血管活性肠肽对RAW264.7小鼠巨噬细胞TREM-1功能的影响
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摘要
目的观察血管活性肠肽(VIP)对RAW264.7小鼠巨噬细胞髓样细胞表达的触发受体-1(TREM-1)功能的影响。方法以小鼠巨噬细胞系RAW264.7为研究对象,细胞接种于培养板后采用TREM-1的单克隆激动抗体激活TREM-1,VIP预处理组采用10 nM的VIP进行干预。1 h后采用Western blot法检测胞内PLC-γ磷酸化改变,采用DCFH-DA荧光探针检测胞内活性氧簇(ROS)的含量;6 h后,采用real-time PCR法检测巨噬细胞TREM-1受体激活后下游靶基因肿瘤坏死因子-α(TNF-α)和单核细胞趋化蛋白-1(MCP-1)的基因表达;12 h后,采用ELISA法检测细胞培养上清液TNF-α和MCP-1的蛋白含量。结果采用TREM-1单克隆激动抗体处理小鼠巨噬细胞后,可显著增高胞内PLC-γ磷酸化水平、ROS含量,以及TNF-α和MCP-1的mRNA与蛋白水平,而VIP预处理可明显抑制单克隆激动抗体所诱导的上述反应。结论 VIP可抑制RAW264.7小鼠巨噬细胞TREM-1激活后诱导的功能改变,从而发挥抗氧化抗炎的作用。
Objective To observe the effect of vasoactive intestinal peptide(VIP) on the function of triggering receptor expressed on myeloid cells-1(TREM-1) in mouse macrophages. Methods The mouse macrophage cell line RAW264.7 was used as the research object. After cells were seeded on the culture plates,TREM-1 was activated by the monoclonal agonistic antibody of TREM-1, and cells in the VIP pretreatment group was treated with 10 nM VIP. After 1 h, the intracellular PLC-γ phosphorylation and intracellular reactive oxygen species(ROS) were detected by Western blot and DCFH-DA, respectively. After 6 h, realtime PCR was used to detect the gene expression of tumor necrosis factor-α(TNF-α) and monocyte chemoattractant protein-1(MCP-1) as the downstream target genes of TREM-1 activation. After 12 h, TNF-αand MCP-1 contentration in the cell culture supernatants were detected by ELISA. Results After treatment of TREM-1 monoclonal agonistic antibody, the intracellular PLC-γ phosphorylation level, ROS content, and mRNA and protein levels of TNF-α and MCP-1 were significantly increased in murine macrophages, while VIP pretreatment significantly inhibited the above reactions induced by monoclonal agonistic antibodies.Conclusion VIP can inhibit the functional changes induced by the activation of TREM-1 in murine macrophages RAW264.7, thereby exerting anti-oxidant and anti-inflammatory effects.
Objective To observe the effect of vasoactive intestinal peptide(VIP) on the function of triggering receptor expressed on myeloid cells-1(TREM-1) in mouse macrophages. Methods The mouse macrophage cell line RAW264.7 was used as the research object. After cells were seeded on the culture plates,TREM-1 was activated by the monoclonal agonistic antibody of TREM-1, and cells in the VIP pretreatment group was treated with 10 nM VIP. After 1 h, the intracellular PLC-γ phosphorylation and intracellular reactive oxygen species(ROS) were detected by Western blot and DCFH-DA, respectively. After 6 h, realtime PCR was used to detect the gene expression of tumor necrosis factor-α(TNF-α) and monocyte chemoattractant protein-1(MCP-1) as the downstream target genes of TREM-1 activation. After 12 h, TNF-αand MCP-1 contentration in the cell culture supernatants were detected by ELISA. Results After treatment of TREM-1 monoclonal agonistic antibody, the intracellular PLC-γ phosphorylation level, ROS content, and mRNA and protein levels of TNF-α and MCP-1 were significantly increased in murine macrophages, while VIP pretreatment significantly inhibited the above reactions induced by monoclonal agonistic antibodies.Conclusion VIP can inhibit the functional changes induced by the activation of TREM-1 in murine macrophages RAW264.7, thereby exerting anti-oxidant and anti-inflammatory effects.
引文
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[16]Solanki A,Bhatt LK,Johnston TP.Evolving targets for the treatment of atherosclerosis[J].Pharmacol Ther,2018,187:1-12.
[17]Tammaro A,Derive M,Gibot S,et al.TREM-1 and its potential ligands in non-infectious diseases:from biology to clinical perspectives[J].Pharmacol Ther,2017,177:81-95.
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[19]Juarranz MG,Santiago B,Torroba M,et al.Vasoactive intestinal peptide modulates proinflammatory mediator synthesis in osteoarthritic and rheumatoid synovial cells[J].Rheumatology(Oxford),2004,43(4):416-422.
[20]Calo G,Sabbione F,Vota D,et al.Trophoblast cells inhibit neutrophil extracellular trap formation and enhance apoptosis through vasoactive intestinal peptide-mediated pathways[J].Hum Reprod,2017,32(1):55-64.
[21]Zhao SJ,Wang DH,Li YW,et al.A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects[J].Int J Nanomedicine,2017,12:2143-2160.
[22]Athari SS,Pourpak Z,Folkerts G,et al.Conjugated Alpha-Alumina nanoparticle with vasoactive intestinal peptide as a Nano-drug in treatment of allergic asthma in mice[J].Eur J Pharmacol,2016,791:811-820.
[2]Bouchon A,Dietrich J,Colonna M.Cutting edge:inflammatory responses can be triggered by TREM-1,a novel receptor expressed on neutrophils and monocytes[J].J Immunol,2000,164(10):4991-4995.
[3]Wang X,Luo B,Lu Y,et al.The triggering receptor expressed by myeloid cells-1 activates TLR4-My D88-NF-kappa B-dependent signaling to aggravate ventilation-induced lung inflammation and injury in mice[J].Cell Tissue Res,2018,374(1):137-148.
[4]Bouchon A,Facchetti F,Weigand MA,et al.TREM-1 amplifies inflammation and is a crucial mediator of septic shock[J].Nature,2001,410(6832):1103-1107.
[5]Liu T,Zhou Y,Li P,et al.Blocking triggering receptor expressed on myeloid cells-1 attenuates lipopolysaccharide-induced acute lung injury via inhibiting NLRP3 inflammasome activation[J].Sci Rep,2016,6:39473.
[6]Croteau-Chonka DC,Raby BA.TREM-1 response signatures common to expression profiles of both asthma affection and asthma control[J].Am J Respir Crit Care Med,2018,198(3):401-404.
[7]宋卓慧,孙国瑛,周勇,等.脂多糖对小鼠巨噬细胞TREM-1表达的影响[J].长治医学院学报,2012,26(2):81-83.
[8]Fu L,Han L,Xie C,et al.Identification of extracellular actin as a ligand for triggering receptor expressed on myeloid cells-1 signaling[J].Front Immunol,2017,8:917.
[9]Zhu J,Duan G,Wang H,et al.TREM-1 activation modulates ds RNAinduced antiviral immunity with specific enhancement of MAPKsignaling and the RLRs and TLRs on macrophages[J].Exp Cell Res,2016,345(1):70-81.
[10]Ramhorst R,Calo G,Paparini D,et al.Control of the inflammatory response during pregnancy:potential role of VIP as a regulatory peptide[J].Ann N Y Acad Sci,2018.doi:10.1111/nyas.13632.
[11]Verma AK,Manohar M,Upparahalli Venkateshaiah S,et al.Neuroendocrine cells derived chemokine vasoactive intestinal polypeptide(VIP)in allergic diseases[J].Cytokine Growth Factor Rev,2017,38:37-48.
[12]Sun GY,Yang HH,Guan XX,et al.Vasoactive intestinal peptide overexpression mediated by lentivirus attenuates lipopolysaccharideinduced acute lung injury in mice by inhibiting inflammation[J].Mol Immunol,2018,97:8-15.
[13]Sun GY,Guan CX,Zhou Y,et al.Vasoactive intestinal peptide rebalances TREM-1/TREM-2 ratio in acute lung injury[J].Regul Pept,2011,167(1):56-64.
[14]宋卓慧.血管活性肠肽对急性肺损伤时肺内TREM-1表达的影响及其机制[D].中南大学,2012.
[15]骆凤,郑浩翔,董良,等.地塞米松对急性肺损伤中TREM-1表达的影响[J].中国临床解剖学杂志,2016,34(5):523-527.
[16]Solanki A,Bhatt LK,Johnston TP.Evolving targets for the treatment of atherosclerosis[J].Pharmacol Ther,2018,187:1-12.
[17]Tammaro A,Derive M,Gibot S,et al.TREM-1 and its potential ligands in non-infectious diseases:from biology to clinical perspectives[J].Pharmacol Ther,2017,177:81-95.
[18]Ran WZ,Dong L,Tang CY,et al.Vasoactive intestinal peptide suppresses macrophage-mediated inflammation by downregulating interleukin-17A expression via PKA-and PKC-dependent pathways[J].Int J Exp Pathol,2015,96(4):269-275.
[19]Juarranz MG,Santiago B,Torroba M,et al.Vasoactive intestinal peptide modulates proinflammatory mediator synthesis in osteoarthritic and rheumatoid synovial cells[J].Rheumatology(Oxford),2004,43(4):416-422.
[20]Calo G,Sabbione F,Vota D,et al.Trophoblast cells inhibit neutrophil extracellular trap formation and enhance apoptosis through vasoactive intestinal peptide-mediated pathways[J].Hum Reprod,2017,32(1):55-64.
[21]Zhao SJ,Wang DH,Li YW,et al.A novel selective VPAC2 agonist peptide-conjugated chitosan modified selenium nanoparticles with enhanced anti-type 2 diabetes synergy effects[J].Int J Nanomedicine,2017,12:2143-2160.
[22]Athari SS,Pourpak Z,Folkerts G,et al.Conjugated Alpha-Alumina nanoparticle with vasoactive intestinal peptide as a Nano-drug in treatment of allergic asthma in mice[J].Eur J Pharmacol,2016,791:811-820.