| ObjectiveTo explore the regulating effect of electroacupuncture on angiogenesis in MCAO rats,using miR-210-3p and AKT/mTOR signaling pathways as the entry point,to explore the three phases of electroacupuncture on miR-210-3p and AKT/mTOR pathway factor interaction regulation,with a view to further elucidate the possible mechanism of electroacupuncture to regulate angiogenesis after cerebral ischemia,and provide new ideas for the discovery of miRNAs as a potential pathway of cerebral ischemia prevention and treatment.Methods160 clean-grade male(Sprague Dawley,SD)rats were randomly selected from the sham operation group of 36,and the remaining 124 rats were established for the MCAO model.The 108 rats that met the scoring standard were randomly divided into 3 groups:model group,electroacupuncture group,electroacupuncture and inhibitor group.The rats in the above 4 groups were divided into three phase groups of 3 d,7 d,and 14 d,with 12 rats in each group.In the electroacupuncture group,select the two acupoints"Baihui"and"Dazhui".Electroacupuncture parameters:density wave,frequency 5～100Hz,1～2 m A,intensity to cause surrounding acupuncture points.The tissue trembles slightly,and the treatment duration is 20 minutes.The first treatment was started 4hours after the operation,once a day,and the treatment was continued for 3 days,7 days,and 14 days,respectively.In the inhibitor group,the specific inhibitor rapamycin was injected intraperitoneally at a concentration of 0.1 mg/ml,0.3 mg/kg each time,once a day,and then electroacupuncture(the same method as the electroacupuncture group).Subsequently,the neurological deficit score(modified neurological severity scores,m NSS)was performed to evaluate the neurological damage of the four groups of rats.The laser doppler blood flow meter was used to measure the local cerebral blood at 5min,3 d,7 d,and 14 d after insertion flow.Use HE staining to detect brain tissue pathology.And use Western Blot to detect AKT/p-AKT/mTOR/p-mTOR protein expression.And use RT-q PCR to detect miR-210-3p,Ephrin A3,AKT,mTOR m RNA expression.Also,the microvessel density(MVD)of the cortex on the ischemic side was counted by the Weidner method.Results1.Modified rat neurological deficit score(mNSS).In the sham operation group,the neurological deficit scores in the four postoperative periods were all 0 points.Model group compared with the electro-acupuncture group,and the electro-acupuncture group had lower nerve function scores than the model Group,the intervention was more significant at 7 d and 14 d(P<0.01).Compared with the inhibitor group,the neurological deficits of the model group were not statistically significant after 3 d,7 d,and 14 d intervention.After 14 d of intervention,compared with the inhibitor group,the scores of neurological deficits in the electroacupuncture group were significantly lower(P<0.01).2.Regional cerebral blood flow(rCBF).The local cerebral blood flow of rats in the sham operation group stabilized at about 130 Pu during the four time periods.Compared with the sham operation group,the blood flow in each time period was significantly lower than that of the sham operation group(P<0.01).Compared with the model group,the electroacupuncture group and the inhibitor group had no statistical significance when they intervened for 5 minutes and 3 days(P>0.05).At 7 d and 14 d of intervention,the local cerebral blood flow of the electroacupuncture group and the inhibitor group was significantly higher than that of the model group,with a significant difference(P<0.05),of which there was a very significant difference at 7 d(P<0.01).Comparing the electroacupuncture group with the inhibitor group,the cerebral blood flow increased at 7 d and 14 d of intervention,and the difference was extremely significant at 7 d(P<0.01),and the local cerebral blood flow continued to increase at 14d,which was significant Difference(P<0.05).3.Brain tissue pathology observation(HE staining).In the sham operation group,the brain tissue of the ischemic cortex was intact in the three time periods,the neurons were arranged in an orderly manner,there were very few vacuoles,and the nuclei were clear and complete.In the model group,a large number of vacuoles were seen in the cerebral tissue of the ischemic side cortex of the rats,the interstitial edema was obvious,the neuronal cells were not arranged tightly,the structure was loose and meshed,the number was significantly reduced,and a large number of neuronal cells were aggregated.The degree of brain tissue damage was further reduced compared with the3rd and 7th days.In different time periods,the brain tissue of the ischemic side cortex of rats in the electroacupuncture and inhibitor groups had different degrees of improvement.Compared with the electroacupuncture group,the injury degree of the inhibitor group was not significantly improved.4.Western Blot to detect AKT/p-AKT/mTOR/p-mTOR protein expression.In the sham operation group,a small amount of AKT,p-AKT,mTOR,and p-mTOR were expressed in the cortex of the rat at each time period.Compared with the sham operation group,the expression of p-AKT in the model group continued to increase in various time periods(P<0.05),reaching a peak at 14 d,and the expression of AKT and mTOR reached a peak at 3 d(P<0.01).The expression of mTOR reached a peak on the7d(P<0.01).Compared with the model group,the expression of AKT/p-AKT/mTOR/p-mTOR was significantly increased in the electroacupuncture group on 3,7 and 14 days(P<0.01),and the expression of AKT and p-AKT reached the peak on the 14th day.The expression of mTOR and p-mTOR increased significantly at each time(P<0.01),and reached a peak at 7 days.The expression of Akt/p-AKT/mTOR/p-mTOR decreased at each time in the inhibitor group(P<0.05).5.RT-q PCR detection of miR-210-3p/Ephrin A3 and AKT/mTORm RNA expression.In the sham operation group,miR-210-3p/Ephrin A3/AKT/mTOR m RNA was slightly expressed in the cortex on the ischemic side of rats.The relative expression of miR-210-3p/Ephrin A3/AKT/mTOR m RNA in the model group was significantly higher than that in the sham operation group on 3,7 and 14 days,and there was a very significant difference(P<0.01).After treatment for 3 d,7 d,and 14 d,the relative expression of miR-210-3p m RNA in the electroacupuncture group was significantly higher than that in the model group,with a very significant difference(P<0.01).The relative expression of Ephrin A3 m RNA was all decreased.There are significant differences(P<0.05 or P<0.01).Comparing the inhibitor group and the electroacupuncture group at different times,the relative expression of miR-210-3p m RNA decreased(P<0.01),and the relative expression of Ephrin A3 m RNA increased(P<0.05).Compared with the model group,the relative expression of AKT/mTOR m RNA in the electroacupuncture group and the inhibitor group was significantly higher than that of the model group(P<0.05 or P<0.01),after 7 d and 14 d intervention in the electroacupuncture group and the inhibitor group.The relative expression of AKT/mTOR m RNA was significantly higher in the treatment group(P<0.01).6.Measurement of microvessel density(MVD).In the sham operation group,only a small amount of positive expression of CD31~+was seen in the three time periods.Compared with the sham operation group,the expression of CD31~+positive cells in the cerebral cortex of the ischemic side of the model group was significantly increased(P<0.05 or P<0.01).The electroacupuncture group compared with the model group,the expression of CD31~+positive cells Significantly increased(P<0.01),and the number of CD31+positive cells reached a peak at 14 d.Compared with the sham operation group,the inhibitor group had no statistical significance at 3 d(P>0.05).Intervene with electroacupuncture on 7 d and 14 d The number of CD31+positive cells in the inhibitor group and the inhibitor group was statistically significant(P<0.05).Conclusion1.Electroacupuncture promotes the reconstruction of neurological function in MCAO rats,improves the local cerebral blood flow in the brain,and improves the pathological changes of brain tissue in the ischemic side cortex of MCAO rats.2.Electroacupuncture can regulate the expression of miR-210-3p and its targeted messenger Ephrin A3 in the cerebral cortex of the ischemic side of MCAO rats.The relative expression of AKT is up-regulate,p-AKT,mTOR,p-mTOR,and CD31~+,suggesting miR-210-3p may specifically regulate the AKT/mTOR signaling pathway,and play a neuroprotective effect by promoting angiogenesis in the recovery of cerebral ischemia.This may be one of the relevant mechanisms of electroacupuncture to promote angiogenesis after cerebral ischemia. |
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