蔡倩, 王晶, 马佳, 马福成, 刘迎雪, 刘晓明. 抑制TLR4/NF-κB通路逆转SiO2诱导的小鼠巨噬细胞炎症反应及M1促炎表型转化[J]. 环境与职业医学, 2022, 39(1): 71-77. DOI: 10.11836/JEOM21184
引用本文: 蔡倩, 王晶, 马佳, 马福成, 刘迎雪, 刘晓明. 抑制TLR4/NF-κB通路逆转SiO2诱导的小鼠巨噬细胞炎症反应及M1促炎表型转化[J]. 环境与职业医学, 2022, 39(1): 71-77. DOI: 10.11836/JEOM21184
CAI Qian, WANG Jing, MA Jia, MA Fucheng, LIU Yingxue, LIU Xiaoming. Reduction of inflammation response and transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge by inhibition of TLR4[J]. Journal of Environmental and Occupational Medicine, 2022, 39(1): 71-77. DOI: 10.11836/JEOM21184
Citation: CAI Qian, WANG Jing, MA Jia, MA Fucheng, LIU Yingxue, LIU Xiaoming. Reduction of inflammation response and transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge by inhibition of TLR4[J]. Journal of Environmental and Occupational Medicine, 2022, 39(1): 71-77. DOI: 10.11836/JEOM21184

抑制TLR4/NF-κB通路逆转SiO2诱导的小鼠巨噬细胞炎症反应及M1促炎表型转化

Reduction of inflammation response and transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge by inhibition of TLR4

  • 摘要: 背景 二氧化硅(SiO2)粉尘可引起肺泡巨噬细胞中的炎症事件和细胞损伤,但具体作用机制不明。

    目的 探究抑制TLR4/NF-κB信号通路在SiO2诱导的小鼠巨噬细胞炎症反应及巨噬细胞表型改变中的作用。

    方法 16只6~8周龄的C57BL/6小鼠(雌雄各半)经50 µL生理盐水或50 µL 50 mg·mL−1的SiO2肺内灌注14、28 d后处死,苏木精伊红染色(HE)对肺组织进行病理学观察,采用免疫印迹法(WB)及免疫荧光法(IF)检测肺组织TLR4信号相关蛋白Toll样受体4(TLR4)、髓样分化因子88(Myd88)和肿瘤坏死因子受体相关因子6(TRAF6)表达。小鼠巨噬细胞系Raw264.7细胞在暴露于SiO2前1 h加入TLR4抑制剂M62812进行预处理,后加入SiO2(100 μg·cm2)协同刺激12 h,采用WB和IF检测TLR4介导的炎性信号关键蛋白TLR4、Myd88、磷酸化的核因子kappaB P65(P-NF-κB P65)、磷酸化的核因子1kappaB抑制蛋白α(P-1κbα)、肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)以及巨噬细胞M1表型标志蛋白一氧化氮合酶(iNOS)、分化抗原决定簇(CD86)和M2表型标志精氨酸酶-1(Arg-1)的表达。酶联免疫吸附实验(ELISA)检测在小鼠Raw264.7细胞中抑制TLR4后细胞培养液上清中炎症因子TNF-α、IL-6的含量。

    结果 小鼠气道灌注SiO2后,HE染色结果显示第14天矽肺小鼠肺组织局部可见明显的肺纤维化结节。WB结果显示,与对照组相比,矽肺小鼠肺组织中TLR4信号相关蛋白TLR4、Myd88和TRAF6表达水平上调(P<0.05)。IF结果显示,与对照组相比,矽肺小鼠肺组织中TLR4和Myd88荧光强度增强,提示TLR4信号激活。体外实验显示与对照组相比,100 µg·cm2 SiO2处理小鼠Raw264.7细胞不同时间(6、12、24、48 h),TLR4、Myd88、P-NF-κB P65和P-1κbα的表达上调,其中TLR4和P-1κbα在6、12及24 h刺激组的上调有统计学意义(P<0.05),Myd88在12、24 h刺激组的上调有统计学意义(P<0.05),P-NF-κB P65在12 h刺激组的上调有统计学意义(P<0.05)。采用抑制剂抑制TLR4表达后,可明显减弱SiO2诱导的TLR4及相关转导通路Myd88、TRAF6、P-NF-κB P65、TNF-α和IL-6表达,同时下调SiO2暴露导致的巨噬细胞M1表型标志iNOS的表达,上调M2表型标志物Arg-1的表达。

    结论 抑制TLR4/NF-κB信号通路可以逆转SiO2诱导的小鼠巨噬细胞炎症反应及向M1促炎表型转化。

     

    Abstract: Background The mechanisms of silicon dioxide (SiO2)-induced inflammation and cell injury in pulmonary macrophages are not fully characterized.

    Objective To investigate the potential roles of inhibition of toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling in inflammation and macrophage polarization in mouse Raw264.7 cells in response to SiO2 stimulation.

    Methods Sixteen 6- to 8-week-old C57BL/6 mice, half male and half female, were intratracheally instilled with 50 µL of SiO2 (50 mg·mL−1 in saline) or normal saline via oropharyngeal route, and the lungs of mice were harvested at 14 d and 28 d post the first challenge of SiO2. HE staining of mouse lung was used for histopathological analysis. The expressions of TLR4 signaling-related proteins were detected by Western blotting (WB) and immunofluorescent (IF) assay, including TLR4, myeloid differentiation factor 88 (Myd88), and TNF receptor associated factor 6 (TRAF6). Raw264.7 cells were stimulated with SiO2 (100 μg·cm2) for 12 h in absence or presence of TLR4 inhibitor M62812 for 13 h before the culture supernatants and cell lysates were harvested for analysis. The expressions of key components of TLR4 signaling cascade including TLR4, Myd88, and phosphorylated nuclear factor-kappa B P65 (P-NF-κB P65), P-1NF-kappa-B inhibitor α (P-1κbα), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), M1 phenotype markers inducible nitric oxide synthase (iNOS) and cluster of differentiation 86 (CD86), as well as M2 phenotype arginase-1 (Arg-1) were accessed by WB and IF. The expressions of inflammation factors IL-6 and TNF-α in supernatants were determined by enzyme-linked immunosorbent assay (ELISA).

    Results After SiO2 intratracheal instillation for 14 d, the HE staining results showed obvious fibrotic nodules in the lung tissues of mice. The results of WB analysis revealed more abundant TLR4, Myd88, and TRAF6 in the silicosis mouse lung samples than in the controls. The results of IF assay showed an increased abundance of TLR4 and Myd88 proteins in the lung samples of silicosis mice at 14 d post the silica challenge, compared to the controls, indicating TLR4 signaling activation. As seen in the in vitro experiment, significant upregulations after the exposure to 100 μg·cm2 SiO2 were observed in TLR4 and P-1κbα at 6, 12, and 24 h (P<0.05); Myd88 at 12 and 24 h (P <0.05); and P-NF-κB P65 at 12 h ( P<0.05). The inhibitor significantly suppressed the expressions of TLR4, Myd88, TRAF6, P-NF-κB P65, TNF-α, and IL-6 in Raw264.7 cells. In addition, the SiO2-induced M1 phenotype marker iNOS was significantly suppressed, but the M2 phenotype marker Arg-1 was increased in the Raw264.7 cells.

    Conclusion The inhibition of TLR4/NF-κB signaling could result in a reduction of the inflammation response and the transition of M1 toward M2 phenotypes of macrophages in response to SiO2 challenge.

     

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