摘要
[背景]二氧化硅(SiO_(2))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SiO_(2)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50μL生理盐水或50μL 50 mg·mL^(-1)的SiO_(2)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SiO_(2)前1 h加入TLR4抑制剂M62812进行预处理,后加入SiO_(2)(100μg·cm^(2))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kappa B P65(P-NF-κB P65)、磷酸化的核因子1kappa B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SiO_(2)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,100μg·cm^(2)SiO_(2)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SiO_(2)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SiO_(2)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SiO_(2)诱导的小鼠巨噬细胞炎症反应及向M1促炎表型转化。
[Background]The mechanisms of silicon dioxide(SiO_(2))-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 SiO_(2)stimulation.[Methods]Sixteen 6-to 8-week-old C57 BL/6 mice,half male and half female,were intratracheally instilled with 50μL of SiO_(2)(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 SiO_(2).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 SiO_(2)(100μg·cm^(2))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-1 NF-kappa-B inhibitorα(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-αin supernatants were determined by enzyme-linked immunosorbent assay(ELISA).[Results]After SiO_(2)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 100μg·cm^(2) SiO_(2)were observed in TLR4 and P-1κbαat 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 SiO_(2)-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 SiO_(2)challenge.
作者
蔡倩
王晶
马佳
马福成
刘迎雪
刘晓明
CAI Qian;WANG Jing;MAJia;MA Fucheng;LIU Yingxue;LIU Xiaoming(Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources of Western China,College of Life Science,Ningxia University,Yinchuan,Ningxia 750021,China;Key Laboratory of Environmental Factors and Chronic Disease Control,School of Public Health,Ningxia Medical University,Yinchuan,Ningxia 750004,China;Department of Pathology,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750004,China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2022年第1期71-77,共7页
Journal of Environmental and Occupational Medicine
基金
国家自然科学基金(31860318,32070863)
