing needling and affected needling.(2)The improvement of motor function in MCAO model rats by opposing needling may be associated with activation of Trk B pathway in the healthy cortex and promotion of axonal growth in C4-C7 segment of spinal cord.(3)The improvement of motor function in MCAO model rats by opposing needling may be related to the regulation of immune pathways such as cytokine-cytokine receptors and Bcell receptor signaling in the healthy cortex,but the molecular mechanism needs to be further verified.Dmrta2,Gnrh1,Lhx3,Rpl10 l,and Dsg1 genes may be involved in the opposing needling to improve neural regeneration in the healthy cortex of the brain after ischemic stroke. |
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