目的 初步探讨LM49(2,4′,5′-三羟基-5,2′-二溴二苯甲酮)对脂多糖(LPS)联合干扰素γ(IFN-γ)诱导的小鼠单核巨噬细胞(RAW264.7)M1/M2极化的影响及其调控机制。方法 四甲基偶氮唑蓝(MTT)法测定LM49对细胞活力的影响;流式细胞术、实时荧光定量聚合酶链反应(PCR)和Western-blot法测定LM49(5,10,20 μmol·L-1)与LPS/INF-γ共同作用于RAW264.7细胞后,巨噬细胞亚型标志物的表达情况及对核因子(NF)-κB和JAK/STAT信号通路的影响。结果 与LPS/INF-γ造模组相比,LM49显著抑制CD16/32+细胞数及诱导型一氧化氮合酶(iNOS)、白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α mRNA的表达,升高CD206+细胞数及Arg-1和IL-10 mRNA的表达,且降低巨噬细胞M1/M2的比值;Western-blot法验证LM49可显著降低TLR4、Myd88、NF-κB和STAT1蛋白的表达量,同时抑制p-JAK2和p-STAT1蛋白磷酸化水平。结论 LM49通过抑制TLR4-Myd88-NF-κB和JAK2-STAT1信号通路,抑制巨噬细胞M1型极化及促进巨噬细胞M2型极化,调节巨噬细胞M1/M2的平衡。
Abstract
OBJECTIVE To investigate the effect of LM49(2,4′, 5′-trihydroxy-5,2′-dibromobenzophenone) on M1/M2 phenotype in RAW264.7 macrophages by LPS plus INF-γ and its regulation mechanism on M1/M2 polarization. METHODS MTT assay was used to determine the effect of LM49 on cell viability. Different concentrations of LM49 (5, 10, 20 μmol·L-1) were used to intervene in LPS combined with IFN-γ induced macrophages, the expression of macrophage subsets of markers and the effect on JAK/STAT of the signaling pathway were examined by flow cytometry, RT-PCR and Western-blot. RESULTS Compared with LPS/INF-γ group, LM49 significantly inhibited the number of CD16/32+ cells and the mRNA expression of iNOS, IL-6 and TNF-α, increased the number of CD206+ cells and the mRNA expression of Arg-1 and IL-10, and decreased the ratio of M1/M2 in macrophage. It was demonstrated that LM49 significantly reduced the proteins expression levels of TLR4, Myd88, NF-κB and STAT1, while inhibiting the phosphorylation levels of p-JAK2 and p-STAT1 by Western-blot. CONCLUSION The study demonstrates that LM49 inhibits M1 polarization and promotes M2 polarization in RAW264.7 macrophages via inhibiting TLR4-Myd88-NF-κB and JAK2-STAT1 pathway, regulating the balance of M1/M2 ratio in macrophages.
关键词
LM49 /
巨噬细胞极化 /
炎症 /
核因子-κB /
JAK/STAT
{{custom_keyword}} /
Key words
LM49 /
macrophage polarization /
inflammation /
NF-κB /
JAK/STAT
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] FLORENT G, STEFFEN J. Monocytes and macrophages:developmental pathways and tissue homeostasis[J]. Nat Rev Immunol, 2014, 14(6):392-404.
[2] MURRAY P J, WYNN T A. Protective and pathogenic functions of macrophage subsets[J]. Nat Rev Immunol, 2011, 11(11):723-737.
[3] LIN H, CAO Y L, DING J X. Effect of saposhnikovan sulphate on the recruitment and re-education of macrophages by MCF-7 cells in vitro through CCL3[J]. Chin Pharm J (中国药学杂志), 2018, 53(20):1755-1761.
[4] VARIN A, GORDON S. Alternative activation of macrophages: immune function and cellular biology [J]. Immunobiology, 2009, 214(7):630-641.
[5] NATHAN C F, MURRAY H W, WIEBE M E. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity [J]. J Exp Med, 1983, 158(3):670.
[6] OLEFSKY J M, GLASS C K. Macrophages, inflammation, and insulin resistance [J]. Annu Rev Physiol, 2010, 72(1):219-246.
[7] HUANG N Q, JIN H, SHI J S, et al. Research progress of resveratrol inhibiting microglial activation in neuroinflammation [J]. Chin Pharm J (中国药学杂志), 2018,53(2):85-91.
[8] MOGHADDAM A S, MOHAMMADIAN S, VAZINI H, et al. Macrophage plasticity, polarization and function in health and disease[J]. J Cell Physiol, 2018, 233(9):6425-6440.
[9] LIU Y C, ZOU X B, CHAI Y F, et al. Macrophage polarization in inflammatory diseases[J]. Int J Biol Sci, 2014, 10(5):520-529.
[10] El SHOUBAKY G A, ABDEL-DAIM M M, MANSOUR M H, et al. Isolation and identification of a flavone apigenin from marine red alga acanthophora spicifera with antinociceptive and anti-inflammatory activities[J]. J Exp Neurosci, 2016, 10(1):21-29.
[11] OLSEN E K, HANSEN E, ISAKSSON J, et al. Cellular antioxidant effect of four bromophenols from the red algae, vertebrata lanosa[J]. Mar Drugs, 2013, 11(8):2769-2784.
[12] ZHAO W Y, FENG X E, BAN S R, et al. Synthesis and biological activity of halophenols as potent antioxidant and cytoprotective agents[J]. Bioorg Med Chem Lett, 2010, 20(14):4132-4134.
[13] FENG X E, LIANG T G, GAO J, et al. Heme Oxygenase-1, a key enzyme for the cytoprotective actions of halophenols by upregulating Nrf2 expression via activating Erk1/2 and PI3K/Akt in EA.hy926 cells[J]. Oxid Med Cell Longev, 2017, 2017(1):7028478.
[14] ZHANG Y L, FENG X E, CHANG R R, et al. Therapeutic effects of 5,2′-dibromo-2,4′,5′-trihydroxydiphenylmethanone (LM49) in an experimental rat model of acute pyelonephritis by immunomodulation and anti-inflammation[J]. Int Immunopharmacol, 2018, 62:155-164.
[15] HUANG J, ZHANG Y, YAN J, et al. Immunomodulatory effect of dendrobium officinale polysaccharide on macrophage RAW264. 7 induced by LPS [J]. Chin Pharm J(中国药学杂志), 2017, 52(7):33-37.
[16] MURRAY P J, ALLEN J E, BISWAS S K, et al. Macrophage activation and polarization:nomenclature and experimental guidelines [J]. Immunity, 2014, 41:14-20.
[17] LAWRENCE T, NATOLI G. Transcriptional regulation of macrophage polarization:enabling diversity with identity [J]. Immunol, 2011(11):750-761.
[18] SZELAG M, PIASZYK-BORYCHOWSK A, PLENS-GALASKAl M, et al. Targeted inhibition of STATs and IRFs as a potential treatment strategy in cardiovascular disease[J]. Oncotarget, 2016, 7(30):48788-48812.
[19] OECKINGHAUS A, HAYDEN M S, GHOSH S. Crosstalk in NF-κB signaling pathways[J]. Nat Immunol, 2011, 12(8):695-708.
[20] JUSTIN I O, ROBERTO R R-G, MATTHEW H G, et al. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance[J]. Nature, 2007, 447(7148):1116-1120.
[21] KRAUSGRUBER T, BLAZEK K, SMALLIE T, et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses[J]. Nat Immunol, 2011, 12(3):231-238.
[22] ZHENG X, WANG H Y. M2 macrophage polarization and the related diseases[J]. Chin Bull Life Sci(生命科学), 2017, 29(9):883-890.
[23] ZHU L N, ZHAO Q J, YANG T, et al. Cellular metabolism and macrophage functional polarization[J]. Int Rev Immunol, 2015, 34(1):82-100.
[24] RUAN J Y,CHEN B C, ZHANG X L, et al. Huang HJ: progress in signaling pathways of macrophage M1/2 polarization[J]. Immunol J(免疫学杂志), 2015(10):911-917.
[25] LUO J R, ZHAO B, TANG L, et al. Inhibitory effect of compound W3D on LPS-induced release of inflammatory mediators of RAW264. 7 cells through TLR4-MyD88-NF-κB signaling pathway [J]. Chin Pharmacol Bull(中国药理学通报), 2018, 34(7):977-982.
[26] DAVID S A,CURT M H. A road map for those who don′t know JAK-STAT[J]. Science, 2002, 296(5573):1653-1655.
[27] YANG H, YANG Y L, CHENG X, et al. Effects and underlying mechanism of extract of scutellaria baicalensis georgi against LPS-induced inflammation in BV2 cells [J]. Chin Pharm J (中国药学杂志), 2018, 53(4):268-272.
[28] FENG L L, SUN Y, SONG P P, et al. Seselin ameliorates inflammation via targeting Jak2 to suppress the proinflammatory phenotype of macrophage[J]. Br J Pharmacol, 2018, 176(2):DOI:10.1111/bph.14521.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
创新药物国家重大科技专项资助(2018ZX09711001-001-017);国家自然科学基金项目资助(81473100);山西省重点研究开发计划资助(重点项目)(201703D111033)
{{custom_fund}}
PDF(4520 KB)
