| Objective:To observe the effect of Acupuncture-rehabilitation method on the expression of apoptosis related factors after cerebral ischemia,and to explore the possible mechanism of its protective effect.Methods:The rats were randomly divided into 5 groups:model group,sham operation group,acupuncture group,rehabilitation group and Acupuncture-rehabilitation method group.The model of permanent middle cerebral artery occlusion was established in rats of model group and all treatment groups.After that,the acupuncture group was selected to simulate the scalp acupuncture with acupuncture points,The rehabilitation group was treated with treadmill training to simulate the clinical rehabilitation treatment,The Acupuncture-rehabilitation method group combined with scalp acupuncture and treadmill training to simulate Acupuncture-rehabilitation method therapy.The model group and sham operation group were not treated.The neurological deficit score(NSS)was improved and used to evaluate the neurological deficits of rats in each group;TTC staining was used to observe the changes of cerebral infarction volume in rats at different time points;Western Blot method was used to detect the expression of cleaved-Caspase-3一8-9 in the ischemic penumbra of cortex at different time points and expression of proteins of cIAP1,cIAP2,XIAP and Survivin who included apoptosis inhibitor IAPs family members.TUNEL method was used to detect the apoptosis of ischemic brain cells.The distribution of cIAP1 in neurons was observed by immunofluorescence double labeling method.Result:1.Neurological Severity Scores:Postoperative 3d,compared with the model group,there was no significant difference between the acupuncture group,the rehabilitation group and the Acupuncture-rehabilitation method group,there was no statistical significance(P<0.05);Postoperative 7d,14d,acupuncture group,rehabilitation group,Acupuncture-rehabilitation method group are more decreased than the model group on the degree of NSS(P<0.05);Compared with the acupuncture group and the rehabilitation group,the degree of neurological impairment of Acupuncture-rehabilitation method group was significantly lower(P<0.05).2.Cerebral infarction volume detection:At each time point,there was no infarction in sham operation group,compared with the model group,the infarct volume of each treatment group was reduced.At the same time point,there was no significant difference between the acupuncture group and the rehabilitation group,which was not statistically significant(P>0.05);Compared with the acupuncture group and the rehabilitation group,the volume of cerebral infarction of Acupuncture-rehabilitation method group was significantly reduced(P<0.05).3.Expression of apoptosis related factors:(1)cleaved-Caspase-3、8、9:At each time point,compared with the sham operation group,the expression of cleaved-Caspase-3,8,9 protein increased in the model group and in the treatment groups;Postoperative 3d,there was no significant difference between the model and the treatment groups(P>0.05);Postoperative 7d,14d,compared with the model group,the expression of cleaved-Caspase-3,8,9 protein from acupuncture group,rehabilitation group and Acupuncture-rehabilitation method group decreased(P<0.05);Compared with the acupuncture group and the rehabilitation group,the expression of cleaved-Caspase-3,8,9protein in the Acupuncture-rehabilitation method group decreased,and the difference was significant(P<0.05).(2)cIAP 1、cIAP2、XIAP、Survivin:At each time point,compared with the sham operation group,the number of cIAP1,cIAP2,XIAP,Survivin positive cells in the model and each treatment groups were decreased;Postoperative 3d,there was no significant difference between the model and the treatment groups(P>0.05);Postoperative 7d,14d,compared with the model group,the expression of cIAP1,cIAP2,XIAP and Survivin protein in acupuncture group,rehabilitation group and acupuncture group were higher(P<0.05);Compared with the acupuncture group and the rehabilitation group,the positive expression of cIAP1,cIAP2,XIAP and Survivin were significantly higher in the Acupuncture-rehabilitation method group(P<0.05).4.Neuronal apoptosis:At each time point,compared with sham operation group,the apoptosis rate of TUNEL staining in model group was significantly increased,and the difference was significant(P<0.05);Compared with the model group,the apoptotic rate of TUNEL staining in each treatment group at each time point was significantly lower(P<0.05);Compared with the rehabilitation group and the acupuncture group,the apoptosis rate of TUNEL cells in the Acupuncture-rehabilitation method group was lower,and the difference was significant(P<0.05).5.Detection of cIAP1 in ischemic cortex neurons by immunofluorescence double labeling method:At each time point,in ischemic brain tissue,the expression of cIAP1(+)and the expression of NeuN(+),it is proved that cIAP1 is indeed located in neurons.At each time point,compared with sham operation group,the expression intensity of cIAP1 and NeuN in model group was significantly lower(P<0.05);Compared with the model group,the expression of cIAP1 and NeuN in the acupuncture group,the rehabilitation group and the Acupuncture-rehabilitation method group were all significantly increased,and the difference was significant(P<0.05);Postoperative 7d,14d,compared with acupuncture group and rehabilitation group,the expression of cIAP1 and NeuN in Acupuncture-rehabilitation method group was increased significantly(P<0.05).Conclusion:1 Acupuncture-rehabilitation method can inhibit the neurological deficit and reduce the infarct volume after cerebral ischemia,better than simple acupuncture and rehabilitation therapy.2 Acupuncture-rehabilitation method can raise the expression of apoptosis inhibitor cIAP1,cIAP2,XIAP,Survivin protein,and inhibit the activation of Pro apoptotic factors Caspase-3,Caspase-8,Caspase-9.3 The mechanism of nerve repair of Acupuncture-rehabilitation method may be related to the upregulation of the expression of protein cIAP1 in ischemic brain tissue,so as to achieve the purpose of inhibiting neuronal apoptosis and brain protection. |
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