肝细胞生长因子活化JAK2/STAT3通路促进巨噬细胞M1型向M2型极化
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摘要
目的研究肝细胞生长因子(HGF)通过Janus激酶2/信号转导和转录激活因子3(JAK2/STAT3)信号通路促进巨噬细胞M1型向M2型极化及其可能机制。方法以正常RAW264.7巨噬细胞为M0对照组;干扰素-γ(IFN-γ)和细菌脂多糖(LPS)诱导的RAW264.7巨噬细胞(M1型)为M1组;不同浓度HGF(5、10、20 ng/ml)干预巨噬细胞M1型为5M1组、10M1组、20M1组;用50μmol/L JAK2特异性阻断剂(AG490)与10 ng/ml HGF共同干预巨噬细胞M1型为AG490+10M1组。CCK8法检测HGF对巨噬细胞增殖的影响。应用Griess reagents试剂盒检测各组培养上清液中亚硝酸盐浓度(NO%)。Western blot法检测各组M1型标志物一氧化氮合酶(iNOS)、白细胞介素-6(IL-6)和M2型标志物精氨酸酶Ⅰ(ArgⅠ)、白细胞介素-10(IL-10)以及JAK2/STAT3信号通路JAK2、P-JAK2、STAT3、P-STAT3的蛋白表达。结果 HGF对巨噬细胞无明显增殖作用。Griess reagents试剂盒检测结果显示,与M0或HGF各干预组相比,M1组培养上清液亚硝酸盐浓度(NO%)明显增高;与M1组相比,HGF各干预组培养上清液亚硝酸盐浓度(NO%)明显降低,并呈剂量依赖性(P<0.05)。Western blot结果显示,与M0组相比,M1组iNOS、IL-6蛋白表达明显增加,ArgⅠ、IL-10蛋白表达显著减少(P<0.05)。与M1组相比,HGF各干预组iNOS、IL-6蛋白表达呈剂量依赖性减少,ArgⅠ、IL-10蛋白表达呈剂量依赖性增加(P<0.05)。与M0或M1组相比,HGF各干预组JAK2/STAT3通路蛋白磷酸化表达呈剂量依赖性增加(P<0.05)。与10M1组相比,AG490+10M1组M2表型相关蛋白ArgⅠ表达明显下降(P<0.05)。结论 HGF通过活化JAK2/STAT3信号通路促进巨噬细胞M1型向M2型极化。
Objective To investigate the effect of hepatocyte growth factor(HGF) on the polarization of macrophage from M1 to M2 via Janus kinase 2/signal transduction and activator of transcription 3(JAK2/STAT3) signaling pathway, and to investigate its possible mechanism behind the effect.Methods The normal RAW264.7 macrophages were used as M0 control group; RAW264.7 macrophages induced by LPS and IFN-γ(M1 phenotype) were M1 group; M1 phenotype macrophages interfered with different concentrations of HGF(5, 10, 20 ng/ml) were 5 M1 group, 10 M1 group, and 20 M1 group, respectively; M1 phenotype macrophages interfered with 50 μmol/L JAK2 specific blocker(AG490) and 10 ng/ml HGF were AG490+10 M1 group. The effect of HGF on the proliferation of macrophages was detected by CCK8 method. The nitrite concentration of culture supernatant(NO%) in each group was measured by the Griess reagents kit. Western blot was used to detect the protein expressions of marker for M1 group, including nitric oxide synthase(iNOS), interleukin-6(IL-6), and that for M2, including arginase Ⅰ(Arg Ⅰ), interleukin-10(IL-10).Theprotein expressions of JAK2, P-JAK2, STAT3, P-STAT3 involved in JAK2/STAT3 signaling pathway were also detected by using Western blot.Results HGF did not significantly inhibited the proliferation of macrophages. The results of the Griess reagents kit showed that the nitrite concentration of culture supernatant(NO%) in the M1 group was higher than that in the M0 or HGF intervention groups; the nitrite concentration of culture supernatant(NO%) in the HGF intervention group was decreased explicitly in a dose-dependent manner(P<0.05). Western blot analysis showed that, compared with M0 group, the protein expressions of iNOS and IL-6 were increased, while the protein expressions of Arg Ⅰ and IL-10 were decreased in M1 group(P<0.05).Compared with M1 group, the protein expressions of iNOS and IL-6 in HGF intervention group were decreased in a dose-dependent manner; the protein expressions of Arg Ⅰ and IL-10 were increased in a dose-dependent manner(P<0.05). Compared with M0 or M1 group, the phosphorylation of JAK2/STAT3 signaling pathway in HGF intervention group was increased in a dose-dependent manner(P<0.05).Compared with the 10 M1 group, the protein expression of M2 phenotype-associated Arg Ⅰ was decreased in the AG490+10 M1 group(P<0.05) after blocking the JAK2/STAT3 signaling pathway.Conclusion HGF promotes the polarization from M1 phenotype macrophage to M2 phenotype by activating JAK2/STAT3 signaling pathway.
Objective To investigate the effect of hepatocyte growth factor(HGF) on the polarization of macrophage from M1 to M2 via Janus kinase 2/signal transduction and activator of transcription 3(JAK2/STAT3) signaling pathway, and to investigate its possible mechanism behind the effect.Methods The normal RAW264.7 macrophages were used as M0 control group; RAW264.7 macrophages induced by LPS and IFN-γ(M1 phenotype) were M1 group; M1 phenotype macrophages interfered with different concentrations of HGF(5, 10, 20 ng/ml) were 5 M1 group, 10 M1 group, and 20 M1 group, respectively; M1 phenotype macrophages interfered with 50 μmol/L JAK2 specific blocker(AG490) and 10 ng/ml HGF were AG490+10 M1 group. The effect of HGF on the proliferation of macrophages was detected by CCK8 method. The nitrite concentration of culture supernatant(NO%) in each group was measured by the Griess reagents kit. Western blot was used to detect the protein expressions of marker for M1 group, including nitric oxide synthase(iNOS), interleukin-6(IL-6), and that for M2, including arginase Ⅰ(Arg Ⅰ), interleukin-10(IL-10).Theprotein expressions of JAK2, P-JAK2, STAT3, P-STAT3 involved in JAK2/STAT3 signaling pathway were also detected by using Western blot.Results HGF did not significantly inhibited the proliferation of macrophages. The results of the Griess reagents kit showed that the nitrite concentration of culture supernatant(NO%) in the M1 group was higher than that in the M0 or HGF intervention groups; the nitrite concentration of culture supernatant(NO%) in the HGF intervention group was decreased explicitly in a dose-dependent manner(P<0.05). Western blot analysis showed that, compared with M0 group, the protein expressions of iNOS and IL-6 were increased, while the protein expressions of Arg Ⅰ and IL-10 were decreased in M1 group(P<0.05).Compared with M1 group, the protein expressions of iNOS and IL-6 in HGF intervention group were decreased in a dose-dependent manner; the protein expressions of Arg Ⅰ and IL-10 were increased in a dose-dependent manner(P<0.05). Compared with M0 or M1 group, the phosphorylation of JAK2/STAT3 signaling pathway in HGF intervention group was increased in a dose-dependent manner(P<0.05).Compared with the 10 M1 group, the protein expression of M2 phenotype-associated Arg Ⅰ was decreased in the AG490+10 M1 group(P<0.05) after blocking the JAK2/STAT3 signaling pathway.Conclusion HGF promotes the polarization from M1 phenotype macrophage to M2 phenotype by activating JAK2/STAT3 signaling pathway.
引文
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[14] Yan D,Wang H W,Bowman R L,et al.STAT3 and STAT6 signaling pathways synergize to promote cathepsin secretion from macrophages via IRE1alphaactivation[J].Cell Rep,2016,16(11) :2914-27.
[15] Gentile A,Trusolino L,Comoglio P M.The Met tyrosine kina se receptor in development and cancer[J].Cancer Metastasis Rev,2008,27(1):85-94.
[2] Weber C,Noels H.Atherosclerosis:current pathogenesisand therapeutic options[J].Nat Med,2011,17(11):1410-22.
[3] Viiri L E,Full L E,Navin T J,et al.Smooth muscle cells inhuman atherosclerosis:proteomic profiling reveals differencesin expression of annexin A1 and mitochondrial proteinsin carotid disease[J].J Mol Cell Cardiol,2013,54:65-72.
[4] Murray P J,Allen J E,Biswas S K,et al.Macrophage activationand polarization:nomenclature and experimentalguidelines[J].Immunity,2014,41(1):14-20.
[5] 圣波,胡泽平,郭影,等.肝细胞生长因子对小鼠巨噬细胞M1、M2亚型极化的影响[J].安徽医科大学学报,2018,53(11):1725-30.
[6] Choi J W,Kwon M J,Kim I H,et al.Pyropia yezoensis glycoprotein promotes the M1 to M2 macrophage phenotypic switch via the STAT3 and STAT6 transcription factors[J].Int J Mol Med,2016,38(2):666-74.
[7] Yuan F,Fu X,Shi H,et al.Induction of murine macrophage M2 polarization by cigarette smoke extract via the JAK2/STAT3 pathway[J].PLoS One,2014,9(9):e107063.
[8] 陈涛,梁萧,贺明,等.RAW264.7细胞 M1、M2亚型的诱导和鉴定[J].中国分子心脏病杂志,2011,11(2):117-20.
[9] Qin H,Holdbrooks A T,Liu Y,et al.SOCS3 deficiency promotes M1 macrophage polarization and inflammation[J].J Immunol,2012,189(7):3439-48.
[10] Gallo S,Sala V,Gaetti S,et al.Cellular and molecular mechanisms of HGF/Met in the cardiovascular system[J].Clin Sci(Lond),2015,129:1173-93.
[11] Aggarwal B B,Kunnumakkara A B,Harikumar K B,et al.Signal transducer and activator of transcription-3,inflammation,and cancer:how intimate is the relationship[J].Ann N Y Acad Sci,2009,1171:59-76.
[12] Yang X O,Panopoulos A D,Nurieva R,et al.STAT3 regulates cytokine mediated generation of inflammatory helper T cells[J].J Biol Chem,2007,282(13):9358-63.
[13] Rodríguez M,Márquez S,de la Rosa J V,et al.Fungal pattern receptors down-regulate the inflammatory response by a cross-inhibitory mechanism independent of interleukin-10 production[J].Immunology,2017,150(2):184-98.
[14] Yan D,Wang H W,Bowman R L,et al.STAT3 and STAT6 signaling pathways synergize to promote cathepsin secretion from macrophages via IRE1alphaactivation[J].Cell Rep,2016,16(11) :2914-27.
[15] Gentile A,Trusolino L,Comoglio P M.The Met tyrosine kina se receptor in development and cancer[J].Cancer Metastasis Rev,2008,27(1):85-94.