The Effect And Mechanism Of Electroacupuncture On Neuroinflammatory Injury Of Focal Cerebral Ischemia/Reperfusion Rats By Regulating ABIN1

In ischemic stroke,there is a transitional region between the infarct core and normal brain tissue known as the penumbra.Since the penumbra contains neurons that are on the verge of death,early restoration of blood perfusion and rescue of penumbra neurons are the focus of stroke treatment.However,reperfusion also contributes to the production of reactive oxygen species,causing the activation of immune responses,which leads to harmful neuroinflammation.After cerebral ischemia/reperfusion,over-activated microglia produce cytotoxic substances such as pro-inflammatory factors,which aggravate neuronal damage.Damaged or dead neurons release"danger signals"that promote microglia activation.Hence,inhibiting intense neuroinflammation in the early stage of cerebral ischemia/reperfusion is the key to reduce the damage of the ischemic penumbra.NF-κB(Nuclear factor kappa B)regulates hundreds of genes related to inflammation and is a critical molecule for the initiation and expansion of neuroinflammation.Suppressing the activation of NF-κB may be one of the effective strategies to prevent neuroinflammation.ABIN1(A20-binding inhibitor of NF-κB activating factor 1)as an inhibitor of NF-κB,is a potential anti-inflammatory molecule.However,little is known about ABIN1,and there is no research report on ABIN1 in cerebral ischemia/reperfusion.EA(Electroacupuncture)is one of the most common forms of treatment in complementary medicine and has been supported by numerous studies for its protective and anti-inflammatory effects on the brain.Although there have been increasing studies on the anti-inflammatory effects of EA by regulating NF-κB,the regulation of ABIN1 by EA has not been reported.Thus,we first established a MCAO/R(Middle cerebral artery occlusion/reperfusion)rat model to detect the expression of ABIN1in the peri-infarct cortex.We through lentivirus injection to silence ABIN1,and then observe the effect of ABIN1 knockdown on neurological deficit,cerebral infarction volume,neuroinflammation in rats.In addition,MCAO/R rats were given EA treatment once a day.EA effects on ABIN1expression were detected.Besides,the role of ABIN1 in EA against neuroinflammation was explored by ABIN1 silencing.PART 1 ABIN1 EXPRESSION AND EFFECT IN THE PERI-INFARCT CORTEX OF FOCAL CEREBRAL ISCHEMIA/REPERFUSION RATSObjective:To detect the expression of ABIN1 in the peri-infarct cortex of focal cerebral ischemia/reperfusion rats and its effect on neuroinflammatory injury after reperfusion.Methods:MCAO/R rat model was established.SD rats were randomly divided into six groups:sham,MCAO/R,MCAO/R+EA,MCAO/R+sham EA,MCAO/R+LV-Scramble,MCAO/R+LV-sh ABIN1.Rats in the EA group were treated with EA once a day.RT-q PCR and western blotting were used to detect the expression of ABIN1 m RNA and protein respectively at different time points after reperfusion.The cell localization of ABIN1 was observed by double immunofluorescence.ABIN1 was silenced by lentivirus.The m NSS,MST and TTC staining were respectively used to observe the neurological deficit and cerebral infarction volume in rats.The activation of microglia and the expression of pro-inflammatory factors TNF-α(Tumor necrosis factor-α),IL-1β(Interleukin-1β),MCP-1(Monocyte chemotactic protein-1)were detected by immunofluorescence and ELISA respectively to further explore the role of ABIN1 in cerebral ischemia/reperfusion neuroinflammatory injury.Results:The levels of ABIN1 m RNA and protein were higher in the MCAO/R group than in the sham group from 12 h to 72 h(P<0.01)and peaked at 24 h,but the expression of ABIN1 at 6 h and 7 d was not significantly different from that in sham group(P>0.05).Moreover,compared with MCAO/R group,EA stimulation further promoted the expression of ABIN1 in MCAO/R+EA group from 6 h to 7 d(P<0.05),but there was no significant difference between the sham EA group and the MCAO/R group.Immunofluorescence showed that the number of ABIN1~+cells in the peri-infarct cortex was significantly increased in the MCAO/R group compared with the sham group(P<0.01),and the number of ABIN1~+cells further increased in rats receiving EA(P<0.01),but there was no significant difference between the sham EA group and the MCAO/R group.ABIN1 was colocalized with neurons and microglia,but not with astrocyte.Moreover,ABIN1~+Neun~+cells accounted for 70.22±1.71%of ABIN1~+cells,and ABIN1~+Iba-1~+cells accounted for 26.83±2.75%of ABIN1~+cells in the peri-infarct cortex at 24 h.The expression of ABIN1 was detected by RT-q PCR and western blotting to prove that ABIN1 gene was successfully knockdown.As compared with the MCAO/R group,the neurological deficit in the MCAO/R+LV-sh ABIN1 group was increased significantly at72 h(P<0.05).TTC showed that no cerebral infarction was found in the sham group at 72 h,and cerebral infarct volume at 72 h was lager in MCAO/R+LV-sh ABIN1 group than in MCAO/R group(P<0.05).ELISA results suggested that the concentrations of pro-inflammatory factors(TNF-α,MCP-1,IL-1β)in the MCAO/R group were significantly higher than that in the sham group(P<0.01),and the expression of these pro-inflammatory factors was further increased after ABIN1 konckdown(P<0.01).Immunofluorescence staining of Iba-1 was used to detect microglia in the peri-infarct cortex 24 h after I/R.The microglia were few in the sham group and exhibited the resting state(hyper-ramification).In the MCAO/R group,the number of microglia was increased and most of microglia were activated(cell body enlargement,branch reducing).The morphological analysis of microglia indicated that the number of endpoints per cell and the length of cell process were decreased in the MCAO/R group compared with the sham group(P<0.01),the number of endpoints per cell and the length of cell process were reduced in the MCAO/R+LV-sh ABIN1 group compared with the MCAO/R group(P<0.01).Conclusion:At the early stage of cerebral ischemia/reperfusion,the expression of ABIN1 in the peri-infarct cortex was increased and was further up-regulated by EA.Neurons and microglia were the main cellular source of ABIN1 in the peri-infarct cortex.ABIN1 knockdown aggravated the neurological impairment,enlarged the infarction volume,promoted the activation of microglia and the expression of pro-inflammatory factors,suggesting that ABIN1 may have an anti-neuroinflammatory effect.PART 2 THE ROLE OF ABIN1 IN ELECTROACUPUNCTURE AGAINST NEUROINFLAMMATION AFTER CEREBRAL ISCHEMIA/REPERFUSIONObjective:To investigated whether EA depends on ABIN1 to attenuate neuroinflammatory injury after cerebral ischemia/reperfusion.Methods:MCAO/R rat model was established and rats in EA group were treated with EA once a day.SD rats were randomly divided into four groups:MCAO/R,MCAO/R+EA,MCAO/R+EA+LV-Scramble,MCAO/R+EA+LV-sh ABIN1.m NSS,MST and TTC staining were respectively used to observe the neurological deficit and cerebral infarction volume in rats.The activation of microglia and the expression of pro-inflammatory factors(TNF-α,MCP-1,IL-1β)were detected by immunofluorescence and ELISA respectively to further explore the effect of ABIN1 knockdown on electroacupuncture against neuroinflammatory injury.The protein expression levels of ABIN1,A20,IκBα(Inhibitor kappa B),p-IκBα,and nuclear/cytoplasmic NF-κB p65 in the peri-infarct cortex at24 h after reperfusion were measured by western blotting.NF-κB p65nuclear translocation and co-location of A20 and ABIN1 were observed by immunofluorescence,and the co-immunoprecipitation was used to detect ABIN1,A20 and NEMO.Results:Neurological impairment of rats in the MCAO/R+EA group was significantly improved at 72 h compared with that of rats in the MCAO/R group(P<0.05).However,MCAO/R+EA+LV-sh ABIN1 group had worse neurological deficits than rats in the MCAO/R+EA group at 72 h(P<0.05).The results of TTC staining at 72 h after reperfusion suggested that the infarct volume was smaller in the MCAO/R+EA group than in the MCAO/R group(P<0.01).ABIN1 knockdown increased the infarct volume compared with that in the MCAO/R+EA group(P<0.05).The expression of proinflammatory cytokines TNF-α,IL-1βand MCP-1 in the MCAO/R+EA group was significantly decreased compare with the MCAO/R group(P<0.01).The expression of these proinflammatory cytokines in the MCAO/R+EA+LV-sh ABIN1 group were higher than those in the MCAO/R+EA group(P<0.01,P<0.05,P<0.05,respectively).Iba-1immunofluorescence staining in the peri-infarct cortex at 24 h after reperfusion indicated that the MCAO/R+EA group had more endpoints per cell and longer cell processes than the MCAO/R group(P<0.01),MCAO/R+EA+LV-sh ABIN1 group had less endpoints per cell and shorter cell processes than the MCAO/R+EA group(P<0.05).The protein expression of A20 and ABIN1 was increased(P<0.05),p-IκBα/IκBαratio was decreased(P<0.01),nuclear/cytoplasmic NF-κB p65 ratio and NF-κB p65 nuclear translocation were decreased(P<0.01)in MCAO/R+EA group compared with MCAO/R group.The protein expression of ABIN1 was decreased(P<0.01)in the MCAO/R+EA+LV-sh ABIN1 group,which exhibited an increased p-IκBα/IκBαratio(P<0.05)and nuclear/cytoplasmic NF-κB p65 ratio(P<0.01),and enhanced NF-κB p65 nuclear translocation compared with the MCAO/R+EA group.The immunofluorescence results showed that A20 and ABIN1 were co-expressed in the cytoplasm.The coimmunoprecipitation results further suggested that ABIN1,A20 and NEMO bind to each other in the peri-infarct cortex at 24 h after reperfusion.Conclusion:In focal cerebral ischemia/reperfusion,EA alleviated neuroinflammation by inhibiting the activation of NF-κB,thus exerting brain protection.The silencing of ABIN1 partially weakened the effect of EA,suggesting that the cerebroprotective effects of EA may depend partially on the the upregulation of ABIN1 expression to inhibit NF-κB activation.In addition,ABIN1 may inhibit NF-κB activation through cooperation with A20.