Electroacupuncture Attenuate The Inflammatory Injury In Cerebral Ischemia/reperfusion Rats Through Upregulation Of Tax1-binding Protein 1 Expression

Objective:It is well known that stroke imposes a heavy burden on family and society.Inflammatory injury is an important injury mechanism after focal cerebral ischemia/reperfusion.NF-?B signaling pathway is the main signal pathway of inflammatory injury.Tax1-binding protein 1(TAX1BP1)is as an important negative regulatory target by recruiting and binding A20 to inhibiting NF-?B signaling pathway.This study investigated the mechanism of TAX1BP1 in electroacupuncture(EA)therapy for inflammatory injury after cerebral ischemia/reperfusion.Methods:This study adopts the method of gene silencing of TAX1BP1.The model was established by right middle cerebral artery occlusion and reperfusion.Rats were treated with EA at the Baihui and left Siguan(Hegu and Taichong)acupoints.Rats were randomly assigned into a sham group,a MCAO/R group,a MCAO/R+EA group,a MCAO/R+EA+LV-TAX1BP1 group,MCAO/R+EA+Vehicle group.Neurobehavioral evaluation,cerebral infarct volume,TAX1BP1 protein expression,activation of NF-kappa B signaling pathway were tested in each group.Results:1.The neurological scores and and the cerebral infarct volume were tested at 72 h after reperfusion.Compared with SHAM group,the neurological scores were decreased(P<0.05),and the cerebral infarct volumes were increased(P<0.01)in MCAO/R group.Compared with MCAO/R group,the neurological scores were increased and the cerebral infarct volumes were decreased in MCAO/R+EA group(P<0.05).Compared with MCAO/R+EA group,the neurological scores were decreased and the cerebral infarct volumes were increased in MCAO/R+EA+LV-TAX1BP1 group(P<0.05).Compared with MCAO/R+EA group,there was no statistically significant difference in the neurological scores and cerebral infarct volume in MCAO/R+EA+Vehicle group(P>0.05).2.Compared the TAX1BP1 protein expression between SHAM group and MCAO/R group at various time points,the TAX1BP1 expression at 12 h,24 h,48 h after reperfusion were increased in MCAO/R group(P<0.05),Compared the TAX1BP1 protein expression between MCAO/R+EA group and MCAO/R group at various time points,the expression of TAX1BP1 at12 h,24 h,48 h,72 h after reperfusion were increased in MCAO/R+EA group(P<0.05),and the expression of TAX1BP1 protein at 24 h after reperfusion achieved the peak.3.TAX1BP1 and A20 were mainly expressed in cytoplasm in cerebral ischemia cortex,and TAX1BP1 was mainly expressed in neurons.4.TAX1BP1 mRNA expression was tested at 24 h after reperfusion.Compared with SHAM group,the TAX1BP1 mRNA expression was increased in MCAO/R group(P<0.01).Compared with MCAO/R group,the TAX1BP1 mRNA expression was increased in MCAO/R+EA group(P<0.01).Compared with MCAO/R+EA group,the TAX1BP1 mRNA expression was decreased in MCAO/R+EA+LV-TAX1BP1 group(P<0.01).Compared with MCAO/R+EA group,there was no statistically significant difference in the TAX1BP1 mRNA expression in MCAO/R+EA+Vehicle group(P>0.05).5.The protein expression of TAX1BP1,A20,P-IKK?,P-I?B?,nuclear NF-?B p65 were tested at 24 h after reperfusion.Compared with MCAO/R group,the TAX1BP1,A20 protein expression were increased and the P-IKK?,P-I?B?,nuclear NF-?B p65 protein expression were decreased in MCAO/R+EA group(P<0.05).Compared with MCAO/R+EA group,the TAX1BP1,A20 protein expression were decreased and the P-IKK?,P-I?B?,nuclear NF-?B p65 protein expression were increased in MCAO/R+EA+LV-TAX1BP1 group(P<0.05).Compared withMCAO/R+EA group,there was no statistically significant difference in the protein expression of TAX1BP1,A20,P-IKK?,P-I?B?,nuclear NF-?B p65 in MCAO/R+EA+Vehicle group(P>0.05).6.Iba-1mRNA and GFAP mRNA expression were tested at 24 h after reperfusion.Compared with SHAM group,the Iba-1 mRNA and GFAP mRNA expression were increased in MCAO/R group(P<0.01).Compared with MCAO/R group,the Iba-1 mRNA and the GFAP mRNA expression were decreased(P<0.01)in MCAO/R+EA group.Compared with MCAO/R+EA group,the Iba-1mRNA and GFAP mRNA expression were increased in MCAO/R+EA+LV-TAX1BP1 group(P<0.01).Compared with MCAO/R+EA group,there was no statistically significant difference in Iba-1 mRNA and GFAP mRNA expression in MCAO/R+EA+Vehicle group(P>0.05).7.Immunofluorescent detection indicated at 24 h after reperfusion.Compared with SHAM group,the TAX1BP1 positive cells counts were increased in MCAO/R group(P<0.001).Compared with MCAO/R group,the TAX1BP1 positive cells counts were increased in MCAO/R+EA group(P<0.001).Compared with MCAO/R+EA group,TAX1BP1 positive cells counts were decreased in MCAO/R+EA+LV-TAX1BP1 group(P<0.001).Compared with MCAO/R+EA group,there was no statistically significant difference in TAX1BP1 positive cells counts in MCAO/R+EA+Vehicle group(P > 0.05).The number of microglial cellsand astrocyte cells in MCAO/R group and MCAO/R+EA+LV-TAX1BP1 group were increased,the morphology became larger,the branches became short.The activation of microglial and astrocyte were decreased in MCAO/R+EA group and MCAO/R+EA+Vehicle group.NF-?B p65 protein were mainly expressed in the nucleus in MCAO/R group and MCAO/R+EA+LV-TAX1BP1 group,NF-?B p65 protein were mainly expressed in cytoplasm in MCAO/R+EA group and MCAO/R+EA+Vehicle group.8.Immunohistochemistry showed that NF-?B p65 protein were mainly expressed in the nucleus in MCAO/R group and MCAO/R+EA+LV-TAX1BP1 group,NF-?B p65 protein were mainly expressed in cytoplasm in MCAO/R+EA group and MCAO/R+EA+Vehicle group at 24 h after reperfusion.Conclusion:Electroacupuncture could significantly inhibit neuronal NF-?B signaling pathway,restrain the activation of microglia and astrocyte,reduce the cerebral infarct volume,promote the focal neural functional recovery in focal cerebral ischemia/reperfusion by upregulating TAX1BP1 protein expression.