Effects Of Electroacupuncture On Cerebral Ischemia Reperfusion Injury In Rats Through AMPK/mTOR Pathway

Background: Stroke is an acute cerebrovascular disease with few effective treatments.Electroacupuncture is a treatment method combining modern electrical stimulation with traditional acupuncture.Autophagy,apoptosis and inflammasome are important pathological changes after stroke.AMPK is a key factor in regulating cellular energy homeostasis and is widely involved in a variety of pathophysiological processes after ischemic stroke.Therefore,it is of great clinical value to explore whether electroacupuncture can regulate neuronal autophagy,apoptosis and NLRP3 inflammasome level following ischemic stroke by mediating AMPK molecule.Objective: We aimed to investigate whether electroacupuncture treatment at two specific acupoints of Quchi(L11)and Zusanli(ST36)could regulate autophagy,apoptosis and NLRP3 inflammasome after ischemic stroke via regulating the AMPK/m TOR signalling pathway.Methods:1.The 90 SD rats were randomly divided into five groups as follows: the sham-operated group(sham);the middle cerebral artery occlusion/reperfusion group(MCAO/R);MCAO/R+EA group(EA);the EA+NC group;the EA+CC group.Electroacupuncture treatment was given at 3 hours after MCAO/R and lasted for 30 minutes.The acupoints of electroacupuncture treatment were Quchi(L11)and Zusanli(ST36)on the affected limbs of rats.After establishment of MCAO/R model,rats in EA+NC group were intraperitoneally injected with solvent,and the EA+CC group rats were intraperitoneally injected with AMPK inhibitor Compound C.The rest of the procedures in EA+NC group and the EA+CC group were the same as that in the EA group.2.At 2h and 24 h following I/R,neurological deficits were evaluated;and then the cerebral infarction volume was measured by TTC staining.The effects of EA on the neurological function and cerebral infarction volume in rats with cerebral ischemia were observed.3.At 30 min following EA treatment,5 rats in each group were executed to detect LC3、CCAS3、Bcl-2、NLRP3、CCAS1、t-AMPK、p-AMPK、t-m TOR and p-m TOR expression level by western blot.Besides,5 rats in each group were used for immunofluorescence analysis to detect LC3 expression.In all rats,a total of 10 rats died during the experimental procedure.Results: The expression level of LC3Ⅱ/Ⅰ,CCAS3,NLRP3(P<0.01)and CCAS1,p-AMPK/t-AMPK(P<0.05)in the MCAO/R group was significantly increased than sham group,whereas the level of Bcl-2、p-m TOR/t-m TOR of MCAO/R group was significantly decreased than sham group(P<0.05).However,in the EA group,EA treatment significantly decreased the expression level of CCAS3,NLRP3,CCAS1,p-m TOR/t-m TOR and increased the expression level of Bcl-2(P<0.05),LC3Ⅱ/Ⅰ(P<0.01)and p-AMPK(P<0.01).The levels of NLRP3 and p-m TOR/t-m TOR in EA+CC group were obviously higher than EA and EA+NC group(P<0.01),and the expression level of CCAS3 and CCAS1 of EA+CC group was significantly increased than EA group(P<0.01)and EA+NC group(P<0.05).While the expression levels of Bcl-2(P<0.05)and LC3Ⅱ/Ⅰ(P<0.01)in EA+CC group were lower than those in EA and EA+NC group,and p-AMPK/t-AMPK was significantly decreased than EA group(P<0.01)and EA+NC group(P<0.05).Conclusions:Our results suggested that the protective effects of electroacupuncture treatment at two points of Quchi(L11)and Zusanli(ST36)in rats following ischemic stroke were associated with the increased level of neuronal autophagy and inhibition of neuronal apoptosis and NLRP3 inflammasome via activating the AMPK/m TOR signalling pathway...