小续命汤提取物下调TLR4/MyD88信号通路减轻脂多糖诱导神经炎症的体内外研究（英文）
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摘要
探讨小续命汤提取物(Xiao-Xu-Ming decoction extract, XXM)对脂多糖(lipopolysaccaride, LPS)诱导的体内外神经炎症的影响。体外实验应用200 ng/mLLPS处理BV2小胶质细胞24小时以诱导炎症反应。体内实验应用5 mg/kgLPS诱导小鼠炎症反应。Griess试剂测定NO含量; ELISA法测定IL-1β、IL-6、TNF-α和MCP-1的含量; WB检测Iba-1、TLR4和MyD88蛋白的表达;RT-PCR测定TLR4和MyD88的mR NA水平。结果发现,在体外细胞上, XXM显著降低LPS诱导的BV2细胞上清液中NO、IL-1β、IL-6和TNF-α的水平增高,抑制LPS诱导的BV2细胞中炎症蛋白TLR4和MyD88的表达。在小鼠脑皮层组织中,XXM显著抑制LPS诱导的小胶质细胞活化,降低LPS诱导的炎症因子和趋化因子IL-1β、IL-6、TNF-α和MCP-1的水平增高,抑制LPS诱导的TLR4和MyD88蛋白的表达。结果表明,XXM可下调TLR4/MYD88信号通路来减轻LPS诱导的神经炎症反应。
In the present study, we aimed to investigate the effects of Xiao-Xu-Ming decoction extract(XXM) on lipopolysaccaride(LPS)-induced neuroinflammation in vitro and in vivo. In vitro, the microglia BV2 cells were treated with 200 ng/mL LPS for 24 h to induce inflammatory responses. In vivo, mice were treated with 5 mg/kg LPS to induce inflammatory responses. The NO level was determined by Griess Reagents. The levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by ELISA. The expressions of Iba-1, TLR4 and MyD88 at the protein levels were determined by Western blotting analysis. The mRNA levels of TLR4 and MyD88 were determined by real-time PCR. In vitro, XXM significantly reduced the levels of various pro-inflammatory factors, including NO, IL-1β, IL-6 and TNF-α, induced by LPS in the supernatant of BV2 cells and suppressed expressions of inflammatory proteins TLR4 and MyD88 induced by LPS in BV2 cells. In vivo, XXM significantly inhibited microglia activation, attenuated LPS-induced inflammatory factors and chemokine production, such as IL-1β, IL-6, TNF-α and MCP-1, and inhibited the expressions of inflammatory proteins including TLR4 and MyD88, in the cortex of LPS-induced mice. Our findings suggested that XXM could attenuate LPS-induced neuroinflammation via down-regulating TLR4/MyD88 signaling pathway.
In the present study, we aimed to investigate the effects of Xiao-Xu-Ming decoction extract(XXM) on lipopolysaccaride(LPS)-induced neuroinflammation in vitro and in vivo. In vitro, the microglia BV2 cells were treated with 200 ng/mL LPS for 24 h to induce inflammatory responses. In vivo, mice were treated with 5 mg/kg LPS to induce inflammatory responses. The NO level was determined by Griess Reagents. The levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by ELISA. The expressions of Iba-1, TLR4 and MyD88 at the protein levels were determined by Western blotting analysis. The mRNA levels of TLR4 and MyD88 were determined by real-time PCR. In vitro, XXM significantly reduced the levels of various pro-inflammatory factors, including NO, IL-1β, IL-6 and TNF-α, induced by LPS in the supernatant of BV2 cells and suppressed expressions of inflammatory proteins TLR4 and MyD88 induced by LPS in BV2 cells. In vivo, XXM significantly inhibited microglia activation, attenuated LPS-induced inflammatory factors and chemokine production, such as IL-1β, IL-6, TNF-α and MCP-1, and inhibited the expressions of inflammatory proteins including TLR4 and MyD88, in the cortex of LPS-induced mice. Our findings suggested that XXM could attenuate LPS-induced neuroinflammation via down-regulating TLR4/MyD88 signaling pathway.
引文
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[2]Zhang,Y.;Zheng,D.P.;Shui,M.Y.;Liu,Y.;Liu,X.Y.,Wang,Y.Y.A new compound W026B alleviates ischemic brain injury through inhibiting the production of inflammatory cytokines in pMCAO and tMCAO,and enhances the beneficial effect of tPA.J.Chin.Pharm.Sci.2018,27,675-685.
[3]Griffiths,M.;Neal,J.W.;Gasque,P.Innate immunity and protective neuroinflammation:new emphasis on the role of neuroimmune regulatory proteins.Int.Rev.Neurobiol.2007,82,29-55.
[4]Kreutzberg,G.W.Microglia:a sensor for pathological events in the CNS.Trends Neurosci.1996,19,312-318.
[5]Dheen,S.T.;Kaur,C.;Ling,E.A.Microglial activation and its implications in the brain diseases.Curr.Med.Chem.2007,14,1189-1197.
[6]Yao,H.;Hu,C.S.;Yin,L.H.;Tao,X.F.;Xu,L.N.;Qi,Y.;Han,X.;Xu,Y.W.;Zhao,Y.Y.;Wang,C.Y.;Peng,J.Y.Dioscin reduces lipopolysaccharide-induced inflammatory liver injury via regulating TLR4/MyD88 signal pathway.Int.Immunopharmacol.2016,36,132-141.
[7]Sun,M.Y.;Deng,B.;Zhao,X.Y.;Gao,C.J.;Yang,L.;Zhao,H.;Yu,D.H.;Zhang,F.;Xu,L.X.;Chen,L.;Sun,X.D.Isoflurane preconditioning provides neuroprotection against stroke by regulating the expression of the TLR4signalling pathway to alleviate microglial activation.Sci.Rep.2015,5,11445.
[8]Wang,S.;Wang,H.;Guo,H.;Kang,L.;Gao,X.;Hu,L.Neuroprotection of Scutellarin is mediated by inhibition of microglial inflammatory activation.Neuroscience.2011,185,150-160.
[9]Kim,S.K.;Kim,H.J.;Choi,S.E.;Park,K.H.;Choi,H.K.;Lee,M.W.Anti-oxidative and inhibitory activities on nitric oxide(NO)and prostaglandin E2(COX-2)production of flavonoids from seeds of Prunus tomentosa Thunberg.Arch.Pharm.Res.2008,31,424-428.
[10]Zhou,H.;Lapointe,B.M.;Clark,S.R.;Zbytnuik,L.;Kubes,P.A requirement for microglial TLR4 in leukocyte recruitment into brain in response to lipopolysaccharide.J.Immunol.2006,177,8103-8110.
[11]Lee,J.C.;Cho,G.S.;Kim,H.J.;Lim,J.H.;Oh,Y.K.;Nam,W.;Chung,J.H.;Kim,W.K.Accelerated cerebral ischemic injury by activated macrophages/microglia after lipopolysaccharide microinjection into rat corpus callosum.Glia.2005,50,168-181.
[12]Shih,R.H.;Wang,C.N.;Yang,C.M.NF-kappa B signaling pathways in neurological inflammation:A mini review.Front.Mol.Neurosci.2015,8,77.
[13]Zhou,X.J.;Sun,X.B.;Gong,X.X.;Yang,Y.;Chen,C.B.;Shan,G.;Yao,Q.S.Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-кB in vivo and in vitro.Int.Immunopharmacol.2017,42,18-24.
[14]Walker,D.G.;Lue,L.F.Immune phenotypes of microglia in human neurodegenerative disease:challenges to detecting microglial polarization in human brains.Alzheimers Res.Ther.2015,7,56.
[15]Patra,M.C.;Choi,S.Recent Progress in the molecular recognition and therapeutic importance of interleukin-1receptor-associated kinase 4.Molecules.2016,21,e1529.
[16]Wang,Y.L.;Ding,C.G.;Du,K.H.;Xiao,Y.;Wu,C.S.;Zhang,J.L.;Qin,H.L.;Du,G.H.Identification of active compounds and their metabolites by high-performance liquid chromatography/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry from Xiao-xu-ming decoction(XXMD).Rapid Commun.Mass Spectrom.2009,23,2724-2732.
[17]Wang,Y.H.;Zhang,H.X.;Li,Q.;Ding,Y.;Hu,J.J.;Du,G.H.High-throughput screening assay for groups of effective components extracted from Xiaoxuming Recipe.Zhong Xi Yi Jie He Xue Bao.2006,4,64-67.
[18]Wang,Y.H.;Qin,H.L.;He,X.L.;Du,G.H.Activity evaluation of components and preparation of effective components group of xiaoxuming decoction for anticerebral ischemic.Zhongguo Zhong Yao Za Zhi.2011,36,2140-2144.
[19]Wang,Y.H.;He,X.L.;Li,X.X.;Qin,H.L.;Du,G.H.Effects of the effective component group of Chinese herbal medicine Xiaoxuming Decoction on brain mitochondria in rats with chronic cerebral ischemia.Zhong Xi Yi Jie He Xue Bao.2012,10,569-576.
[20]Wang,Y.H.;He,X.L.;Yang,H.G.;Qin,H.L.;Du,G.H.Effects of the active components of Chinese herbal medicine Xiaoxuming Decoction on memory behavior and brain injury in rats with chronic cerebral ischemia.Zhong Xi Yi Jie He Xue Bao.2012,10,91-99.
[21]Yang,H.;Cheng,X.;Yang,Y.L.;Wang,Y.H.;Du,G.H.Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells.Neural.Regen.Res.2017,12,1860-1864.
[22]Bussi,C.;Peralta,Ramos,J.M.;Arroyo,D.S.;Gaviglio,E.A.;Gallea,J.I.;Wang,J.M.;Celej,M.S.;Iribarren,P.Autophagy down regulates pro-inflammatory mediators in BV2 microglial cells and rescues both LPS and alpha-synuclein induced neuronal cell death.Sci.Rep.2017,7,43153.
[23]Yang,H.;Cheng,X.;Yang,Y.L.;Wang,Y.H.;Du,G.H.Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/MyD88 signaling pathway in BV2 cells.Neural.Regen.Res.2017,12,1860-1864.
[24]Cheng,X.;Yang,Y.L.;Yang,H.;Wang,Y.H.;Du,G.H.Kaempferol alleviates LPS-induced neuroinflammation and BBB dysfunction in mice via inhibiting HMGB1release and down-regulating TLR4/MyD88 pathway.Int.Immunopharmacol.2018,56,29-35.
[25]Wang,C.H.;Wu,C.S.;Qin,H.L.;Zhang,J.L.Rapid discovery and identification of 68 compounds in the active fraction from Xiao-Xu-Ming decoction(XXMD)by HPLC-HRMS and MTSF technique.Chin.Chem.Lett.2014,25,1648-1652.
[26]Du,K.H.;Wu,C.S.;Ding,C.G.;Zhao,S.L.;Qin,H.L.;Zhang,J.L.Simultaneous LC-MS analysis and of wogonin and oroxylin A in rat plasma,and pharmacokinetic studies after administration of the active fraction from Xiao-Xu-Ming decoction.Chromatographia.2009,69,1259-1266.
[27]Wang,C.H.;Jia,Z.X.;Wang,Z.;Hu,T.;Qin,H.L.;Du,G.H.;Wu,C.S.;Zhang,J.L.Pharmacokinetics of 21active components in focal cerebral ischemic rats after oral administration of the active fraction of Xiao-Xu-Ming decoction.J.Pharm.Biomed.Anal.2016,122,110-117.
[28]Lan,R.;Xiang,J.;Wang,G.H.;Li,W.W.;Zhang,W.;Xu,L.L.;Cai,D.F.Xiao-Xu-Ming Decoction Protects against Blood-Brain Barrier Disruption and Neurological Injury Induced by Cerebral Ischemia and Reperfusion in Rats.Evid.Based Complement.Alternat.Med.2013,2013,629782.
[29]Lan,R.;Zhang,Y.;Xiang,J.;Zhang,W.;Wang,G.H.;Li,W.W.;Xu,L.L.;Cai,D.F.Xiao-Xu-Ming decoction preserves mitochondrial integrity and reduces apoptosis after focal cerebral ischemia and reperfusion via the mitochondrial p53 pathway.J.Ethnopharmacol.2014,151,307-316.
[30]Li,Y.;Lv,O.;Zhou,F.G.;Li,Q.S.;Wu,Z.C.;Zheng,Y.R.Linalool inhibits LPS-induced inflammation in BV2microglia cells by activating Nrf2.Neurochem.Res.2015,40,1520-1525.
[31]Chamorro,A.;Hallenbeck,J.The harms and benefits of inflammatory and immune responses in vascular disease.Stroke.2006,37,291-293.
[32]Kim,I.D.;Ha,B.J.The effects of paeoniflorin on LPS-induced liver inflammatory reactions.Arch.Pharm.Res.2010,33,959-966.
[33]Liu,B.;Du,L.;Hong,J.S.Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation.J.Pharmacol.Exp.Ther.2000,293,607-617.
[34]Sawada,M.;Kondo,N.;Suzumura,A.;Marunouchi,T.Production of tumor necrosis factor-alpha by microglia and astrocytes in culture.Brain Res.1989,491,394-397.
[35]Wang,S.X.;Jing,H.R.;Yang,H.Y.;Liu,Z.D.;Guo,H.;Chai,L.J.;Hu,L.M.Tanshinone I selectively suppresses pro-inflammatory genes expression in activated microglia and prevents nigrostriatal dopaminergic neurodegeneration in a mouse model of Parkinson’s disease.J.Ethnopharmacol.2015,164,247-255.
[36]Kim,H.;Bae,S.;Kwon,K.Y.;Hwang,Y.I.;Kang,J.S.;Lee,W.J.A combinational effect of acetaminophen and oriental herbs on the regulation of inflammatory mediators in microglia cell line,BV2.Anat.Cell Biol.2015,48,244-250.
[37]Moniruzzaman,M.;Lee,G.;Bose,S.;Choi,M.;Jung,J.K.;Lee,H.;Cho,J.Antioxidant and antiinflammatory activities of N-((3,4-Dihydro-2H-benzo[h]chromene-2-yl)methyl)-4-methoxyaniline in LPS-induced BV2 microglial cells.Biol.Pharm.Bull.2015,38,1831-1835.
[38]Henn,A.;Lund,S.;Hedtj?rn,M.;Schrattenholz,A.;P?rzgen,P.;Leist,M.The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation.ALTEX.2009,26,83-94.
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