Mitochondrial Uncoupling Reduces Microglia-mediated Inflammatory Injury After Intracerebral Hemorrhage And The Underlying Mechanism

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Endogenous mitochondrial uncoupling protein 2 inhibits microglia-mediated inflammatory responses following intracerebral hemorrhageObjective:To investigate whether endogenous uncoupling protein 2(UCP2)plays a role in microglia-mediated neuroinflammation following cerebral hemorrhage.Methods:1)BV2 microglia and primary microglia were treated with red blood cell lysate(RBC lysate)to mimic the activation of microglia after cerebral hemorrhage.Mitochondrial superoxide(ROS)is the key endogenous activator of endogenous UCPs.To investigate whether microglia enhanced mitochondrial ROS production after the treatment with red blood cell lysate,we used mitochondrial ROS probe MitoSOX to assess mitochondrial ROS levels,and mitotracker Green and Hoechst to label mitochondria and nuclei respectively.2)To investigate whether RBC lysate activated microglial UCP2,the mitochondrial membrane potential of microglia was detected by mitochondrial membrane potential fluorescent probe tetramethylrhodamine ethyl ester(TMRM),and intracelluar ATP levels and ADT/ATP ratios were assessed with a commercial ATP detection kit.3)By using small interfering RNA(UCP2-siRNA)to knock down endogenous UCP2 in microglia,we investigated whether RBC lysate reduces the mitochondrial membrane potential in a UCP2-depending manner.4)After knockdown of UCP2 in BV2 microglia,enzyme-linked immunosorbent assay(ELISA)was used to detect whether knockdown of UCP2 aggravated microglial inflammation induced by RBC Lysate.5)Lentivirus-mediated shRNA(UCP2-shRNA)approach was used to knock down endogenous UCP2 in the striatum of mice.Then,intracerebral hemorrhage(ICH)was induced in the striatum with the striatal injection of collagenase and the striatal levels of inflammatory factors IL-1?,IL-6 and TNF-? were assessed with ELISA at 3 days following ICH.By using the approaches,we investigated whether UCP2 knockdown aggravated neuroinflammation in mice following ICH.Results:1)The MitoSOX red fluorescence and co-localized MitoTracker green fluorescence showed that mitochondrial ROS levels were significantly elevated in BV2 microglia and primary microglia 6 h-24 h after the treatment with RBC lysate compared with the control group.2)RBC Lysate treatment reduced mitochondrial membrane potential and intracellular ATP levels in BV2 microglia.3)Endogenous expression of UCP2 in BV2 microglia was significantly decreased after transfection with UCP2-siRNA,and knockdown of UCP2 prevented RBC Lysate from reducing mitochondrial membrane potential in microglia.4)Knockdown of UCP2 in BV2 microglia aggravated microglial inflammation induced by RBC lysate.5)UCP2 expression in the striatum was decreased after 14 days after the striatal injection of lentivirus expressing UCP2-shRNA.UCP2 knockdown with lentiviral shRNA led to aggravation of brain edema,increased expression of inflammatory factors IL-1?,IL-6,TNF-? in the striatum,and aggravated neuroinflammatory response.Conclusion:Endogenous UCP2 was activated by blood components after intracerebral hemorrhage.Activation of endogenous UCP2 inhibited microglia-mediated neuroinflammatory responses after ICH.Part ? Exogenous mitochondrial uncouplers reduce microglia-mediated inflammatory injury after intracerebral hemorrhageObjective:To study the effects of exogenous mitochondrial uncouplers on microglia-mediated neuroinflammation after intracerebral hemorrhage(ICH)and the therapeutic effects of mitochondrial uncouplers on cerebral hemorrhage.Methods:1)In the cellular model,BV2 microglia were treated with red blood cell lysate(RBC Lysate)to mimic the activation of microglia following intracerebral hemorrhage.ELISA was used to assess the effects of exogenous uncoupler chloramine sulphate(Niclosamide,Nic).Can inhibit cell inflammation caused by RBC Lysate.2)To explore whether Nic can act on the mitochondrial uncoupling pathway,intracellular ATP levels and ADP/ATP ratios were assessed,and the fluorescent probe tetramethylrhodamine ethyl ester(TMRM)was used to assess mitochondrial membrane potential in BV2 microglia.3)To explore whether Nic can alleviate inflammatory injury after intracerebral hemorrhage in mice,brain tissue water content,neurological deficits and inflammatory factor(IL-1?,IL-6 and TNF-?)levels were determined at 3 days following ICH.4)To further explore whether structurally unrelated mitochondrial uncouplers also alleviated inflammatory injury after ICH in mice,we examined the effects of FCCP on brain tissue water content,neurological deficits and the expression of inflammatory factors at 3 days after cICH.Results:1)Nic significantly reduced the levels of inflammatory mediators IL-1?,TNF-a and IL-6 released from BV2 microglia treated with RBC lysate in a dose-dependent manner.2)Nic reduced intracellular ATP levels and increased the ADP/ATP ratios in microglia cells,Moreover,Nic decreased the mitochondrial membrane potential of BV2 microglia.3)Nic reduced brain edema,decreased the expression levels of inflammatory mediators in the hemorrhagic striatum,and alleviated neurological deficits at 3 days following ICH.4)The structurally unrelated uncoupler FCCP also reduced brain edema,decreased the expression levels of inflammatory mediators in the hemorrhagic striatum,and alleviated neurological deficits,at 3 days following ICH.Conclusion:Exogenous mitochondrial uncouplers inhibited microglia-mediated neuroinflammation and improved neurological deficits following ICH.Part ? Exogenous mitochondrial uncoupler inhibits microglia-mediated neuroinflammation after intracerebral hemorrhage by activating AMPKObjective:Since mitochondrial uncouplers reduce intracellular ATP levels and ATP reduction is a major mechanism underlying the activation of AMP-activated protein kinase(AMPK),we investigated whether the mitochondrial uncoupler Nic inhibited neuroinflammation by activating AMPK after intracerebral hemorrhage.Methods:1)To explore whether the uncoupler Nic reduced the microglia-mediated neuroinflammation by activating AMPK following ICH in vitro,western blot was used to assess AMPK activation(phosphorylation)in BV2 microglia after treating with RBC lysate and/or Nic.AMPK activation levels were also determined in the hemorrhagic striatum of mice subjected to ICH and/or Nic treatment in vivo.2)To investigate whether AMPK was required for the suppressive effect of Nic on microglia-mediated neuroinflammation,small interfering RNA targeting AMPK(AMPK-siRNA)was used to knock down AMPK in BV2 microglia.After siRNA transfection for 2 days,BV2 microglia were treated with RBC Lysate and/or Nic,and then enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of inflammatory factors(IL-1?,IL-6,and TNF-?).3)To explore whether Nic reduced the inflammatory response by activating AMPK following intracerebral hemorrhage in vivo,lentivirus expressing AMPK-shRNA was injected into the striatum to knock down endogenous AMPK in the striatum,and the efficiency of AMPK knockdown in the striatum was examined.4)At 14 days after injection of lentivirus expressing AMPK-shRNA,ICH was induced in mice by injecting collagenase into the striatum.Brain tissue water content,neurological deficits and inflammatory factor(IL-1?,IL-6 and TNF-?)levels were determined at 3 days following ICH.5)To investigate whether Nic inhibited the mTOR activation,the signaling pathway downstream of AMPK,western blot was used to detect the activation of mTOR substrate S6 in the striatum and contralateral striatum at 3 days following intracerebral hemorrhage.Results:1)Compared with the vehicle-treated control group,AMPK activation levels were enhanced in Nic-treatedBV2 microglia after RBC lysate.Compared to vehicle treatment,AMPK activation was also enhanced in the hemorrhagic striatum of mice subjected to Nic treatment following ICH.2)Compared with the control group transfected with Ctrl-siRNA,the levels of inflammatory factor IL-1?,IL-6 and TNF-? were significantly increased in BV2 microglia transfected with AMPK-siRNA after treatment with RBC lysate and niclosamide.3)Endogenous AMPK expression was significantly decreased in the striatum injected with lentivirus expressing the AMPK-shRNA vs the striatum injected with the control shRNA(Ctrl-shRNA)Knockdown of AMPK blocked the suppressive effect of niclosamide on neuroinflammatory responses following ICH.4)knockdown of AMPK blocked the therapeutic effects of niclosamide on brain edema and neurological deficits following ICH.5)At 3 days after ICH,niclosamide reduced the phosphorylation level of S6 in the hemorrhagic striatum.Conclusion:Exogenous mitochondrial uncoupler niclosamide inhibited microglial-induced neuroinflammation by activating AMPK following ICH.