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

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.

Sonic Hedgehog信号通路在大鼠牙髓炎中的作用

目的:观察Sonic Hedgehog(Shh)信号分子在大鼠牙髓炎中的免疫定位,探讨Shh信号通路在牙髓防御和修复中所起的作用。方法:将15只SD大鼠随机分成1d、3d和7d组,每组5只,用穿髓开放法建立大鼠牙髓炎模型,3组大鼠分别于术后1,3,7d处死,取出下颌磨牙,常规组织学处理,免疫组化方法检测Shh,Smo,Ptc,Gli1的表达,并对Shh信号通路在大鼠牙髓炎中的表达进行半定量分析,对照组为大鼠正常牙髓。结果:大鼠牙髓炎模型1,3,7dShh广泛表达于穿髓孔下方牙髓间充质细胞及远离穿髓孔的根髓细胞中,表达量随炎症的进展无显著性提高(P>0.05)。Ptc、Smo、Gli1在大鼠牙髓损伤后1d未见阳性表达,3d和7d均表达于穿髓孔下方牙髓间充质细胞中,且表达量均有显著性提高(P<0.05)。空白对照组中Shh信号通路阴性表达。结论:Shh信号通路在牙髓炎过程中表达,提示其可能被激活,参与牙髓炎症反应。