| Part one Electroacupuncture stimulation at acupionts of Quchi(LI11) nd Zusanli(ST36)may inhibit nerve cell apoptosis after ishemic stroke.Objective: we aim to explore whether EA therapy at points of Quchi(LI11)and Zusanli(ST36)may alleviate nerve apoptosis and exert neuroprotective effect after ischemic stroke.Methods:The 72 rats were divided into three groups(n=6/group)randomly as follows:(i)sham-operated group(sham);(ii)middle cerebral artery occlusion(MCAO)/reperfusion group(MCAO);(iii)MCAO + electroacupuncture(EA)group(EA).EA therapy was performed at the points of Quchi(LI11)and Zusanli(ST36)of the right affected limbs via an EA apparatus and was initiated at 24 h after reperfusion once a day for 3 consecutive days.At 72 h after ischemia/reperfusion(I/R),neurological deficits were evaluated.6 rats in each group were executed to assess the infarct area by TTC method and 6 rats in each group were decapitated to detect the percentage of TUNEL-positive cells by TUNEL staining;6 rats in each group were executed to detect apoptosis-related protein Bcl-2,Caspase-3,Cleaved caspase-3,Bim expresion level by western blot;remaining rats were decapitated to detect apoptosis-related protein Bcl-2,Caspase-3,Bim expresion level by immmunohistochemistry.Results:The neurological deficits score of MCAO group was obviously higher than sham group(P<0.01),whereas EA group was lower than MCAO group at 72 h after I/R injury(P<0.05)?The rats in sham group demonstrated no infarct volumes.However,the infarct size of rats in MCAO group was higher than EA group(P<0.05).The percentage of TUNEL-positive cells number in the MCAO group was significantly elevated compared to the sham group at 72 h following ischemic stroke(P<0.01).EA markedly decreased the percentage of TUNEL-positive cells number compared with MCAO group(P<0.01).The results of western blot showed that the expression of caspase-3(P<0.05),cleaved-caspase-3(P<0.01)and Bim(P<0.01)were elevated in the MCAO group compared to sham group,and the expression level of Bcl-2(P<0.01)were decreased;however,expression level of caspase-3(P<0.05),cleaved-caspase-3(P<0.01)and Bim(P<0.01)were down-regulated and expression level of Bcl-2(P<0.05)were up-regulated in the EA group compared to MCAO group.The results of immmunohistochemistry demonstrated that EA treatment significantly inhibited the up-regulation of caspase-3 and Bim-positive cells,and reversed the reduction of Bcl-2-positive cells induced by the ischemic brain injury(P<0.01).Conclusion: Electroacupuncture stimulation at Quchi(LI11)and Zusanli(ST36)points decreased neurological deficits score,cerebral infact volume and apoptosis of cerebral cortex,indicating that electroacupuncture stimulation may inhibit nerve cell apoptosis following ischemic stroke so as to exerting neuroprotective effect.Part two Electroacupuncture at Quchi(LI11)and Zusanli(ST36)points ay regualte PTEN,ERK signal pathway.Objective: we aimed to evaluate whether EA stimulation at points of Quchi(LI11)and Zusanli(ST36)may regulate PTEN?ERK signaling pathway and Midkine(MK)after ischemic stroke.Methods: The 36 rats were divided into three groups randomly as follows:(i)sham-operated group(sham);(ii)middle cerebral artery occlusion(MCAO)group(MCAO);(iii)MCAO + EA group(EA).EA therapy was performed at the points of Quchi(LI11)and Zusanli(ST36)of the right affected limbs via an EA apparatus and was initiated at 24 h after reperfusion once a day for 3 consecutive days.At 72 h following ischemia/reperfusion,6 rats in each group were executed to assess p-PTEN,p-PDK1,p-GSK-3?,Thr308,Ser473,tAkt,Midkine,p-ERK1/2,p-JNK,p-P38,ERK1/2,JNK,P38 expression level by western blot;6 rats in each group were executed to detect the Midkine level by immmunohistochemistry.Results: The expression level of p-PTEN?Ser473 of MCAO group was significantly increased than sham group,whereas the level of Thr308?p-PDK1 and p-GSK-3? of MCAO group was significantly decreased than sham group(P<0.01).However,in the EA group,EA treatment significantly decreased the expression level of p-PTEN?Ser473(P<0.01)and increased the expression level of p-PDK1?p-GSK-3??Thr308 than MCAO group(P<0.01).There were not significant differences of t-Akt levels among three groups(P>0.05).Express level of p-ERK1/2,p-JNK and p-P38 in MCAO group were obviously elevated compared with Sham group(P<0.01).However,compared with MCAO group,expression levels of p-ERK1/2(P<0.01),p-JNK(P<0.05)and p-p38(P<0.05)in EA group were significantly decreased.There were not significant differences of ERK1/2?JNK?P38 levels among three groups(P>0.05).There was a significant differences of MK levels among three groups by western blot and immmunohistochemistry analysis.The expression of MK in MCAO group(P<0.01)and EA group(P<0.01)may significantly increase compared to sham group.Furthermore,The MK levels of rats in EA group were obvious higher than MCAO group(P<0.01).Conclusion: Electroacupuncture at Quchi(LI11)and Zusanli(ST36)points may exert neuroprotective effect via regulating PTEN,ERK pathway and MK after ischemic stroke. |
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