| Objective:To observe the effect of electroacupuncture combined with enriched rehabilitation training on oxidative stress related factor Silent information regulator 1(SIRT1)and peroxisome proliferator-activated receptorγ coactivator-1α(PGC-1α).Explore the correlation between neurofunctional recovery and oxidative stress in rats with focal cerebral ischemia under different treatment modalities.To explore the protective mechanism of SIRT1/PGC-1α axis on nerve cells in rats with focal cerebral ischemia.Method:A total of 36 SPF rats were randomly selected from 200 SPF rats in sham operation group,and the remaining 164 rats were established MCAO model according to the modified Zea Longa suture bolting method.According to the rejection criteria,144 qualified rats were screened and divided into the following groups according to random number table method: Model group(36 rats),electroacupuncture group(36rats),enriched rehabilitation training group(36 rats)and combined electroacupuncture enriched rehabilitation training group(36 rats)were divided into 3 days,7 days and 14 days according to different courses of treatment,with 12 rats in each subgroup.Rats in each treatment group received intervention treatment 4 hours after postoperative anesthesia and wakefulness.In the electroacupuncture group and the combined group,"Baihui" and "Dazhui" were selected as the main acupoints,supplemented with "Neiguan" and "Taixi" for treatment.The rehabilitation group and the combined group received enriched environment and rehabilitation training.Each treatment group was treated once a day for 3 days,7 days and 14 days.The sham-operation group and the model group were fed routinely without treatment intervention,and the samples were collected at 3,7 and 14 days for observation and comparison.The neurological function deficit of rats in each group at different time periods(4 h after operation,3 days after intervention,7 days after intervention,14 days after intervention)was observed by m NSS18 score.Doppler cerebral blood flow meter was used to observe the changes of cerebral blood flow in each group at different time periods(5 minutes after interception,3,7 and 14 days after intervention).He staining was used to observe the pathological changes in the ischemic cortex of rats in three time periods.The changes of MDA and SOD expression in three time periods were determined by biochemical analysis.Western Blot and RT-q PCR were used to observe the protein and gene expression of SIRT1 and PGC-1α in five groups.Factorial analysis of interaction between electroacupuncture and enrichment rehabilitation training.Result:1.Defects in neurological functionThe neurological function scores of rats in the sham operation group were 0 at 4 h,3 d,7 d and 14 d.Compared with the sham operation group,the scores of the model group were higher than those of the sham operation group at four different time periods(P<0.01).Compared with the model group,there was no significant difference in the scores of the treatment groups at 4 hours and 3 days(P>0.05),while the scores of the treatment groups at 7 days and 14 days were significantly decreased(P>0.01).Compared with the electroacupuncture group and the rehabilitation group,the combined group had no significant difference in the 4-hour and 3-day period(P>0.05),and the score of the combined group was significantly decreased in the 7-day and14-day period(P<0.01).There was no statistical significance between the electroacupuncture group and the rehabilitation group in four time periods(P>0.05).2.Analysis of changes in local cerebral blood flowThe cerebral blood flow in the sham operation group was stable at different time periods.The cerebral blood flow was significantly decreased in all groups except the sham operation group(P<0.01)at 5 min after suture insertion.Compared with the sham operation group,the number of patients in the model group decreased at 5 min,3d,7 d and 14 d intervals(P<0.01);Compared with the model group,there was no significant difference between the treatment groups after 5 minutes of suturing(P>0.05).After 3 days of treatment,there was no significant difference between the electroacupuncture group and the rehabilitation group(P>0.05),but there was a significant increase in the combination group(P<0.01).The cerebral blood flow in the treatment group increased at 7 days and 14 days(P<0.01).Compared with the electroacupuncture group and the rehabilitation group,the cerebral blood flow in the combined group was significantly increased at 3,7 and 14 days after treatment(P<0.01).There was no statistical significance between the electroacupuncture group and the rehabilitation group in four time periods(P>0.05).3.Histopathological observationIn the sham operation group,there was no significant difference at different time periods.The structure of nerve cells in the ischemic cortex was complete,with clear nucleoli,compact and orderly arrangement,and few vacuoles.The arrangement of nerve cells in the model group was obviously disordered and dispersed,with a large number of vacuoles,nuclear pyknosis and chromophilic substance.However,with the extension of treatment time,the decrease of vacuoles and chromophilic substance could be observed.Compared with model group,treatment group in three days with a certain degree of improvement,although have different levels of interstitial edema,but cortex addicted to dyeing quality is relatively small,cellular structure damage relative ease,especially joint group,in the period of 7 days,14 days,the treatment group improved significantly,the number,structure,arrangement of nerve cell gradually returned to normal,dense structure by loose gradually,The changes of chromophilic matter,nuclear pyropysis and vacuolation were reduced,and the improvement was most obvious in the electroacupuncture group and the combination group at 14 days.4.Analysis of MDA and SOD expressionThere was no significant difference in MDA and SOD expression in the sham operation group at three different time periods.Compared with sham operation group,MDA content in model group was significantly increased at 3,7 and 14 days(P<0.01),and SOD activity was significantly decreased(P<0.01).Compared with model group,MDA in treatment groups decreased significantly(P<0.01)and SOD increased to some extent(P<0.01)in three time periods.Compared with electroacupuncture group and rehabilitation group,MDA content in combined group decreased(P<0.05 or P<0.01)and SOD activity increased(P<0.01)at three time periods.There was no significant difference between the electroacupuncture group and the rehabilitation group in each time period(P>0.05).5.Analysis of expression of SIRT1 and PGC-1αproteinThere were no significant changes in the contents of SIRT1 and PGC-1α in the ischemic cortex of the sham operation group at 3,7 and 14 days.Compared with the sham operation group,the protein expression levels of SIRT1 and PGC-1α in the model group were significantly decreased at three time periods(P<0.01);Compared with the model group,the expression of SIRT1 and PGC-1α in the treatment group were increased at different time periods(P<0.01).Compared with treatment groups,the contents of SIRT1 and PGC-1α protein in combination group were higher than those in electroacupuncture group and rehabilitation group(P<0.01).There was no significant difference in protein expression between the electroacupuncture group and the rehabilitation group at three different time periods(P>0.05).6.Expression analysis of SIRT1 m RNA and PGC-1αm RNASIRT1m RNA and PGC-1αm RNA didn’t change significantly in the ischemic cortex of rats in the sham operation group at different time periods.Compared with the sham operation group,the m RNA expressions of SIRT1 and PGC-1α in the model group were significantly decreased at three time periods(P<0.01);Compared with model group,the m RNA expression levels of SIRT1 and PGC-1α in all treatment groups were increased(P<0.01);Compared with the treatment group,the m RNA levels of SIRT1 and PGC-1α in the combined group were significantly increased(P<0.05 or P<0.01)compared with the electroacupuncture group and the rehabilitation group.There was no significant difference between the electroacupuncture group and the rehabilitation group in different time periods(P>0.05).7.Analysis of factors associated with EA and enriched prehabilitation The main effect of each treatment method on MDA expression level in different time periods was statistically significant(P<0.01)and electroacupuncture combined with rich rehabilitation training had an interactive effect on MDA expression level(P<0.01).At the 3-day stage and the 7-day stage,the main effect of each treatment on SOD expression level was different(P<0.05 or P<0.01);Electroacupuncture combined with enrichment rehabilitation training had an interactive effect on SOD expression(P<0.05 or P<0.01),and there was no significant difference in SOD expression between the 14 days of combined treatment(P<0.05).At the 3rd and 7th day stages,the main effect of electroacupuncture and rehabilitation on the expression level of SIRT1 m RNA was statistically significant(P<0.01).Electroacupuncture combined with rehabilitation training had an interaction effect(P<0.05),and there was no significant interaction effect between the two treatment methods in the 14-day period(P>0.05).At three different time segments,the main effect of electroacupuncture and rehabilitation on PGC-1α m RNA expression level was statistically significant(P<0.01).Both electroacupuncture and rehabilitation combined therapy had interaction effects(P<0.05).Conclusion:1.Electroacupuncture,enriched rehabilitation training and combined treatment of both can promote the recovery of neurological function,cerebral blood flow recanalization,inhibit oxidative stress and improve the pathological condition of ischemic area in MCAO rats.Electroacupuncture combined with enrichment rehabilitation training is significantly better than monotherapy.2.Electroacupuncture combined with enrichment rehabilitation training may play a neuroprotective role by activating the expression of SIRT1 in the SIRT1/PGC-1α axis,thereby upregulating the expression of downstream PGC-1α,inhibiting oxidative stress response in MCAO rats. |
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