Electroacupuncture Inhibits Neuronal Autophagy And Apoptosis Via The PI3K/AKT Pathway Following Ischaemic Stroke

Part one Electroacupuncture stimulation at acupionts of Quchi(LI11) and Zusanli(ST36)may relieve neurological injury after ishemic stroke by inhibiting nerve cell aotophagyObjective: The main purpose of the study was to explore whether EA could improve cerebral ischemia/reperfusion(I/R)injury via autophagy,and to determine the best frequency of EA treatment.Methods:Ninety male Sprague-Dawley rats(250g-280g)were divided into five groups,consisting of sham group,MCAO/R group,EA group one(2Hz,1.0m A),EA group two(50 Hz,1.0m A)and EA group three(100 Hz,1.0m A).EA treatment was performed at 24 h after MCAO/R in rats of EA group once per day for 3 days.EA treatment was performed at the acupoints of Quchi(LI11)and Zusanli(ST36)on the right affected limbs of rats.At 72 h following ischemia/reperfusion,neurological deficits were evaluated;and then the infarct area was assessed by TTC staining.The expressions of related protein LC3,P62,LAMP-1 and Atg5 were measured by western blot.Results : The rats in EA group and MCAO/R group had obvious neurological dysfunction at 2 hours and 72 hours after I / R injury,while the rats in sham operation group had no neurological symptoms.The neurological deficit score of MCAO/R group was significantly higher than sham group at 72 h after I/R injury(P < 0.01),while the neurological deficit degree of EA1 group was lower than MCAO/R group(P < 0.05).The scores of neurological defect score in EA group with different frequencies were lower than those in MCAO/R group.There was no infarct volume in sham operated group,but the infarct volume in MCAO/R group was significantly higher than EA group(P < 0.05).Moreover,the infarction volume and neurological scores in EA2 and EA3 groups were significantly higher than in EA1 group.Therefore,the treatment parameters of EA1 group were the best treatment parameters.The results of western blot showed that the expression levels of LC3 ? / ?(P < 0.01)and Atg5(P < 0.05)in MCAO/R group were higher than sham group,the expression of P62(P < 0.05)and LAMP-1(P < 0.01)were decreased.However,the expression of LC3?/ I(P < 0.01)and Atg5(P < 0.01)in EA1 group were decreased compared to MCAO/R group,the expression of P62(P < 0.05)and LAMP-1(P <0.05)were increased compared to MCAO/R group.The levels of LC3II/I in EA2(P<0.05)and EA3(P<0.05)group were significantly decreased compared with MCAO/R group.The level of LAMP-1 in EA3 group(P<0.05)was obviously increased compared with MCAO/R group.Conclusion: Our results demonstrated that EA treatment had a significant therapeutic effect on cerebral ischemic rats by regulating autophagy,and low frequency EA treatment can significantly relieve neurological injury,reduce infarct volumes and lower neurological deficit scores.The treatment parameters of 1.0 m A and 2 Hz are the best recommendation combination to reduce the injury after ischemic stroke.Part two Effects of electroacupuncture stimulation at acupionts of Quchi(LI11)and Zusanli(ST36)on PI3K/AKT pathway in rats with cerebral infarctionObjective: We aimed to evaluate whether EA treatment at the acupoints of Zusanli(ST36)and Quchi(LI11)exerted a neuroprotective effect on ischaemic stroke rats by modulating autophagy and apoptosis via the PI3K/AKT/m TOR signalling pathway.Methods: The 90 rats were randomly divided into five groups(n=18/group)as follows:(i)sham-operated group(sham);(ii)middle cerebral artery occlusion(MCAO)/reperfusion group(MCAO/R);(iii)the EA group,EA treatment was given at 24 h after MCAO in rats of EA group once per day for 3 days.EA treatment was performed at the points of Quchi(LI11)and Zusanli(ST36)on the right affected limbs of rats;(iv)the EA + NC group,NC was provided by intraperitoneally injection daily for 3 days,and the last injection was performed at 30 min before surgery.The rest of the procedures were the same as the EA group;(v)in the EA+D group,the PI3 K inhibitor Dactolisib was provided to the rats by intraperitoneally injection daily for 3 days,and the last injection was performed at 30 min before surgery.The other processing methods were the same as that in the EA+NC group.Results: The results of western blot showed that the expression levels of LC3-? / I,CCAS3 and Atg7 of MCAO/R group were significantly increased than sham group,while the expression levels of P62,LAMP-1,PI3 K,P-m TOR and P-Akt of MCAO/R group were significantly decreased than sham group(P < 0.01).However,EA treatment significantly decreased the levels of LC3-? / I(P < 0.01),CCAS3(P < 0.01)and Atg7(P < 0.05)and increased the expression levels of P62,LAMP-1,PI3 K,P-m TOR and P-Akt compared to MCAO/R group(P<0.01).The levels of LC3 - ? / I,CCAS3(P < 0.01)and Atg7(P < 0.05)in EA + D group were obviously higher than EA and EA + NC group,while the expression levels of P62,LAMP-1,PI3 K and P-m TOR in EA + D group were lower than those in EA and EA + NC group(P < 0.01),and the expression of P-Akt in EA + D group was significantly lower than EA group(P < 0.01).The expression level of LC3 protein in EA(P < 0.05)and EA + NC(P < 0.05)groups was significantly lower than that in MCAO/R group,and the level of LC3 in EA + D group was significantly higher than that in EA group(P < 0.05)and EA + NC group(P < 0.05)by immmunohistochemistry analysis.Conclusion: Our results indicated that the protective effects of EA treatment at points of Quchi(LI11)and Zusanli(ST36)in rats following cerebral I/R injury were associated with the inhibition of neuronal apoptosis and autophagy via activating the PI3K/AKT/m TOR signalling pathway.