| Purpose: This study aimed to clarify the role of IKKβ in the electroacupuncture-mediated inhibition of inflammatory injury after focal cerebral ischemia/reperfusion and the mechanism by gene interference approach.IKKβ is an essential IKK subunit in activating the nuclear factor kappa B classic signaling pathway by proinflammatory cytokines.Methods: The model was established by middle cerebral artery occlusion(MCAO).205 male SD rats were randomly divided into sham group(n=30),ischemia/reperfusion(I/R)group(n=50),I/R+vehicle group(n=20),EA group(n=30),sh IKKβ group(n=25),IKKβ over expression group(n=25),IKKβ exprssion+EA group(n=25)which were randomized into ischemia of 2h after reperfusion at 6h,12 h,24h,48 h,72h.Electroacupuncture stimulation was performed in the Sprague-Dawley rat model in “Baihui”(GV 20)and “Siguan”(Hegu LI4/Taichong LR3)acupoints.Gene silencing and overexpression approaches were used for IKKβ interference.The infarct volume was measured by2,3,5-triphenyltetrazolium chloride monohydrate(TTC).The protein levels of IKKβ、p IKKβ、p IκBα and nuclear NF-κB p65 were detected by Western blot.The location and expression of IKKβ in microglia were observed using the confocal immunofluorescence approach.The level of inflammatory cytokines(IL-6 and IL-10)was detected by enzyme linked immunosorbent assay(ELISA).Results:1.The nerve function score of the I/R group was lower than that of the sham group 24 h after reperfusion(P(27)0.01).The score of the sh IKKβ group was higher than that of the I/R group(P(27)0.05).Meanwhile,the score of the IKKβ over-expression group was lower than that of the I/R group(P(27)0.05).However,no obvious effect of electroacupuncture treatment was observed in 24h(P>0.05).The neurological defect symptoms of the EA group significantly improved compared with the I/R group 72 h after reperfusion(P(27)0.05).The neurologic function score of the sh IKKβ group was higher than that of the I/R group(P(27)0.05).Meanwhile,the score of the IKKβover-expression group was lower than that of the I/R group(P(27)0.05).The nerve function score significantly improved by electroacupuncture treatment as indicated by IKKβ over-expression(P(27)0.05).2.The infarction volume of the I/R group significantly increased compared with the sham group 72 h after reperfusion(P<0.01)..The infarction volume significantly decreased after electroacupuncturetreatment(P(27)0.05).The infarction volume of the sh IKKβ group was lower than that of the I/R group(P(27)0.01).Meanwhile,the score of the IKKβover-expression group increased compared with the I/R group(P(27)0.01).The infarction volume of the IKKβ over-expression+EA group significantly reduced compared with the IKKβ over-expression group(P(27)0.05).3.The value of p IKKβ in the I/R group was found to increase at each time point compared with the sham group(P(27)0.01)using Western blot.However,the expression of p IKKβ significantly decreased compared with the I/R group at 24 h,48 h,and 72 h after electroacupuncture treatment(P(27)0.05).The ratio of p IKKβ obviously decreased(P(27)0.01)and the ratio of p IκBα significantly decreased in the sh IKKβ group(P(27)0.05)24 h after interference compared with the I/R group;the nuclear translocation of downstream protein NF-κB p65 obviously reduced(P(27)0.01).Both p IKKβand p IκBα in the IKKβ over-expression group significantly increased compared with the I/R group(P(27)0.01);the nuclear translocation of downstream protein NF-κB p65 obviously increased(P<0.05).IKKβover-expression+EA group vs IKKβ over-expression group,both p IKKβand p IκBα decreased(P<0.05),and the nuclear translocation of downstream protein NF-κB p65 obviously decreased(P(27)0.05).4.Immunofluorescence double-labeling showed that the microglia were obviously activated in the cerebral ischemic cortex 24 h after reperfusion,with larger cell body and axonal retraction presented in somecells.IKKβ expression significantly decreased in the cytoplasm of microglia in the sh IKKβ group(P<0.01).Most of the microglia were activated,and IKKβ expression in microglia significantly increased in the IKKβ overexpression group(P(27)0.01).However,the activation of microglia decreased,and IKKβ expression in microglia also significantly decreased after electroacupuncture interference(P(27)0.05).5.The ELISA showed that the content of IL-6 in the sh IKKβ group was significantly lower than that in the I/R group(P(27)0.05),whereas the content of IL-10 in the sh IKKβ group was higher than that in the I/R group(P<0.05).Meanwhile,the content of IL-6 in the IKKβ over-expression group increased compared with the I/R group(P(27)0.05),whereas the content of IL-10 in the IKKβ over-expression group was lower than that in the I/R group(P(27)0.05).The content of IL-6 reduced while that of IL-10 increased in the IKKβ over-expression+EA group compared with the IKKβover-expression group(P(27)0.05).Conclusions: IKKβ silencing showed obvious inhibition and improvement of the NF-κB p65 pathway–mediated inflammatory response after focal cerebral ischemia/reperfusion in the ischemic cortex.This study confirmed that electroacupuncture was capable of inhibiting inflammatory response at the early stage after focal cerebral ischemia/reperfusion by downregulating IKKβ expression as a target. |
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