法舒地尔(Fasudil)抑制脂多糖诱导的小鼠星形胶质细胞活化和炎性反应及其机制
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摘要
目的探讨法舒地尔(Fasudil)对脂多糖(LPS)诱导的星形胶质细胞活化和炎症反应及Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路的影响。方法体外培养新生C57BL/6小鼠大脑皮质星形胶质细胞,细胞分为PBS对照组、1μg/m L LPS刺激组、1μg/m L LPS联合15μg/m L盐酸法舒地尔处理组,Griess法检测培养细胞上清液一氧化氮(NO)的水平,ELISA检测肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-10和IL-4的水平,免疫荧光细胞化学染色检测星形胶质细胞胶质原纤维酸性蛋白(GFAP)及TLR4的表达,Western blot法检测GFAP、TLR4和磷酸化的NF-κBp65(p-NF-κBp65)蛋白水平。结果与PBS组比较,LPS组NO、TNF-α和IL-6水平显著升高,IL-10和IL-4水平降低;法舒地尔能抑制LPS诱导的NO、TNF-α和IL-6的分泌,增加IL-10和IL-4的分泌。法舒地尔处理组星形胶质细胞GFAP表达显著降低,同时TLR4和NF-κB蛋白的水平也降低。结论法舒地尔阻断TLR4/NF-κB信号通路抑制LPS诱导的星形胶质细胞活化及炎性反应。
Objective To evaluate the influence of Fasudil on the lipopolysaccharide( LPS)-induced activation and inflammatory response of astrocytes and TLR4/NF-κB signaling pathway. Methods Astrocytes were separated from the cerebral cortex of newly-born C57 BL/6 mice and cultured in vitro. The cells were divided into PBS control group,1 μg/m L LPS stimulation group,1 μg/m L LPS combined with 15 μg/m L Fasudil treatment group. The production of NO in the cell supernatant was detected by Griess reagent. The levels of tumor necrosis factor-α( TNF-α),interleukin-6( IL-6),IL-10 and IL-4 were measured by ELISA. The expressions of glial fibrillary acidic protein( GFAP) and TLR4 in the astrocytes were detected by immunofluorescence cytochemistry. The protein levels of GFAP,TLR4 and p-NF-κBp65 were assessed by Western blot analysis. Results Compared with the PBS control group,the levels of NO,TNF-α and IL-6 in the LPS group significantly increased,but the levels of IL-10 and IL-4 decreased. Fasudil treatment significantly inhibited LPS-induced the secretion of NO,TNF-α and IL-6 and enhanced the production of IL-10 and IL-4. The expression of GFAP significantly decreased in the Fasudil treatment group,and the protein levels of TLR4 and p-NF-κBp65 also decreased. Conclusion Fasudil can inhibit LPS-induced astrocyte activation and inflammatory response by blocking TLR4/NF-κB signaling pathway.
Objective To evaluate the influence of Fasudil on the lipopolysaccharide( LPS)-induced activation and inflammatory response of astrocytes and TLR4/NF-κB signaling pathway. Methods Astrocytes were separated from the cerebral cortex of newly-born C57 BL/6 mice and cultured in vitro. The cells were divided into PBS control group,1 μg/m L LPS stimulation group,1 μg/m L LPS combined with 15 μg/m L Fasudil treatment group. The production of NO in the cell supernatant was detected by Griess reagent. The levels of tumor necrosis factor-α( TNF-α),interleukin-6( IL-6),IL-10 and IL-4 were measured by ELISA. The expressions of glial fibrillary acidic protein( GFAP) and TLR4 in the astrocytes were detected by immunofluorescence cytochemistry. The protein levels of GFAP,TLR4 and p-NF-κBp65 were assessed by Western blot analysis. Results Compared with the PBS control group,the levels of NO,TNF-α and IL-6 in the LPS group significantly increased,but the levels of IL-10 and IL-4 decreased. Fasudil treatment significantly inhibited LPS-induced the secretion of NO,TNF-α and IL-6 and enhanced the production of IL-10 and IL-4. The expression of GFAP significantly decreased in the Fasudil treatment group,and the protein levels of TLR4 and p-NF-κBp65 also decreased. Conclusion Fasudil can inhibit LPS-induced astrocyte activation and inflammatory response by blocking TLR4/NF-κB signaling pathway.
引文
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[30]Gorina R,Font-Nieves M,Márquez-Kisinousky L,et al.Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between My D88-dependent NF-κB signaling,MAPK,and JAK1/STAT1 pathways[J].Glia,2011,59(2):242-255.
[2]Fakhoury M.Immune-mediated processes in neurodegeneration:where do we stand[J].J Neurol,2016,263(9):1683-1701.
[3]Wilhelmsson U,Bushong E A,Price D L,et al.Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury[J].Proc Natl Acad Sci U S A,2006,103(46):17513-17518.
[4]Faulkner J R,Herrmann J E,Woo M J,et al.Reactive astrocytes protect tissue and preserve function after spinal cord injury[J].J Neurosci,2004,24(9):2143-2155.
[5]Liddelow S A,Guttenplan K A,Clarke L E,et al.Neurotoxic reactive astrocytes are induced by activated microglia[J].Nature,2017,541(7638):481-487.
[6]Li N,Zhang X,Dong H,et al.Lithium ameliorates LPS-induced astrocytes activation partly via inhibition of Toll-like receptor 4expression[J].Cell Physiol Biochem,2016,38(2):714-725.
[7]Samy Z A,Al-Abdullah L,Turcani M,et al.Rat astrocytes during anoxia:Secretome profile of cytokines and chemokines[J/OL].Brain Behav,2018,8(7):e01013.DOI:10.1002/brb3.1013.Epub 2018 Jun 4.
[8]Guo M F,Meng J,Li Y H,et al.The inhibition of Rho kinase blocks cell migration and accumulation possibly by challenging inflammatory cytokines and chemokines on astrocytes[J].J Neurol Sci,2014,343(1/2):69-75.
[9]Okun E,Griffioen K J,Mattson M P.Toll-like receptor signaling in neural plasticity and disease[J].Trends Neurosci,2011,34(5):269-281.
[10]Zhang Z,Qin P,Deng Y,et al.The novel estrogenic receptor GPR30 alleviates ischemic injury by inhibiting TLR4-mediated microglial inflammation[J/OL].J Neuroinflammation,2018,15(1):206.DOI:10.1186/s12974-018-1246-x.
[11]Martorana F,Guidotti G,Brambilla L,et al.Withaferin A inhibits nuclear factor-κB-dependent pro-inflammatory and stress response pathways in the astrocytes[J/OL].Neural Plast,2015,2015:381964.DOI:10.1155/2015/381964.Epub 2015 Jul 21.
[12]Rosciszewski G,Cadena V,Murta V,et al.Toll-like receptor 4(TLR4)and triggering receptor expressed on myeloid cells-2(TREM-2)activation balance astrocyte polarization into a proinflammatory phenotype[J].Mol Neurobiol,2018,55(5):3875-3888.
[13]Li Y H,Xie C,Zhang Y,et al.FSD-C10,a Fasudil derivative,promotes neuroregeneration through indirect and direct mechanisms[J/OL].Sci Rep,2017,7:41227.DOI:10.1038/srep41227.
[14]尉杰忠,谷青芳,闫玉清,等.法舒地尔通过抑制脑内小胶质细胞活化并促进其M2极化而改善APP/PS1双转基因小鼠认知功能[J].细胞与分子免疫学杂志,2017,33(12):1585-1593.
[15]刘春云,郭尚德,张年萍,等.Fasudil对实验性自身免疫性脑脊髓炎小鼠致脑炎性T细胞的免疫调节作用[J].中南大学学报(医学版),2016,41(3):225-232.
[16]Ponath G,Park C,Pitt D.The role of astrocytes in multiple sclerosis[J/OL].Front Immunol,2018,9:217.DOI:10.3389/fimmu.2018.00217.e Collection 2018.
[17]González-Reyes R E,Nava-Mesa M O,Vargas-Sánchez K,et al.Involvement of astrocytes in Alzheimer's disease from a neuroinflammatory and oxidative stress perspective[J/OL].Front Mol Neurosci,2017,10:427.DOI:10.3389/fnmol.2017.00427.e Collection 2017.
[18]Costantini E,D’Angelo C,Reale M.The role of immunosenescence in neurodegenerative diseases[J/OL].Mediators Inflamm,2018,2018:6039171.DOI:10.1155/2018/6039171.e Collection 2018.
[19]Buffo A,Rolando C,Ceruti S.Astrocytes in the damaged brain:molecular and cellular insights into their reactive response and healing potential[J].Biochem Pharmacol,2010,79(2):77-89.
[20]Eng L F,Ghirnikar R S.GFAP and astrogliosis[J].Brain Pathol,1994,4(3):229-237.
[21]Deng Y T,Wang X S,Zhao M G,et al.Gentiopicroside protects neurons from astrocyte-mediated inflammatory injuries by inhibition of nuclear factor-κB and mitogen-activated protein kinase signaling pathways[J].Neuroreport,2018,29(13):1114-1120.
[22]孟健,郭敏芳,尉杰忠,等.硫辛酸抑制脂多糖诱导的星形胶质细胞细胞因子及趋化因子的表达[J].细胞与分子免疫学杂志,2014,30(4):346-350.
[23]Calabrese V,Mancuso C,Calvani M,et al.Nitric oxide in the central nervous system:neuroprotection versus neurotoxicity[J].Nat Rev Neurosci,2007,8(10):766-775.
[24]Miljkovic D,Momcilovic M,Stojanovic I,et al.Astrocytes stimulate interleukin-17 and interferon-gamma production in vitro[J].J Neurosci Res,2007,85(16):3598-3606.
[25]Zhang Y,Huang R,Zhang Y,et al.IL-17 induces MIP-1αexpression in primary mouse astrocytes via TRPC channel[J].Inflammopharmacology,2016,24(1):33-42.
[26]Qiu D,Li X N.Pioglitazone inhibits the secretion of proinflammatory cytokines and chemokines in astrocytes stimulated with lipopolysaccharide[J].Int J Clin Pharmacol Ther,2015,53(9):746-752.
[27]Brodie C,Goldreich N,Haiman T,et al.Functional IL-4 receptors onmouse astrocytes:IL-4 inhibits astrocyte activation and induces NGF secretion[J].J Neuroimmunol,1998,81(1/2):20-30.
[28]Pasare C,Medzhitov R.Toll-like receptors:Linking innate and adaptive immunity[J].Adv Exp Med Biol,2005,560:11-18.
[29]Marinelli C,Di Liddo R,Facci L,et al.Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes[J/OL].J Neuroinflammation,2015,12:244.DOI:10.1186/s12974-015-0458-6.
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