Nrf2 Regulates Phagocytosis And Phenotypic Transformation Of Microglia Following Intracerebral Hemorrhage

Objective:Explore the mechanism of endogenous hematoma clearance following intracerebral hemorrhage,and investigate the microglial phagocytic activation and phenotypic transformation and neuroprotective effects using nuclear factor erythroid 2-related factor2（Nrf2）agonists monacsin（Xuezhikang）and siRNA-Nrf2（Nrf2-/-）via in vitro experiments or via in vivo experiments.Methods:In vitro experiments,BV-2 cells were cultured in a humidified incubator with 5%CO₂ at 37 °C,and grew to logarithmic phase.In order to screen the best sequence of Nrf2-siRNA,and to screen the best drugs concentration of monascin or Xuezhikang,RT-PCR was used to detect endogenous control Nrf2-mRNA levels.BV-2 microglia were randomly divided into four groups: normal control group,microglia + Nrf2-siRNA（100?nmol/L）,microglia + monascin（15?μM）,microglia + Xuezhikang（200?μg/m L）.we examined the effect of monascin/Xuezhikang（Nrf2-agonist）or Nrf2-siRNA on phagocytic clearance of erythrocytes or bioparticles under a microscope.Meanwhile we observed the expressions of CD80（proinflammatory phenotype: M1 subtype marker）/CD206（anti-inflammatory phenotype: M2 subtype marker）in the above different groups.Simultaneously,pro-inflammatory and anti-inflammatory cytokines（e.g.Trem1/TNF-α,and Trem2/BDNF）were measured by using flow cytometry,immunofluorescence,and western blot,respectively.In vivo experiments,experimental animal Groups: sham Group,ICH+vehicle Group,ICH+Nrf2-/-Group,ICH+monascin Group（10mg/kg/day,twice）,ICH+Xuezhikang Group（0.2g/kg/day,twice）.ICH was induced by stereotactic,intrastriatal injection of type Ⅳ collagenase.At 24 h and 3d after ICH induction,all animals were subjected to neurofunctional assessments using the Garcia score,and we further measured brain water content and hematoma content of the mice（see Supplementary Materials）.Meanwhile we observed the expressions of CD80/CD206 in the above different groups.Simultaneously,pro-inflammatory and anti-inflammatory cytokines（e.g.Trem1/TNF-α,and Trem2/BDNF）were measured by using immunofluorescence and western blot,respectively.Results:The administration of Nrf2 agonist-monascin/Xuezhikang enhanced the phagocytic clearance of erythrocytes or bioparticles by up-regulating Nrf2,which is consistent with the results of promoting hematoma clearance in vivo experiments.In vivo or vitro experiments,monascin/Xuezhikang promoted the expressions of Trem2,CD206 and BDNF,while inhibited the expressions of Trem1,CD80 and TNF-α.Conversely,Nrf2 inhibition showed the opposite results following ICH.That is,upregulation of Nrf2 can attenuate neuroinflammatory,strengthen phagocytic function,promote the phenotypic conversion of microglia to M2 subtype,and exhibit its neuroprotective role;while Nrf2 inhibition can exacerbate neuroinflammatory,reduce phagocytic activity,and promote the phenotypic transformation of microglia to M1 subtype.Conclusion:Nrf2 can regulate the phagocytic function,neuroinflammatory and the phenotypic transformation of microglia,and further clarify the mechanism of Nrf2 neuroprotective role on clearing hematoma,reducing brain edema following intracerebral hemorrhage.