| ObjectiveBased on the acupoints of meridians and collaterals and modern rehabilitation theory,this project uses the electro-acupuncture combined treadmill training and endoplasmic reticulum stress as the starting point,with the targets of GRP78,GADD153 and caspase-12.The purpose of this project was to observe the effect of electroacupuncture combined treadmill training on the repair of ischemic lesions and neurological function in early stage of cerebral ischemia,as to explore the effect of electroacupuncture combined treadmill training on endoplasmic reticulum stress in order to provide a new experimental basis and theoretical basis for the clinical combination of traditional rehabilitation therapy and modern rehabilitation techniques.MethodsMiddle cerebral artery occlusion(MCAO)was performed on SD rats(male,clean grade).2 hours after cerebral ischemia,the 120 rats(score 1-3 in Neurological deficit 5points system)were randomly divided into model group(group B),electroacupuncture treatment group(group C),rehabilitation group(group D),electroacupuncture combined treadmill training group(group E)with 30 rats in each group,and sham operation group(group A)30 rats(only carotid separation).Each group is divided into three subgroups after 3d,7d and 14 d with cerebral ischemia after 2 hours.Group A and group B were not treated.Group C and group D were treated with electroacupuncture at 4h after operation and the acupoints were selected as "Baihui" and "Fengfu".The parameters for the density of waves is 5 to 100 times/ s with the intensity of the rat limbs mild jitter not screaming and struggling for the degree(usually 3 ~ 5V),20 min one day.The rats in group D and group E underwent treadmill training 4h after operation at a speed of 15 m / min for 30 min every day for 3 days,7 days and 14 days.Animals in those five groups were drawn at time points: 3,7 and 14 days after intervention.After intervention for 3,7 and 14 days,the neurological deficit score(NDS)was used to observe the neurological deficit in each group.And the behavioralfunction of each group was observed by using the behavioral function scale,the levels of cerebral infarction were measured by TTC staining in different time.The changes of the apoptosis was observed by Tunel test in the hippocampus at the time points of 3,7and 14 days.The expression of GRP78 ? GADD153 and caspase-12 protein in the endoplasmic reticulum stress response of the ischemic in hippocampus was detected by using two methods(Western blotting and RT-PCR).Results1.Neurological deficit score:The NDS of five experimental rats were made at 3,7,7 and 14 days after intervention.The results were as follows: The NDS scores of rats in sham operation group at 4 time points were all 0;in model group,NDS in the three intervention groups were significantly higher than those in the sham operation group(P <0.01),while those in the three intervention groups were lower than those in the untreated group on the 1st,3rd,and 7th day after intervention);At the 14 th day of intervention,the NDS in the model group was significantly higher than that in the three intervention groups(P<0.05 or P <0.01).Compared with the acupuncture group,the NDS in the other two intervention groups was significantly higher than that in the acupuncture group The difference was statistically significant(P <0.01)infarction volume test.2.The volume detection of cerebral infarction:After TTC staining,the ischemic area of rats showed pale color.Ischemic and normal tissue boundaries clear,able to distinguish between the normal area and the boundaries of the ischemic area,forehead,roof,temporal lobe and basal ganglia infarction may occur.The postoperative staining showed that there was no pale area in the sham operation group and the pale area in the model group.Compared with the model group,the volume of the pale area in each intervention group was larger than that in the model group,while the volume of the pale area(P> 0.05).Compared with the model group,the volume of the pale area of the two intervention groups was smaller than the model group at 7 days after intervention,the difference wasstatistically significant(P <0.05 or P <0.01).Intervention 14 d,three intervention group compared with the model group,the intervention group pale volume larger than the latter,and the difference between the volume of the pale area was statistically significant(P <0.01);acupuncture group pale area volume and the remaining two Compared with the intervention group,the difference was statistically significant(P<0.01).3.Apoptosis detectionTunel staining was used to observe the apoptosis of hippocampus at 3,7 and 14 days after ischemia.The results showed that the number of apoptotic cells in each group was the highest and the apoptosis rate was the highest at 3 days after modeling,Apoptosis rate decreased.At 3,7 and 14 days after intervention,the apoptosis rate of rats in sham operation group was stable at three time points.Compared with sham operation group,the apoptosis rate in model group at each time point was significantly higher than that in sham operation group <0.01).Compared with the three intervention groups,the apoptosis rate of the model group was significantly higher than the former(P <0.01).Intervention 3d,acupuncture group apoptosis rate was lower than the EA group,the difference between the two groups was statistically significant(P <0.01).Intervention 14 d,acupuncture group apoptosis rate compared with the other two intervention groups were smaller,the difference was statistically significant(P <0.05).4.GRP78,GADD153 and caspase-12 Protein expression in Hippocampus area:The expression of GRP78,GADD153 and caspase-12 in ischemic hippocampus after focal cerebral ischemia / reperfusion were found to be enhanced by western blot.The expression of GRP78,GADD153 and caspase-12 were significantly increased in sham operation group,Compared with the latter group,all the protein expressions were lower than the latter(P <0.01).Intervention 3d,GRP78 expression in acupuncture group was stronger than the other two intervention groups and sham operation group,with statistical significance(P <0.05).The expression of GADD153 and caspase-12 in model group was significantly higher than that in sham operation group and the otherthree intervention groups(P <0.01 or P <0.05).The expression of GADD153 in acupuncture group was significantly lower than that in the other two intervention groups at 3d,with significant difference(P <0.01).The expression of GADD153 in acupuncture group was significantly decreased at 3d,14 d compared with the other two intervention groups,the difference was statistically significant(P <0.01 or P <0.05).5.GRP78,GADD153 and caspase-12 gene expression in Hippocampus area:The expression of RGRP78,GADD153 and caspase-12 in hippocampus of hippocampus after cerebral ischemia were detected by RT-PCR.The expression of GRP78,GADD153 and caspase-12 in sham-operation group was small,Compared with the sham operation group,the gene expression of three groups at each time point was significantly higher(P <0.01).At each time point,the expression of GRP78 in the acupuncture group was stronger than those in the other two intervention groups and the model group.The difference between the two groups was statistically significant(P<0.05 or P <0.01).Compared with the other two intervention groups,the expression of GADD153 in acupuncture group was lower than that in the other two groups(P <0.05 or P <0.01).Compared with the other two intervention groups,the expression of caspase-12 in acupuncture group was lower than that in acupuncture group for 7 days(P <0.05 or P <0.01).Conclusion1.Compared with simple electroacupuncture or rehabilitation therapy,the combination therapy of the two can effectively reduce the endoplasmic reticulum stress reaction caused by focal cerebral ischemia and enhance limb coordination and balance ability.2.Compared with the simple electroacupuncture and rehabilitation therapy,the combination of the two can reduce infarct volume and reduce pathological changes in brain tissue.3.Electroacupuncture combined with treadmill training can up-regulate the expression of PERK pathway GRP78 in hippocampal ER stress and down-regulatethe endoplasmic reticulum stress response CHOP / GADD153 and apoptosis factor Caspase-12.This may be one of the related mechanisms that acupuncture and Kang therapy can relieve cell apoptosis after cerebral ischemia and reperfusion and protect nerve function. |
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