| Objective: To observe the effects of Jiaji electroacupuncture on motor function,apoptosis of neural cells of the spinal cord tissue and ERK5 signaling pathway-related factors in rats with spinal cord injury,by which to elucidate the mechanism of ERK5 signaling pathway in the treatment of SCI with Jiaji electroacupuncture,and to reveal the anti-apoptotic mechanism of Jiaji electroacupuncture.Methods:Experiment 1: To observe the effects of Jiaji electroacupuncture on behavior,histopathology and apoptosis of SCI rats.Fifty-four rats were randomly divided into three groups: Sham Operation Group(Sham,18 rats),Model Group(Model,18 rats)and Jiaji Electroacupuncture Group(EA,18rats).Each group was divided into three subgroups according to the time point of 1d,3d and 7d after operation.Six rats in each subgroup and each group was given different intervention methods.The motor function of the lower limbs of each group was observed by BBB score;Using HE staining to observe the pathological changes of spinal cord tissue;and the apoptosis of neural cells in spinal cord was evaluated by TdT-mediated dUTp nick-end labeling(TUNEL).Experiment 2: To observe the effect of Jiaji electroacupuncture on ERK5 signaling pathway-related factors.Experimental grouping was the same as Experiment 1.The expressions of ERK5,p-ERK5,CREB,p-CREB,Bcl-2 and Bax proteins were observed by immunohistochemistry and Western Blot.Experiment 3: To observe the role of ERK5 signaling pathway in the treatment of spinal cord injury with Jiaji electroacupuncture.Twenty-four rats with successful intrathecal tubes were randomly divided into Sham Group(Sham,6 rats),Model Group(Model,6 rats),Jiaji Electroacupuncture Group(EA,6 rats)and Jiaji Electroacupuncture + Inhibitor Group(EA + EI,6 rats).Different interventions were given to each group.The motor function of both lower limbs was observed by BBB score,the pathological changes of spinal cord tissues were observed by HE staining,and the apoptosis of neural cells inspinal cord was detected by TUNEL.The expressions of ERK5,p-ERK5,CREB,p-CREB,Bcl-2 and Bax proteins were observed by immunohistochemistry and Western Blot.Results:Experiment 1:(1)BBB score was used to observe the motor function of both lower limbs of rats in each group: The Sham Group had no obvious abnormality in the motor function,the Model Group had obvious motor dysfunction of both lower limbs,BBB score was significantly lower in each time point than that in the Sham Group.The BBB score of the EA Group was significantly higher than that of the Model Group at the same time point(p< 0.05).(2)Observation of pathological changes by HE staining: In the Sham Group,the structure of spinal cord tissue was intact,the structure of white matter,gray matter,anterior and posterior horn of the spinal cord,and central tube were clearly visible,normal cell morphology with round cell body and normal cell nuclear morphology,and no bleeding,neuronal edema was observed.In the 1d Model Group,there was focal patchy shaped hemorrhage,increased interstitial space,destroyed structure of normal tissue,neuronal edema,cell body shrinkage,nucleus pyknosis,and decreased number of neurons.There were no significant differences of the cell morphology in the EA Group compared with the Model Group,also could see the patchy hemorrhage and neuronal edema.In the cell of the 3d Model Group still showed bleeding,the tissue interspace became larger,there was vacuolization,the cell morphology and structure changed more obviously,the cell body shrinked,the cell nucleus condensed into a mass,the nerve cells were obviously reduced,and a large number of inflammatory cell infiltration could be seen,the number of glial cells increased.In the EA Group compared with the Model Group,the degree of tissue damage was slightly lighter,the number of nerve cells increased slightly,and the incidence of inflammatory infiltration and glial cells increased.In the cell of the 7d Model Group,theintercellular space became larger,a large number of vacuoles were observed in the visual field,nerve cells were further reduced,and a large number of inflammatory cells infiltrated,and the increased number of glial cells.Compared with the Model Group,the interstitial space was significantly reduced,and the cell morphology was more superior,and the nerve cells were increased,the increasing of the number of inflammatory infiltrates and glial cells was significantly reduced in the EA Group.(3)TUNEL detection of neuronal apoptosis in spinal cord tissue: The Sham Group had no obvious apoptosis.There were significant differences between the Model Group and the EA Group at the same time point(p< 0.05).In the 1d Model Group,the number of neurons was significantly reduced,apoptosis increased,and diffuse distribution,and there was no significant difference between the EA Group and the Model Group(p > 0.05).In the 3d Model Group,almost no normal neuronal cells were observed,and the number of apoptotic cells increased significantly,mostly concentrated around the injured area.Compared with the Model Group,the number of apoptotic cells decreased significantly in the EA Group(p < 0.05).Compared with the 3d Model Group,the apoptotic neurons in the 7d Model Group were less and scattered.The EA Group neuron apoptosis was significantly reduced,and the normal neurons were significantly increased compared with the Model Group(p< 0.05).Experiment 2:(1)Immunohistochemical results: Compared with the Sham Group,the expression of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 was significantly increased in the Model Group at the same time point(p< 0.05),and with the increase of injury time,the expression increases.Compared with the Model Group,the expressions of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 were significantly increased in the EA Group at the same time point(p< 0.05),and with the increase of injury time,the expression increases.Compared with the Sham Group,the expression level of Bax in the Model Group wassignificantly increased(p< 0.05),and the expression level of the group of 3d was the highest.The expression level of Bax in the EA Group was significantly lower than that in the Model Group and had significant statistical difference(p< 0.05).(2)Western Blot results: The expression of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 protein in the Model group was significantly higher than that in the Sham Group(p < 0.05).Compared with the Model Group,the expressions of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 were significantly increased in the EA Group at the same time point(p < 0.05),and with the increase of injury time,the expression increases.Compared with the Sham Group,the expression level of Bax in the Model Group was significantly increased(p < 0.05),and the expression level of the group of 3d was the highest.The expression level of Bax in the EA Group showed a downward trend compared with the Model Group and had statistical difference(p <0.05).Experiment 3:(1)BBB score: Compared with Sham Operation Group,BBB score of the Model Group,the EA Group and the EA+EI Group were significantly different(p < 0.05).BBB score in the EA Group was significantly higher than that in the Model Group(p< 0.05),and BBB score in the EA Group was significantly higher than that in the EA+EI Group(p< 0.05).(2)HE staining results: In the Sham Group,the structure of spinal cord tissue was intact,the structure of white matter,gray matter,anterior and posterior horn of the spinal cord,and central tube were clearly visible,normal cell morphology with round cell body and normal cell nuclear morphology.In the Model Group,the interstitial space was significantly increased,numerous vacuole-like changes,nerve cells were significantly reduced,and a large number of inflammatory cells and glial cells were observed.Compared with the Model Group,the EA Group had smaller intercellular space,the cell morphology was more superior,the number of nerve cells increased,theinflammatory infiltration and the increase of glial cells were significantly reduced.Compared with the EA Group,the damage was more serious and the number of the neuron was decreased in the EA+EI Group.(3)TUNEL detection results: There was no obvious apoptosis in the Sham Group.The gliocytes and neuron cells in the Model Group were obviously apoptotic compared with the Sham Group,and compared with the Model Group,the number of apoptotic cells in the EA Group was significantly less than that in the Model Group(p< 0.05).The number of apoptotic cells in the EA+EI Group was significantly higher than that in the EA Group(p <0.05).(4)Immunohistochemical test results: Compared with the Sham Group,the expression levels of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 were significantly higher in the Model Group(p < 0.05).The expression levels of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 in the EA Group were significantly higher than those in the Model Group(p < 0.05).The expression levels of p-ERK5,CREB,p-CREB and Bcl-2 in the EA+EI Group were significantly lower than those in the EA Group(p < 0.05).There was no significant difference in the expression of ERK5(p>0.05).The expression level of Bax in the Model Group was significantly higher than that in the Sham Group(p <0.05).The expression level of Bax in the EA Group was lower than that in the Model Group(p < 0.05).Compared with the EA Group,there was no significant change of Bax expression in the EA+EI Group(p>0.05).(5)Western Blot results: The expression levels of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 were significantly higher in the Model Group than those in the Sham Group(p< 0.05).The expression levels of ERK5,p-ERK5,CREB,p-CREB and Bcl-2 protein in the EA Group were significantly increased than those in the Model Group(p < 0.05).Compared with the EA Group,the expression levels of p-ERK5,CREB,p-CREB and Bcl-2 in the EA+EI Group were significantly decreased(p< 0.05).There was no significant difference in the expression level of ERK5(p>0.05).Compared with the Sham Group,theexpression level of Bax in the Model Group increased significantly(p <0.05),while compared with the Model Group,the expression level of Bax in the EA Group showed a downward trend and had statistical difference(p <0.05).Compared with the EA Group,the expression level of Bax in the EA+EI Group had no significant change(p>0.05).Conclusion:1.Jiaji electroacupuncture can improve motor function of both lower limbs in rats with spinal cord injury;2.Jiaji electroacupuncture can reduce the apoptosis of neurons and promote the recovery of the function of the injured tissue;3.After spinal cord injury,ERK5 signaling pathway is activated and the expression and activation of CREB and Bcl-2 is increased in the downstream of ERK5 signaling pathway;4.Jiaji electroacupuncture can regulate ERK5 signaling pathway-related proteins,thereby inhibiting neuronal apoptosis in rats with spinal cord injury.It is one of the mechanisms of Jiaji electroacupuncture to promote spinal cord injury repair. |
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