Notch1信号激活核因子κB(NF-κB)参与小鼠RAW264.7巨噬细胞炎症介质释放
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摘要
目的观察Notch1信号在脂多糖(LPS)诱导巨噬细胞炎症介质表达中的作用并探讨其作用机制。方法给予100 ng/m L LPS处理小鼠RAW264.7细胞8 h,反转录PCR观察RAW264.7细胞Notch1和发状分裂相关增强子1(Hes1)mRNA表达;LPS处理前给予Notch1信号抑制剂10μmol/L(3,5-二氟苯乙酰基)-L-丙氨酰基-L-2-苯基甘氨酸叔丁酯(DAPT)预处理1 h,ELISA检测细胞培养液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6、一氧化氮(NO)和前列腺素E2(PGE2)含量,反转录PCR检测诱导型一氧化氮合酶(iNOS)和环加氧酶2(COX-2)mRNA水平,Western blot法检测iNOS、COX-2、核因子κBp65(NF-κBp65)、磷酸化的核因子κB抑制蛋白α(p-IκBα)蛋白水平。结果 LPS刺激RAW264.7细胞后明显提高细胞Notch1和Hes1 mRNA水平,DAPT明显抑制LPS诱导RAW264.7细胞TNF-α、IL-1β、IL-6、NO和PGE2的表达。DAPT阻断Notch1信号后明显降低LPS诱导RAW264.7细胞的iNOS和COX-2 mRNA和蛋白水平,DAPT能够抑制LPS诱导的IκBα磷酸化和NF-κBp65核转位。结论 Notch1通过激活NF-κB参与LPS诱导巨噬细胞炎症介质释放。
Objective To study the effect of Notch1 signaling on the release of inflammatory mediators in lipopolysaccharide(LPS)-induced macrophages and the related mechanism. Methods The expressions of Notch1 and hairy and enhancer of split 1( Hes1) mRNAs were investigated by reverse transcription PCR( RT-PCR) in mouse RAW264. 7 cells after stimulated with 100 ng/m L LPS for 8 hours. Prior to stimulation with LPS,mouse RAW264. 7 cells were treated with DAPT(10 μmol/L),an inhibitor of Notch1 signaling,for 1 hour. The concentrations of tumor necrosis factor( TNF-α),interleukin 1β( IL-1β),IL-6,nitric oxide( NO) and prostaglandin E2(PGE2) in cell culture media were measured by ELISA. The mRNA levels of nitric oxide synthase( i NOS) and cyclooxygenase-2( COX-2) were examined by RT-PCR. The protein levels of i NOS,COX-2,nuclear factor kappa Bp65( NF-κBp65) and phosphorylated nuclear factor κB inhibitor α( p-IκBα) were detected by Western blotting. Results The expressions of Notch1 and Hes1 mRNAs significantly increased in mouse RAW264. 7 cells after stimulated with LPS. The levels of TNF-α,IL-1β,IL-6,NO and PGE2 were significantly up-regulated in cell culture media after stimulated with LPS, but the levels of those inflammatory mediators were reduced by DAPT. The mRNA and protein levels of i NOS and COX-2 were significant raised in mouse RAW264. 7 cells after stimulated with LPS,while they were inhibited by DAPT. Both IκBα-phosphorylation and NF-κBp65 translocation into nuclear in LPS-induced RAW264. 7 cells were also inhibited by DAPT. Conclusion Notch1 signaling activates NF-κB to participate in LPS-induced inflammatory mediator release in macrophages.
Objective To study the effect of Notch1 signaling on the release of inflammatory mediators in lipopolysaccharide(LPS)-induced macrophages and the related mechanism. Methods The expressions of Notch1 and hairy and enhancer of split 1( Hes1) mRNAs were investigated by reverse transcription PCR( RT-PCR) in mouse RAW264. 7 cells after stimulated with 100 ng/m L LPS for 8 hours. Prior to stimulation with LPS,mouse RAW264. 7 cells were treated with DAPT(10 μmol/L),an inhibitor of Notch1 signaling,for 1 hour. The concentrations of tumor necrosis factor( TNF-α),interleukin 1β( IL-1β),IL-6,nitric oxide( NO) and prostaglandin E2(PGE2) in cell culture media were measured by ELISA. The mRNA levels of nitric oxide synthase( i NOS) and cyclooxygenase-2( COX-2) were examined by RT-PCR. The protein levels of i NOS,COX-2,nuclear factor kappa Bp65( NF-κBp65) and phosphorylated nuclear factor κB inhibitor α( p-IκBα) were detected by Western blotting. Results The expressions of Notch1 and Hes1 mRNAs significantly increased in mouse RAW264. 7 cells after stimulated with LPS. The levels of TNF-α,IL-1β,IL-6,NO and PGE2 were significantly up-regulated in cell culture media after stimulated with LPS, but the levels of those inflammatory mediators were reduced by DAPT. The mRNA and protein levels of i NOS and COX-2 were significant raised in mouse RAW264. 7 cells after stimulated with LPS,while they were inhibited by DAPT. Both IκBα-phosphorylation and NF-κBp65 translocation into nuclear in LPS-induced RAW264. 7 cells were also inhibited by DAPT. Conclusion Notch1 signaling activates NF-κB to participate in LPS-induced inflammatory mediator release in macrophages.
引文
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[16]Foldi J,Chung A Y,Xu H,et al.Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jagged1[J].J Immunol,2010,185(9):5023-5031.
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[18]Cheng P,Wang T,Li W,et al.Baicalin alleviates lipopolysaccharideinduced liver inflammation in chicken by suppressing TLR4-mediated NF-κB pathway[J/OL].Front Pharmacol,2017,18,8:547.doi:10.3389/fphar.2017.00547.e Collection 2017.
[19]Wu J T,Yang G W,Qi C H,et al.Anti-inflammatory activity of platycodin D on alcohol-induced fatty liver rats via TLR4-My D88-NF-κBsignal path[J].Afr J Tradit Complement Altern Med,2016,13(4):176-183.
[20]Phan N T,Cabot P J,Wallwork B D,et al.Cellular and molecular mechanisms of chronic rhinosinusitis and potential therapeutic strategies:review on cytokines,nuclear factor kappa B and transforming growth factor beta[J].J Laryngol Otol,2015,129(Suppl 3):S2-S7.
[21]Lawrence T.The nuclear factor NF-kappa B pathway in inflammation[J/OL].Cold Spring Harb Perspect Biol,2009,1(6):a001651.doi:10.1101/cshperspect.a001651.Epub 2009 Oct 7.
[22]Durand J K,Baldwin A S.Targeting IKK and NF-κB for therapy[J].Adv Protein Chem Struct Biol,2017,107:77-115.
[23]Brady G,Haas D A,Farrell P J,et al.Molluscum contagiosum virus protein MC005 inhibits NF-κB activation by targeting NEMO-regulated IκB kinase activation[J].J Virol,2017,91(15).pii:e00545-17.doi:10.1128/JVI.00545-17.Print 2017 Aug 1.
[24]Napetschnig J,Wu H.Molecular basis of NF-κB signaling[J].Annu Rev Biophys,2013,42:443-468.
[25]Rumzhum N N,Ammit A J.Cyclooxygenase 2:its regulation,role and impact in airway inflammation[J].Clin Exp Allergy,2016,46(3):397-410.
[26]Sakthivel K M,Guruvayoorappan C.Acacia ferruginea inhibits inflammation by regulating inflammatory i NOS and COX-2[J].J Immunotoxicol,2016,13(1):127-135.
[27]Yamada H,Kikuchi S,Inui T,et al.Gentiolactone,a secoiridoiddilactone from Gentianatriflora,inhibits TNF-α,i NOS and Cox-2 mRNAexpression and blocks NF-κB promoter activity in murine macrophages[J/OL].PLo S One,2014,9(11):e113834.doi:10.1371/journal.pone.0113834.e Collection 2014.
[28]Zhao F,Chen L,Zhang M,et al.Inhibition of lipopolysaccharideinduced i NOS and COX-2 expression by indole alkaloid,3-(hydroxymethyl)-6,7-dihydroindolo[2,3-a]quinolizin-(12H)-one,via NF-κB inactivation in RAW264.7 macrophages[J].Planta Med,2013,79(9):782-787.
[2]Yoshimura Y,Barua A.Female reproductive system and immunology[J].Adv Exp Med Biol,2017,1001:33-57.
[3]Cheng L,Zhang Z,Li G,et al.Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system[J].Vaccine,2017,35(45):6143-6153.
[4]Gr9sch S,Niederberger E,Geisslinger G.Investigational drugs targeting the prostaglandin E2 signaling pathway for the treatment of inflammatory pain[J].Expert Opin Investig Drugs,2017,26(1):51-61.
[5]Rose-John S.The soluble interleukin 6 receptor:advanced therapeutic options in inflammation[J].Clin Pharmacol Ther,2017,102(4):591-598.
[6]Lind M,Hayes A,Caprnda M,et al.Inducible nitric oxide synthase:Good or bad?[J].Biomed Pharmacother,2017,93:370-375.
[7]Borthwick L A.The IL-1 cytokine family and its role in inflammation and fibrosis in the lung[J].Semin Immunopathol,2016,38(4):517-534.
[8]Maillard I,Fang T,Pear W S.Regulation of lymphoid development,differentiation,and function by the Notch pathway[J].Annu Rev Immunol,2005,23:945-974.
[9]高春辰,秦鸿雁,韩骅.Notch信号调控巨噬细胞激活分子机制的研究进展[J].细胞与分子免疫学杂志,2014,30(1):108-111.
[10]丰帆,徐光辉,同李平,等.Notch信号与树突状细胞发育分化及功能调控[J].细胞与分子免疫学杂志,2012,28(11):1214-1216.
[11]Palaga T,Buranaruk C,Rengpipat S,et al.Notch signalling is activated by TLR stimulation and regulates macrophage functions[J].Eur J Immunol,2008,38(1):174-183.
[12]Okamoto M,Takeda K,Lucas J J,et al.Low-dose lipopolysaccharide affects lung allergic responses by regulating Jagged1 expression on antigen-pulsed dendritic cells[J].Int Arch Allergy Immunol,2012,157(1):65-72.
[13]González M J,Ruiz-García A,Monsalve E M,et al.DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR-activated murine macrophages[J].Eur J Immunol,2015,45(9):2615-2627.
[14]Zhang Q,Wang C,Liu Z,et al.Notch signal suppresses Toll-like receptor-triggered inflammatory responses in macrophages by inhibiting extracellular signal-regulated kinase 1/2-mediated nuclear factorκB activation[J].J Biol Chem,2012,24,287(9):6208-6217.
[15]Tsao P N,Wei S C,Huang M T,et al.Lipopolysaccharide-induced Notch signalling activation through JNK-dependent pathway regulates inflammatory response[J].J Biomed Sci,2011,18(1):56.
[16]Foldi J,Chung A Y,Xu H,et al.Autoamplification of Notch signaling in macrophages by TLR-induced and RBP-J-dependent induction of Jagged1[J].J Immunol,2010,185(9):5023-5031.
[17]Cao Q,Lu J,Kaur C,et al.Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells[J].Glia,2008,56(11):1224-1237.
[18]Cheng P,Wang T,Li W,et al.Baicalin alleviates lipopolysaccharideinduced liver inflammation in chicken by suppressing TLR4-mediated NF-κB pathway[J/OL].Front Pharmacol,2017,18,8:547.doi:10.3389/fphar.2017.00547.e Collection 2017.
[19]Wu J T,Yang G W,Qi C H,et al.Anti-inflammatory activity of platycodin D on alcohol-induced fatty liver rats via TLR4-My D88-NF-κBsignal path[J].Afr J Tradit Complement Altern Med,2016,13(4):176-183.
[20]Phan N T,Cabot P J,Wallwork B D,et al.Cellular and molecular mechanisms of chronic rhinosinusitis and potential therapeutic strategies:review on cytokines,nuclear factor kappa B and transforming growth factor beta[J].J Laryngol Otol,2015,129(Suppl 3):S2-S7.
[21]Lawrence T.The nuclear factor NF-kappa B pathway in inflammation[J/OL].Cold Spring Harb Perspect Biol,2009,1(6):a001651.doi:10.1101/cshperspect.a001651.Epub 2009 Oct 7.
[22]Durand J K,Baldwin A S.Targeting IKK and NF-κB for therapy[J].Adv Protein Chem Struct Biol,2017,107:77-115.
[23]Brady G,Haas D A,Farrell P J,et al.Molluscum contagiosum virus protein MC005 inhibits NF-κB activation by targeting NEMO-regulated IκB kinase activation[J].J Virol,2017,91(15).pii:e00545-17.doi:10.1128/JVI.00545-17.Print 2017 Aug 1.
[24]Napetschnig J,Wu H.Molecular basis of NF-κB signaling[J].Annu Rev Biophys,2013,42:443-468.
[25]Rumzhum N N,Ammit A J.Cyclooxygenase 2:its regulation,role and impact in airway inflammation[J].Clin Exp Allergy,2016,46(3):397-410.
[26]Sakthivel K M,Guruvayoorappan C.Acacia ferruginea inhibits inflammation by regulating inflammatory i NOS and COX-2[J].J Immunotoxicol,2016,13(1):127-135.
[27]Yamada H,Kikuchi S,Inui T,et al.Gentiolactone,a secoiridoiddilactone from Gentianatriflora,inhibits TNF-α,i NOS and Cox-2 mRNAexpression and blocks NF-κB promoter activity in murine macrophages[J/OL].PLo S One,2014,9(11):e113834.doi:10.1371/journal.pone.0113834.e Collection 2014.
[28]Zhao F,Chen L,Zhang M,et al.Inhibition of lipopolysaccharideinduced i NOS and COX-2 expression by indole alkaloid,3-(hydroxymethyl)-6,7-dihydroindolo[2,3-a]quinolizin-(12H)-one,via NF-κB inactivation in RAW264.7 macrophages[J].Planta Med,2013,79(9):782-787.