7 RAW α TNF-α -1β IL-1β -1 monocyte chemotactic protein MCP-1-2 macrophage DMEM GIBCO inflammatory protein 2 MIP-2 No C1345 3% No

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 740 DOI /j cnki cjbmb RAW264 7 * H 2 O 2 RAW264 7 H 2 O 2 RAW264 7 MTT PCR qrt-pcr ELISA RAW264 7 H 2 O 2 40 μmol /L RAW μmol /L 40 μmol /L H 2 O 2 RAW264 7 TNF-α IL-1β MCP-1 MIP U /ml H 2 O 2 TNF-α IL-1β MCP-1 MIP-2 H 2 O 2 -α -1β -1-2 Q291 Q786 R Hydrogen Peroxide Stimulates Expression of Cytokine and Chemokine Genes in Murine RAW264 7 Cells CUI Hai-Yan HAN Chen-Lu XIAO Peng LI Wei-Guo * WANG Kun-Ying College of Life Sciences Henan Normal University Xinxiang Henan China Abstract Hydrogen peroxide H 2 O 2 as a major form of reactive oxygen species ROS produced from cell respiratory burst functions as signal molecules in promoting the expression of pro-inflammatory cytokine /chemokine genes in macrophage We investigated H 2 O 2 stimulation on the expression of proinflammatory cytokine /chemokine genes in the murine RAW264 7 cells From the results of MTT with qrt-pcr and ELISA assays H 2 O 2 was showed to markedly increase Tnf-α Il-1β Mcp-1 and Mip-2 gene expression at 20 ~ 40 μmol / L without obvious cytotoxicity Catalase treatment at 200 U / ml reduced H 2 O 2 -stimulated expression of the up-mentioned gene These data suggested that H 2 O 2 was an important stimulator regulating the expression of pro-inflammatory cytokines / chemokines in macrophages Key words hydrogen peroxide tumor necrosis factor-α interleukin-1β monocyte chemotactic protein 1 macrophage inflammatory protein 2 macrophages ROS H 2 O 2 ROS No No 2010A * Tel liwg0618@ htu cn Received January Accepted April Supported by Key Program of Science and Technology of Henan Province 1 No Natural Science Basic Research Plan of Educational Bureau of Henan Province No 2010A * Corresponding author Tel liwg0618@ htu cn

2 7 RAW α TNF-α -1β IL-1β -1 monocyte chemotactic protein MCP-1-2 macrophage DMEM GIBCO inflammatory protein 2 MIP-2 No C1345 3% No TNF-α IL-1β Sigma-Aldrich 3-4 MCP-1 PCR Real SYBR Mixture with ROX MIP-2 - cdna Genecopoeia PCR 5-6 TNF-α IL-1β MCP-1 MIP-2 ELISA TNF-a IL-1β Long 7 3% H 2 O 2 TNF-α DMEM 100 μmol /L 1 mmol /L ROS Hsu 8 H 2 O 2 DMEM lipopolysaccharides LPS IL-1β ROS Mokgobu 9 - TNF-α IL-1β ROS 10% catalase CAT 100 U /ml 100 μg /ml DMEM GPx H 2 O % CO 2 TNF-a MTT ROS RAW /ml MCP μl / 96 MIP-2 TNF-a MCP μmol /L H LPS 2 O 2 DMEM 9 12 h 24 h 36 h 48 h MTT NR h 10 μl MTT RAW h DMSO 2 4- DNFB RAW Mip-2 15 ROS A 492 % MCP-1 MIP-2 % = 珔 A / 珔 A 492 ROS 100% 1 4 H 2 O 2 RAW264 7 RAW264 7 TNF-α IL-1β /ml MCP-1 MIP-2 3 ml / 6 RAW H 2 O 2 H 2 O μmol /L H 2 O 2 PCR qrt-pcr 200 ELISA H 2 O 2 U /ml 3 TNF-α IL-1β MCP-1 MIP RAW264 7 ATCC 150 μl 5 min H 2 O U /ml 30 min H 2 O 2

3 μmol /L μl dntp 25 mmol /L 1 μl RNase 25 U / 24 h 3 μl 1 μl M-MLV 200 U /μl 1 μl ELISA RNase-free 25 μl RNA qrt-pcr 1 5 RAW264 7 Tnf-α Il-1β Mcp-1 Mip-2 PCR RAW264 7 RNA qrt- PCR 10 μmol /L 0 5 μl cdna 1 PBS 2-3 Trizol μlrnase-free 20 μl qrt-pcr RNA 260 nm 280 nm 95 A RNA 10 min s s 40 RNA cdna RNA 1 μg Oligo Tnf-α Il-1β Mcp-1 Mip-2 β-actin PCR PCR Primer 5 0 Table 1 qrt-pcr 2 Real SYBR Mixture with ROX 10 μl ABI 7500 Real Time PCR Applied Biosystems USA dt μmol /L 1 μl 5 RT ct Table 1 qrt-pcr primer design for mouse Tnf-α Il-1β Mcp-1 Mip-2 and β-actin Primer Primer sequence Product length bp Tnf-α upper primer CTGAACTTCGGGGTGATCGGT Tnf-α lower primer TCCTCCACTTGGTGGTTTGCTAC 158 Il-1β upper primer CAGGCAGGCAGTATCACTCATTGT Il-1β lower primer AACGTCACACACCAGCAGGTTATC 175 Mcp-1 upper primer TCTGCCCTAAGGTCTTCAGCA Mcp-1 lower primer GCATCACAGTCCGAGTCACACTA 287 Mip-2 upper primer TGTCAATGCCTGAAGACCCTGCC Mip-2 lower primer AACTTTTTGACCGCCCTTGAGAGTGG 126 β-actin upper primer CAGGTCATCACTATTGGCAACGAG β-actin lower primer GATGCCACAGGATTCCATACCC RAW264 7 TNF-α IL-1β MCP-1 MIP-2 ELISA r /min 10 min TNF-α IL-1β MCP-1 MIP-2 ELISA 2 2 H 2 O 2 RAW264 7 A 450 TNF-α IL-1β MCP-1 MIP ± means ± qrt-pcr ELISA SD SPSS14 0 RAW ~ 40 μmol /L H 2 O 2 Excel P < h Tnf-α Il-1β Mcp-1 Mip-2 mrna P < 0 01 Fig 1 RAW μmol /L 160 μmol /L H 2 O 2 24 h RAW264 7 Fig 2 RAW μmol /L 2 H 2 O 2 Tnf-α Il-1β Mcp-1 Mip-2 mrna 2 1 H 2 O 2 RAW264 7 RAW264 7 MTT 0 ~ 160 μmol /L H 2 O 2 20 μmol /L H 2 O 2 Tnf-α Il-1β RAW264 7 Mcp-1 Mip-2 mrna 2 03 RAW264 7 H 2 O 2 40 μmol /L TNF-α IL-1β MCP-1 24 h MIP ng /L 163 ng /L 177 ng /L

4 7 RAW Fig 1 Effect of hydrogen peroxide H 2 O 2 on murine RAW264 7 cell viability The RAW264 7 cells were incubated with DMEM absence or presence H 2 O 2 0 ~ 160 μmol /L for 24 hours Cell viability were analyzed with MTT method * P < 0 05 and ** P < 0 01 compared to cells in control group TNF-α IL-1β MCP-1 MIP ng /L 307 ng /L 275 ng /L 133 ng /L H 2 O 2 RAW H 2 O 2 RAW264 7 TNF-α IL-1β qrt-pcr ELISA 200 U /ml RAW264 7 TNF-α IL-1β 40 μmol /L H 2 O 2 RAW264 7 TNF-α IL-1β RAW U /ml 30 min 40 μmol /L H 2 O 2 24 h 40 μmol /L H 2 O 2 TNF-α 63 3% 56% IL-1β 52% Fig 3 RAW264 7 TNF-α 87 ng /L IL-1β μmol /L H 2 O 2 TNF-α IL- H 2 O 2 RAW264 7 TNF-α 1β MCP-1 MIP-2 mrna 5 35 IL-1β 2 4 H 2 O 2 RAW264 7 MCP-1 MIP-2 qrt-pcr ELISA 200 U/mL RAW264 7 Fig 2 Hydrogen peroxide H 2 O 2 stimulates pro-inflammatory cytokine TNF-α and IL-1β and chemokines MCP-1 and MIP-2 expression in murine macrophage RAW264 7 The RAW264 7 cells were incubated with DMEM absence or presence H 2 O and 40 μmol /L for 24 hours The level of Tnf-α Il-1β Mcp-1 and Mip-2 mrna were tested with qrt-pcr The level of TNF-α IL-1β MCP-1 and MIP-2 were analyzed with ELISA ** P < 0 01 compared to unstimulated cells

5 Fig 3 Catalase reduces H 2 O 2 -stimulated the expression and production of cytokines TNF-α and IL-1β in murine macrophage RAW264 7 The RAW264 7 cells were incubated with 200 U /ml catalase CAT 40 μmol /L H 2 O 2 or coincubated with 200 U /ml catalase pretreatmentd for 30 min and 40 μmol /L H 2 O 2 for 24 hours respectively The mrna level of cytokines Tnf-α A and Il-1β C were analyzed with qrt-pcr The secretion of chemokines TNF-α B and IL-1β D were measured with ELISA ** P < 0 01 compared to control group cells ## P < 0 01 compared to H 2 O 2 stimulation group cells MCP-1 MIP-2 40 μmol /L H 2 O 2 H 2 O 2 RAW264 7 MCP-1 MIP-2 TNF-α IL-1β RAW U /ml 30 mins 40 μmol / L H 2 O 2 24 h 40 μmol /L H 2 O 2 ROS TNF-α IL- MCP-1 1β 45% 42% MIP-2 59% 31% Fig 4 NF-κB 17 H 2 O 2 RAW264 7 MCP-1 MIP-2 AP-1 NF-κB 18 H 2 O 2 RAW264 7 MCP-1 MIP-2 MCP-1 MIP-2 1 ROS TNF-α p38 JNK H 2 O 2 - TNF-α IL-1β NF-κB p38mapk 9 ROS 3 NF-κB TLR ROS TNF-α IL-1β MCP-1 MIP-2 H 2 O 2 ROS MCP-1 MIP-2 H 2 O 2 H 2 O 2 Mip-2 mrna

6 7 RAW Fig 4 Catalase reduces H 2 O 2 - stimulated the expression and production of chemokines MCP-1 and MIP-2 in murine macrophage RAW264 7 The RAW264 7 cells were incubated with 200 U /ml catalase CAT 40 μmol /L H 2 O 2 or coincubated with 200 U /ml catalase pretreatmentd for 30 min and 40 μmol /L H 2 O 2 for 24 hours respectively The mrna level of chemokines Mcp-1 A and Mip-2 C were analyzed with qrt-pcr The secretion of chemokines MCP-1 B and MIP-2 D were measured with ELISA ** P < 0 01 compared to control group cells ## P < 0 01 compared to H 2 O 2 stimulation group cells H 2 O 2 Mip-2 D 16 References H 2 O 2 Mcp-1 Mip-2 extracellular signal-regulated kinase ERK NF-κB AP-1 CREB 21 ROS RAW Mip-2 MAPK 15 H 2 O 2 view J RAW264 7 TNF-α IL-1β 4 Stow JL Murray RZ Mcp-1 Mip H 2 O 2 TNF-α IL-1β H 2 O 2 Mcp-1 1 Rhee SG Kang SW Jeong W et al Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins J Curr Opin Cell Biol Reuter S Gupta SC Chaturvedi MM et al Oxidative stress inflammation and cancer how are they linked J Free Radic Biol Med Schulte W Bernhagen J Bucala R Cytokines in sepsis potent immunoregulators and potential therapeutic targets-an updated Mediators Inflamm Intracellular trafficking and secretion of inflammatory cytokines J Cytokine Growth Factor Rev Deshmane SL Kremlev S Amini S et al Monocyte chemoattractant protein-1 MCP-1 an overview J J Interferon Cytokine Res Mip-2 MCP-1 MIP-2 CD49d-dependent manner J H 2 O Burdon PC Martin C Rankin SM The CXC chemokine MIP-2 stimulates neutrophil mobilization from the rat bone marrow in a Blood Long H Shi T Borm PJ et al ROS-mediated TNF-α and MIP- 2 gene expression in alveolar macrophages exposed to pine dust H 2 O 2

7 J Part Fibre Toxicol Hsu HY Wen MH Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression J J Biol Chem Mokgobu MI Cholo MC Anderson R et al Oxidative induction of pro-inflammatory cytokine formation by human monocytederived macrophages following exposure to manganese in vitro J J Immunotoxicol Carter AB Tephly LA Venkataraman S et al High levels of catalase and glutathione peroxidase activity dampen H 2 O 2 signaling in human alveolar macrophages J Am J Respir Cell Mol Biol Sato M Miyazaki T Nagaya T et al Antioxidants inhibit TNFa mediated stimulation of interleukin-8 monocyte chemoattractant protein-1 and collagenase expression in cultured human synovial cells J J Rheumatol Wang JC Zhao Y Chen SJ et al AOPPs induce MCP-1 expression by increasing ROS- mediated activation of the NF-κB pathway in rat mesangial cells inhibition by sesquiterpene lactones J Cell Physiol Biochem Kopydlowski KM Salkowski CA Cody MJ et al Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo J J Immunol Chong IW Lin SR Hwang JJ et al Expression and regulation of macrophage inflammatory protein-2 gene by vanadium in mouse macrophages J Inflammation Kim D Kim YJ Seo JN et al 2 4-Dinitrofluorobenzene modifies cellular proteins and induces macrophage inflammatory protein-2 gene expression via reactive oxygen species production in RAW cells J Immunol Invest Shi MM Chong I Godleski JJ et al Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages J Immunology Ndengele MM Muscoli C Wang ZQ et al Superoxide potentiates NF-kB activation and modulates endotoxin-induced cytokine production in alveolar macrophages J Shock Nakao N Kurokawa T Nonami T et al Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW macrophage cells via activation of p38 and stress-activated protein kinase J Innate Immun Asehnoune K Strassheim D Mitra S et al Involvement of reactive oxygen species in Toll- like receptor 4-dependent activation of NF-kappa B J J Immunol Lorne E Zmijewski JW Zhao X et al Role of extracellular superoxide in neutrophil activation interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production J Am J Physiol Cell Physiol C985-C Jaramillo M Olivier M Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signalregulated kinase- and cyclic adenosine 5'-monophosphate camp -dependent pathways involvement of NF-kappa B activator protein 1 and camp response element binding protein J J Immunol

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环球中医药 2016 年 5 月第 9 卷第 5 期摇 Global Traditional Chinese Medicine, May 2016,Vol 郾 9, No 郾 5 摇 557 两种电针对脊髓损伤 14 天后大鼠运动功能神经元及 MEK2 p 鄄 ERK1 表达的影响论著宋良玉摇吕威摇景泉凯摇莫雨