The Roles Of Mitochondrial Uncoupler And MTOR Inhibitor In Lipopolysaccharide-induced Neuroinflammation

Part Ⅰ.Mild mitochondrial uncoupling induced by FCCP suppresses microglial activation through reducing ROSAim: Neuroinflammation accompanied by excessive microglial activation plays a significant role in the pathgenesis of Parkinson’s disease(PD).Therefore,effective regulation of microglial activation or neuroinflammation is a promising therapeutic way for the treatment of PD.This study is thought to investigate the role of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone(FCCP)in the regulation of neuroinflammation in PD,which is dependent on characteristic of FCCP for decreasing reactive oxygen species(ROS)generation by mild mitochondrial uncoupling in microglia.Methods: The BV2(a microglial cell line)cells were pretreated with FCCP(10 μM)which was removed after treatment,followed by the treatment of lipopolysaccharide(LPS).The protein level of iNOS or COX-2 that indicates microglial activation in cell lysates was detected through Western Blot assays.The levels of TNFα and IL-6 that were secreted into the culture medium were determined using enzyme-linked immunosorbent assay(ELISA)to evaluate the inflammatory factors in BV2 cells.The effects of FCCP on inflammatory factor expressions were further determined using real-time quantitative PCR(qPCR)to detect mRNA levels of iNOS,COX-2,TNFα and IL-6.Furthermore,we detected the influences of conditioned medium(CM)from FCCP-treated BV2 cells on dopaminergic(DA)neuronal cell line SH-SY5 Y in vitro.To investigate whether mild mitochondrial uncoupling induced by FCCP affects mitophagy in our system,we detected the mitochondrial components in BV2 cells using Western blot.The decreased mitochondrial membrane potential(ΔΨm)and ROS were visualized by probes TMRM and DCFH-DA individually.The influences of FCCP on NF-κB activation were detected using Western blot,immunocytofluorescence and subcellular fractionation assays.Finally,we performed stereotaxic injections of FCCP and LPS into the sabstandia nigra(SN)of mouse brains and immunohistochemistry assays(IHC)using coronal sections prepared with a freezing microtome to evaluate the effects of FCCP on LPS-induced neuroinflammory reaction and DA neuronal death in vivo.Results: The mild mitochondrial uncoupling induced by the pretreatment of BV2 cells with FCCP predominantly suppressed the expressions of inflammatory factors including iNOS,COX-2,TNFα and IL-6 both in mRNA and protein levels,in cells that were stimulated with LPS.Further,the conditioned medium assays indicated that a treatment of FCCP protected DA neurons from cell death that was induced by LPS-activated microglia.FCCP did not affect the mitochondrial content but decreased the ROS production through mild mitochondrial uncoupling.Thus,our data suggested that the repressive effects of FCCP on microglial activation results from mild mitochondrial uncoupling that effectively decreases the ROS production by FCCP.Furthermore,the phosphorylation and nuclear translocation of the NF-κB p65 subunit were inhibited by FCCP in LPS-stimulated BV2 cells,suggesting that FCCP decreases ROS generation to inhibit microglial activation.In LPS-treated mice,FCCP protected DA neurons from LPS-induced.Thus,our study demonstrates that FCCP inhibits ROS generation by uncoupling mitochondria to repress LPS-induced microglial activation both in vitro and in vivo.Conclusion: FCCP suppresses microglial activation through the mild mitochondrial uncoupling effect of FCCP leading to the decline in ROS generation to inhibit NF-κB activation.Further,FCCP exhibits anti-inflammatory and neuroprotective efficacy in LPS-induced cellular and mouse models of PD.Part Ⅱ.Rapamycin inhibits neuroinflammation in an autophagy-independent wayAim: As a key modulator in age-related diseases,m TOR plays a siginificant role in neurodegenerative diseases and inflammation.We employ m TOR inhibitor Rapamycin in the model of Parkinson’s disease(PD)to investigate the effects of Rapamycin on neuroinflammation.Methods: The BV2 cells(a microglial cell line)were pretreated with mTOR inhibitor(Rapamycin(10 n M)or Torin1(1 n M))and then stimulated using lipopolysaccharide(LPS).The protein levels of pro-inflammatory cytokines Inducible Nitro Oxide Synthase(i NOS)and Cyclooxygenase-2(COX-2)were detected using Western Blot.The amount S of Nitro Oxide(NO),Interleukin-6(IL-6),Tumor Nucrosis Factor α(TNFα)and Prostaglandin E2(PGE2)that were released into culture medium were measured via specific kits.The m RNA levels of pro-inflammatory cytokines including i NOS,COX-2,IL-6 and TNFα were detected using real-time quantitative PCR(q PCR)to estimate the effects of m TOR inhibitors on microglial activation.To identify the dependence of Rapamycin and Torin1 anti-inflammatory effects on their inhibition of m TOR,microglial activation status was detected in cells in which m TOR was knocked down using RNA interference.Further,similar experiments were performed using Atg-5-knocked down cells to address whether the anti-inflammatory effects of Rapamycin and Torin1 are dependent on autophagic induction.The NF-κB-or AP-1-luciferase reporter assays were performed using stable transfected BV2 cell line that was treated with Rapamycin and LPS to investigate the impact of Rapamycin on these two main inflammation-related transcriptional factors.Conditioned medium(CM)assays in vitro were performed to illustrate the effects of Rapamycin on microglial activation-induced neuronal death using the primary cultured neurons.Experiments including stereotaxic injections of Rapamycin and LPS into the sabstandia nigra(SN)of mouse brains and q PCR or Immunohistochemistry assays(IHC)were used to evaluate the effects of Rapamycin on neuroinflammation and DA neuronal death in vivo.Results: In BV2 cells,low concentrations of mTOR inhibitors Rapamycin and Torin1 inhibit the expression of pro-inflammatory factors including i NOS,NO,COX-2,PGE2,IL-6 and TNFα at mRNA and protein levels in the cells that were stimulated with LPS. Similar results were obtained in m TOR-or Atg-5-knocked down BV2 cells.Rapamycin significantly suppresses the transcriptional activity of AP-1 rather than NF-κB.Using CM assays in vitro and brain stereotaxis injection experiments in vivo demonstrated that Rapamycin protects DA neurons from LPS-induced cell death through inhibition of microglial activation.Conclusion: m TOR inhibitor Rapamycin inhibits microglial activation through suppressing AP-1 transcriptional activity in an autophagy-independent manner.And it exhibits significant protective effects towards neurons both in vitro and in vivo through inhibiting microglial activation.