| Experiment 1 Effects of Acupuncture-rehabilitation Therapy on Motor Function and Neuronal Apoptosis in Rats with Cerebral IschemiaObjective:To observe the effects of acupuncture-rehabilitation therapy on the motor function and neuronal apoptosis in rats with cerebral ischemia,and to explore the relationship between cIAP1 expression and neuron survival.Methods:90 SPF adult male Sprague-Dawley rats were randomly divided into five groups,namely sham group,model group,acupuncture group,rehabilitation group and acupuncture-rehabilitation group,and 18 in each group.Each group was further divided into 3 days,7 days and 14 days subgroups(n=6).All rats except the sham group received permanent middle cerebral artery occlusion(pMCAO)surgery.Rats in the sham and model group received no treatment,while the acupuncture group accepted scalp acupuncture,rehabilitation group accepted treadmill training,and the acupuncture-rehabilitation group accepted both scalp acupuncture and treadmill training.The motor function of rats was assessed with forelimb grip strength test and Rota-rod test.Nissl staining was used to observe neuronal damage in the cerebral ischemic penumbra.The apoptosis(TUNEL-DAPI co-localization)and neuronal apoptosis(TUNEL-NeuN co-localization)in the cerebral ischemic penumbra using TUNEL-NeuN immunofluorescence double staining.The cIAP1-NeuN immunofluorescence double staining co-localization technique was used to observe the fluorescence intensity of cIAP1 and NeuN,neuron survival and neuronal cIAP1 expression in the cerebral ischemic penumbra.The correlation between the fluorescence intensity of cIAP1 and NeuN,and the number of cIAP1-NeuN and NeuN-DAPI co-localized cells was analyzed,respectively.Result:1.Forelimb grip strength test:At each time point after surgery,compared with the model group,the grasping power of rats in each treatment group was significantly increased(P<0.05).At 7d and 14d after surgery,the grip strength of the rats in the acupuncture-rehabilitation group further increased compared with the acupuncture and rehabilitation group(P<0.05).2.Rota-rod test:At each time point after surgery,compared with the model group,the stay time of the rats in each treatment group was significantly longer(P<0.05).At 7d and 14d after surgery,the stay time in the acupuncture-rehabilitation group was further prolonged compared with the acupuncture and rehabilitation group(P<0.05).3.Nissl staining:3 days after surgery,the number of cortical nerve cells in the right brain tissue of the model group decreased,the cell structure was significantly damaged,normal neurons were rare,the cell bodies were atrophic,the number of Nissl bodies was significantly reduced,and the dissolution phenomenon was obvious;The nerve cells were tight,the cytoplasm was darkly stained,and the number of Nissl bodies was increased.It was seen that there was an increase in the number of perinuclear Nissl bodies of some neurons and the neuronal damage gradually recovered at 7 days after surgery;At 14 days after surgery,neurons were still tight,the number of Nissl bodies was further increased,and cytoplasm staining was deepened.A small number of neurons were rich in Nissl bodies and the morphology was further restored.At each time point,the number of nerve cells in each treatment group increased,the morphology improved,the atrophy of the cell bodies eased,the number of Nissl bodies increased,and the deep staining of cytoplasm was patchy.With the prolongation of treatment time,the number of normal neurons was increased.Increased,and the improvement in the acupuncture-rehabilitation group was the most significant.4.Apoptosis:At each time point after surgery,compared with the model group,the number of apoptosis(TUNEL-DAPI co-localization cells)in each treatment group was significantly reduced(P<0.05);the number of apoptosis in the acupuncture-rehabilitation group were further decreased compared with those in the acupuncture and rehabilitation group(P<0.05).5.Neuronal apoptosis:At each time point after surgery,compared with the model group,the number of neuronal apoptosis(TUNEL-NeuN co-localization cells)in each treatment group was significantly reduced(P<0.05);the number of neuronal apoptosis in the acupuncture-rehabilitation group were further decreased compared with those in the acupuncture and rehabilitation group(P<0.05).6.cIAP1 fluorescence intensity:At each time point after surgery,compared with the model group,the cIAP1 fluorescence intensity in each treatment group was increased(P<0.05);compared with the acupuncture group,the cIAP1 fluorescence intensity in the acupuncture-rehabilitation group was further increased(P<0.05).<0.05).At 7d and 14d after surgery,the cIAP1 fluorescence intensity in the acupuncture-rehabilitation group was further increased compared with the rehabilitation group(P<0.05).At 7 days after surgery,the cIAP1 fluorescence intensity in the acupuncture group further increased compared with the rehabilitation group(P<0.05).7.NeuN fluorescence intensity:At each time point after surgery,compared with the model group,the NeuN fluorescence intensity in each treatment group was increased(P<0.05);the NeuN fluorescence intensity in the acupuncture-rehabilitation group was further increased compared with those in the acupuncture and rehabilitation group(P<0.05).At 7 days after surgery,NeuN fluorescence intensity in the rehabilitation group further increased compared with the acupuncture group(P<0.05).At 14 days after surgery,NeuN fluorescence intensity in the acupuncture group further increased compared with the rehabilitation group(P<0.05).8.Surviving neurons:At each time point after surgery,compared with the sham group,the surviving neurons(NeuN-DAPI co-localization cells)in the model group were reduced(P<0.05);the surviving neurons in each treatment group was increased compared with the model group(P<0.05);the surviving neurons in acupuncture-rehabilitation group was further increased compared with those in the acupuncture and rehabilitation group(P<0.05).9.Coexpression of cIAP1 and NeuN:At each time point after surgery,compared with the sham group,the cIAP1-NeuN co-localization cells in the model group decreased(P<0.05);the cIAP1-NeuN co-localization cells in each treatment group was increased compared with the model group(P<0.05);the cIAP1-NeuN co-localization cells in the acupuncture-rehabilitation group was further increased compared with those in the acupuncture and rehabilitation group(P<0.05).10.Correlation analysis of cIAP1 and NeuN fluorescence expression:Pearson analysis of cIAP1 and NeuN fluorescence intensity in sham group and model group at each time point after surgery showed that the fluorescence intensity of cIAP1 and NeuN showed a strong linear positive correlation(P<0.0001)under ischemic conditions;Pearson analysis of cIAP1 and NeuN fluorescence intensity in the model group and the treatment group showed that under the intervention conditions,the fluorescence intensity of cIAP1 and NeuN showed a strong linear positive correlation(P<0.0001).11.Correlation analysis of cIAP1-NeuN and NeuN-DAPI co-localization cells:Pearson analysis of cIAP1-NeuN and NeuN-DAPI co-localization cells in sham group and model group at each time point after surgery showed that the co-localization cells of cIAP1-NeuN and NeuN-DAPI showed a strong linear positive correlation(P<0.0001)under ischemic conditions;Pearson analysis of cIAP1-NeuN and NeuN-DAPI co-localization cells in the model group and the treatment group showed that under the intervention conditions,the co-localization cells of cIAP1-NeuN and NeuN-DAPI showed a strong linear positive correlation(P<0.0001).Conclusion:1.Acupuncture-rehabilitation therapy can increase the forelimb grip strength,improve the motor coordination,and promote motor function reconstruction after cerebral ischemia in rats,better than only acupuncture or only rehabilitation treatment;2.Acupuncture-rehabilitation therapy can reduce the neurons apoptosis in ischemic penumbra of cerebral ischemic rats,reduce the number of apoptotic cells,and exert a neuroprotective effect;3.The expression of cIAP1 on neurons is closely related to the survival of neurons.After cerebral ischemia,the expression of cIAP1 is down-regulated and the survival of neurons is decreased.After acupuncture-rehabilitation therapy intervention,the expression of cIAP1 is up-regulated and the survival of neurons is increased.Experiment 2 The Mechanism of cIAP1 in Acupuncture-rehabilitation Therapy-induced Reducing Neuronal Apoptosis in Cerebral Ischemic RatsObjective:To construct a lentivirus shRNA expression vector for the cIAP1 gene and explore the mechanism of cIAP1 in Acupuncture-rehabilitation therapy-induced reduing neuronal apoptosis in cerebral ischemic rats.Methods:Three shRNAs for cIAP1 and one negative control sequence were transfected into SH-SY5Y cells to establish an SH-SY5Y cell line with stable cIAP1 shRNA expression.The untransfected group(Control),negative control transfection group(shRNA NC group),shRNA-1 transfection group,shRNA-2 transfection group,and shRNA-3 transfection group were established.Real-time PCR and Western blotting techniques were used to detect the interference efficiency of three cIAP1 shRNAs at mRNA and protein levels.The cIAP1 shRNA with the highest interference efficiency was screened for constructing a lentivirus shRNA expression vector for the cIAP1 gene.180 SPF adult male Sprague-Dawley rats were randomly divided into five groups,namely sham group,model group,acupuncture-rehabilitation group,acupuncture-rehabilitation+Lv-cIAP1 NC group and acupuncture-rehabilitation+Lv-cIAP1 group,and 36 in each group.Each group was further divided into 3 days,7 days and 14 days subgroups(n=12).All rats except the sham group received permanent middle cerebral artery occlusion(pMCAO)surgery,while the rats in acupuncture-rehabilitation+Lv-cIAP1 NC group and acupuncture-rehabilitation+Lv-cIAP1 group were injected with Lv-cIAP1 NC and Lv-cIAP1 lentivirus interfering vectors respectively in the lateral ventricle within 30 min before the pMCAO model.Rats in the sham and model group received no treatment,while the other three groups received scalp acupuncture combined treadmill training.Western blotting was used to detect the expression of cIAP1,cleaved caspase-9,cleaved caspase-8,cleaved caspase-3 and NeuN protein in each experimental group.TUNEL-NeuN immunofluorescence double-staining co-localization technique was used to observe the apoptosis and neuronal apoptosis in the cerebral ischemic penumbra.Result:1.cIAP1 mRNA:Compared with the control group,cIAP1 mRNA expression on SH-SY5Y cells in shRNA-1,shRNA-2,shRNA-3 groups were down-regulated by 28.76%,71.91%,and 10.37%,respectively(P<0.05),the shRNA-2 has the highest interference efficiency.2.cIAP1 protein:Compared with the control group,the cIAP1 protein expression on SH-SY5Y cells in shRNA-1,shRNA-2,shRNA-3 groups were down-regulated by 46.89%,74.43%,and 38.03%,respectively(P<0.05),the shRNA-2 has the highest interference efficiency.3.cIAP1 protein expression:At each time point after surgery,the expression of cIAP1 in the model group was decreased compared with the sham group(P<0.05);compared with the model group,the expression of cIAP1 in the acupuncture-rehabilitation group was increased(P<0.05);compared with acupuncture-rehabilitation group,the expression of cIAP1 was decreased in acupuncture-rehabilitation+Lv-cIAP1 group(P<0.05),while the expression of cIAP1 in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significantly different(P>0.05).4.cleaved caspase-9 protein expression:At each time point after surgery,the expression of cleaved caspase-9 in the model group was increased compared with the sham group(P<0.05);compared with the model group,the expression of cleaved caspase-9 in the acupuncture-rehabilitation group was decreased(P<0.05);compared with acupuncture-rehabilitation group,the expression of cleaved caspase-9 was increased in acupuncture-rehabilitation+Lv-cIAP1 group(P<0.05),while the expression of cleaved caspase-9 in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significantly different(P>0.05).5.cleaved caspase-8 protein expression:At each time point after surgery,the expression of cleaved caspase-8 in the model group was increased compared with the sham group(P<0.05);compared with the model group,the expression of cleaved caspase-8 in the acupuncture-rehabilitation group was decreased(P<0.05);compared with acupuncture-rehabilitation group,the expression of cleaved caspase-8 was increased in acupuncture-rehabilitation+Lv-cIAP1 group(P<0.05),while the expression of cleaved caspase-8 in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significantly different(P>0.05).6.cleaved caspase-3 protein expression:At each time point after surgery,the expression of cleaved caspase-3 in the model group was increased compared with the sham group(P<0.05);compared with the model group,the expression of cleaved caspase-3 in the acupuncture-rehabilitation group was decreased(P<0.05);compared with acupuncture-rehabilitation group,the expression of cleaved caspase-3 was increased in acupuncture-rehabilitation+Lv-cIAP1 group(P<0.05),while the expression of cleaved caspase-3 in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significantly different(P>0.05).7.NeuN protein expression:At each time point after surgery,the expression of NeuN in the model group was decreased compared with the sham group(P<0.05);compared with the model group,the expression of NeuN in the acupuncture-rehabilitation group was increased(P<0.05);compared with acupuncture-rehabilitation group,the expression of NeuN was decreased in acupuncture-rehabilitation+Lv-cIAP1 group(P<0.05),while the expression of NeuN in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significantly different(P>0.05).8.Apoptosis:At each time point after surgery,compared with the sham group,the number of apoptosis(TUNEL-DAPI co-localization cells)in the model group increased significantly(P<0.05);compared with the model group,the number of apoptotic cells in acupuncture-rehabilitation group was significantly reduced(P<0.05);compared with the acupuncture-rehabilitation group,the number of apoptotic cells was increased in the acupuncture-rehabilitation+Lv cIAP1 group(P<0.05),while the number of apoptotic cells in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significant different(P>0.05).9.Neuronal apoptosis:At each time point after surgery,compared with the sham group,the number of neuronal apoptosis(TUNEL-NeuN co-localization cells)in the model group increased significantly(P<0.05);compared with the model group,the number of neuronal apoptosis in acupuncture-rehabilitation group was significantly reduced(P<0.05);compared with the acupuncture-rehabilitation group,the number of neuronal apoptosis was increased in the acupuncture-rehabilitation+Lv cIAP1 group(P<0.05),while the number of neuronal apoptosis in acupuncture-rehabilitation+Lv-cIAP1 NC group was not significant different(P>0.05).Conclusion:Acupuncture-rehabilitation therapy can inhibite the activation of caspase-9,caspase-8,and caspase-3 by regulating the expression of cIAP1,then reduce neuronal apoptosis in the ischemic penumbra of cerebral ischemic rats and reduce the number of apoptosis. |
PDF Full Text Request