Botch Protects Neurons From Ischemic Insult By Antagonizing Notch-mediated Neuroinflammation

Objective:Owing to the continued high morbidity and high mortality rate after stroke,it is important to seek treatments other than conventional thrombolysis.Notchl up-regulation participates in inflammatory responses after cerebral ischemia-reperfusion(I/R)injury,and it has been reported that Botch binds to and blocks Notchl maturation.In this study,we investigated the role of Botch during I/R injury and explored its potential mechanisms.Methods:A middle-cerebral-artery occlusion/reperfusion(MCAO/R)model was established in adult male Sprague-Dawley rats in vivo,animals experiencing MCAO/R were sacrificed at different intervals(1,3,6,12,24,48,or 72 h after MCAO/R).We detected the protein levels of Botch and Notch intracellular domain(NICD)by western blotting,and cellular localization of Botch protein was detected by immunofluorescence staining.After selecting the time point with the most significant changes in protein levels,Sprague-Dawley(SD)rats were divided into the following six groups:sham group(sham),MCAO/R group,MCAO/R+Si-negative control group(CtrSiRNA),MCAO/R+SiRNA-Botch group(SiRNA),MCAO/R+vector group(Vector),and MCAO/R+Botch-overexpression group(Over-Botch).After detecting the intervention of each group by Western blotting,observe the following indicators after modeling:1)Behavioural tests such as the Rotarod test and the Adhesive-removal test were performed on days 0,1,3,7,and 14 after modeling,and data and statistics were recorded.2)Behavioral scoring was performed 24 hours after modeling.3)Fluoro-Jade B(FJB)staining was used to detect neuronal apoptosis.4)TTC staining was used to analyze the infarct volume.5)The immunofluorescence staining was used to observe the activation and infiltration of microglia in injured hemispheres.6)ELISA was used to detect the levels of IL-6 and TNF-? in rat serum.7)The changes of Botch and NICD in rats and the relationship with Notch1 were detected by Western blot.Cultured neurons and microglia were exposed to oxygen-glucose deprivation/reoxygenation(OGD/R)to mimic I/R injury in vitro,we detected the protein levels of Botch and Notch intracellular domain(NICD)by western blotting at different time points.After selecting the time point with the most significant changes in protein levels,the primary cultured neurons or microglia were divided into the following six groups:normal group(Normal),OGD/R group,OGD/R+Si-negative control group(CtrSiRNA),OGD/R+SiRNA-Botch group(SiRNA),OGD/R+vector group(Vector),and OGD/R+Botch-WT group(Over-Botch).At 12 h after OGD/R,cells were used for testing.Result:1)After MCAO/R modeling in rats,protein levels of Botch were significantly elevated at 6 h after MCAO/R and peaked at 48 h after MCAO/R in vivo.In cultured primary neurons after OGD/R modeling,Botch expression was also significantly increased at 12 h and peaked by 12-24 h.2)Immunostaining of brain tissue 24 h after MCAO/R demonstrated that Botch was expressed in NeuN+ neurons and Iba1+ microglia,but not in GFAP+astrocyte.3)At the cellular level,FJB staining confirmed that Botch knockdown increased neuronal death as compared with those of the siRNA-Botch and CtrSiRNA groups.4)At the tissue level,showed by TTC staining,siRNA knockdown of Botch led to an enlarged ischemic volume after MCAO/R,while overexpression of Botch reduced the ischemic volume.5)Botch upregulation alleviated the sensory and motor deficits caused by MCAO/R,as suggested by results of the rotarod test,adhesive-removal test,and neurobehavioral scores,while Botch downregulation exacerbated the neurological dysfunction following ischemic stroke.6)After ischemic stroke,microglia were activated(Iba1+)and distributed in a cluster-like pattern.Upregulation of Botch reduced the number of activated microglia,and downregulation of Botch increased microglial activation.7)We found that,consistent with the activation pattern of microglia,upregulation of Botch reduced the expression levels of the pro-inflammatory cytokines,IL-6 and TNF-?,after MCAO/R,whereas downregulation of Botch led to higher levels of pro-inflammatory cytokines in vivo and in vitro.8)Our in vivo and in vitro western blotting data showed that there was an increased expression of the Notch intracellular domain(NICD)after MCAO/R and OGD/R.Knockdown of Botch upregulated the NICD protein level in rat brains after MCAO/R,while overexpression of Botch downregulated the NICD maturation.9)As indicated by immunofluorescent staining,NICD was found to translocate to the mitochondria in neurons and microglia after OGD/R.9)Using a fluorescent microplate reader,we confirmed that when Botch was overexpressed,the accumulation of oxidizing substances in neurons was significantly reduced;when Botch was knocked down,the accumulation of oxidizing substances increased.After detecting the intervention of each group by Western blotting,observe the following indicators after modeling:1)ELISA was used to detect the levels of inflammatory factors IL-6 and TNF-?in the supernatant of rat primary microglia.2)The levels of Botch and NICD in primary neuron cells and their relationship with Notchl were detected by Western blotting.3)The confocal immunofluorescence staining technique was used to detect the localization of NICD in the subcellular organelle mitochondria.4)MitoSox technology was used to detect the translocation of NICD to mitochondria using a fluorescence microplate reader to stimulate the accumulation of superoxide(mtROS)in mitochondria.Conclusions:1)Botch expression increases after ischemic injury.2)Botch exerts neuroprotection in a rat model of MCAO/R.3)Botch alleviates microglial infiltration and neuroinflammation after MCAO/R.4)Botch antagonizes the activation of the Notchl signaling pathway.5)Botch decreases neuroinflammation and accumulation of reactive oxygen species by inhibiting NICD translocation.