Small-molecule inhibitor of smoothend suppress neuroinflammation through hedgehog signaling independent mechanisms

摘要

OBJECTIVE Microglial activation mediated neuroinflammation plays an important role in the progression of neurodegerative diseases.The purpose of this study was to investigate the effect of small-molecule inhibitors of smoothend(Smo) on microglial activation mediate neuroinflammation.METHODS To search for the novel anti-neuroinflammatory agents,the BV-2 cel-based nitric oxide(NO) assay was used to screen a series of small-molecule inhibitors of Smo.The production of NO and cell viability were detected by Griess and MTT assay respectively.The expression of inflammatory factors were evaluated by Real-time quantitative PCR or Western blotting or ELISA assay,and activation of signal molecules were detected by Western blotting.The dopaminergic neuronal cell apoptosis were measured by flow cytometry.The stereotaxic injection of LPS into the mice Substantia Nigra(SN) were employed to establish animal model of neuroinflammation and immunofluorescence staining on brain slices sections were used to verify microglial activation and dopaminergic cell loss.RESULTS To search for the compounds that inhibit microglial activation,we have screened a series of Smo inhibitors(0.01-40 μmol·L^(-1)) for the activity to inhibit NO production in BV-2 microglia cells.Among the Smo inhibitors tested,Hh-079 strongly inhibited LPS-induced microglial NO production without affecting the cell viability.Hh-079 significantly inhibited the expression of proinflammatory factors in LPSstimulated BV-2 microglial cells.Hh-079 inhibited phosphorylation of IKKα/β,phosphorylation and degradation of IκBα,phosphorylation of P65 subunit of NF-κB and NF-κB transcriptional activity in LPS-stimulated BV-2 microglial cells.Hh-079 reduced cytotoxicity of conditioned media of activated microglia toward HT-22 neuroblastoma cells in a co-culture system.Mechanistic studies demonstrated that among the Shh-Ptch-Gli pathway,microglia cells did not express detectable levels of Shh,Smo and Gli.Furthermore,neither Smo inhibitor cyclopamine no Gli inhibitor GANT61 exhibited inhibitory properties on proinflammatory genes expression in LPS activated microglia cells.In addition,co-treatment of recombinant Shh or Smo agonist SAG did not block inhibitory effects of Hh-079 on proinflammatory gene expression in LPS activated microglia cells.In vivo study demonstrated that pretreatment of Hh-079(25 and 50 mg·kg-1) significantly reduced TH-positive cells loss and microglia cells activation in the LPS induced neuroinflammation mouse model.CONCLUSION Smo inhibitor Hh-079 suppress neuroinflammation through hedgehog signaling independent mechanisms.