TREM-1促进巨噬细胞的迁移并参与脂多糖诱导的小鼠心肌功能障碍

TREM-1 promotes macrophage migration and participates in lipopolysaccharide-induced myocardial dysfunction in mice

目的探讨髓系细胞触发受体-l(triggering receptor expressed oil myeloid cells-1,TREM-1)对脂多糖(lipopolysaccharide,LPS)诱导的小鼠心肌功能障碍的作用以及对巨噬细胞RAW264.7迁移的影响。方法将健康C57BL/6J雄性小鼠随机分为对照组、LPS组、LPS+LP17组、LPS+TAK-242组并进行相应处理,6 h后超声心电图检测小鼠心功能指标,12 h取心肌组织HE染色观察病理改变,免疫组化染色观察巨噬细胞表面分子F4/80阳性表达,ELISA检测血清中炎症因子TNF-α、IL-6、IL-1β及IFN-γ的表达情况,实时荧光定量PCR检测TREM-1、BNP以及炎症因子TNF-α、IL-6、IL-1β、IFN-γ的mRNA水平;培养巨噬细胞RAW264.7,将细胞随机分为正常组、LPS组、LP17+LPS组、TAK-242+LPS组并进行对应处理,实时荧光定量PCR检测TREM-1与TLR4的mRNA水平,ELISA检测细胞培养液中炎症因子TNF-α、IL-6、IL-1β及IFN-γ的表达情况,Transwell小室观测RAW264.7细胞迁移情况,蛋白免疫印迹检测RAW264.7中TLR4/NF-κB信号通路相关蛋白水平。结果与对照组相比,超声心电图检测结果显示LPS组小鼠的EF与FS下降,LVD增大,IVS减小,LVPW变薄,变化均显著(P<0.05),实时荧光定量PCR结果显示TREM-1 mRNA与BNP mRNA表达水平上升(P<0.05),免疫组化染色法显示F4/80有大量阳性表达(P<0.01),ELISA检测显示炎症因子TNF-α、IL-6、IL-1β、IFN-γ的含量增加(P<0.05),实时荧光定量PCR检测也表明这些炎性因子mRNA水平升高(P<0.05);与LPS组小鼠相比,注射TREM-1抑制剂LP17使EF、FS上升,有效抑制了LVD增大、IVS减小以及LVPW变薄的现象(P<0.05),TREM-1 mRNA与BNP mRNA表达水平均降低(P<0.05),心肌损伤减轻,F4/80阳性表达减少(P<0.01),炎症因子水平也明显下降(P<0.05),与注射TLR4抑制剂TAK-242的效果一致。与对照组相比,LPS诱导RAW264.7细胞后,细胞中TREM-1 mRNA与TLR4 mRNA水平升高(P<0.05),炎症因子TNF-α、IL-6、IL-1β、IFN-γ的含量增加(P<0.05),细胞大量迁移(P<0.01),细胞中TLR4、MyD88、NF-κBp65、p-IκBα蛋白水平升高(P<0.05);与LPS组相比,TREM-1抑制剂LP17预处理后再给予LPS诱导,细胞中TREM-1 mRNA与TLR4 mRNA水平降低(P<0.05),炎症因子的含量下降(P<0.05),RAW264.7细胞迁移被抑制(P<0.01),TLR4、MyD88、NF-κBp65、p-IκBα蛋白水平也明显降低(P<0.05)。结论给小鼠注射TREM-1抑制剂可有效减轻LPS诱导的心肌功能障碍,抑制RAW264.7细胞TREM-1表达可抑制细胞的迁移,TLR4/NF-κB途径可能参与了这一过程。

To investigate the effect of triggering receptor expressed oil myeloid cells-1(TREM-1) on macrophage RAW264.7 migration and myocardial dysfunction induced by lipopolysaccharide(LPS) in mice, healthy C57 BL/6 J male mice were recruited and randomly divided into control group, LPS group, LPS+LP17 group, LPS+TAK-242 group and treated accordingly. Six hours after the corresponding treatment, echocardiography was used to detect cardiac function indicators in mice;myocardial tissue was taken for HE staining at 12 h to observe pathological changes. Immunohistochemical staining was used to observe the positive expression of F4/80, while ELISA to detect the expression of inflammatory factors TNF-α, IL-6, IL-1β and IFN-γ in serum. Real-time fluorescence quantitative PCR was used to detect mRNA levels of TREM-1, BNP and inflammatory factors TNF-α, IL-6, IL-1β, IFN-γ. The macrophage RAW264.7 was cultured and the cells were randomly divided into normal group, LPS group, LP17+LPS group, TAK-242+LPS group and treated accordingly. Real-time PCR was applied to detect the mRNA levels of TREM-1 and TLR4, while ELISA to detect the expression of inflammatory factors TNF-α, IL-6, IL-1β and IFN-γ in cell culture fluid. Transwell was employed to observe the migration of RAW264.7 cells;Western blot was used to detect the levels of TLR4/NF-κB signaling pathway-related proteins in RAW264.7. The results of echocardiography showed that the EF and FS of mice in the LPS group decreased, LVD increased, IVS decreased, LVPW became thinner(P<0.05), as compared with the control group;real-time quantitative PCR results showed TREM-1 mRNA and BNP mRNA expression levels increased(P<0.05), immunohistochemical staining showed there was much positive expression of F4/80(P<0.01), and ELISA showed the levels of inflammatory factors TNF-α, IL-6, IL-1β and IFN-γwere increased(P<0.05), while real-time quantitative PCR detection also showed that the mRNA levels of these inflammatory factors were increased(P<0.05). Injection of the TREM-1 inhibitor LP17 could reverse the changes in mice of LPS group, which was consistent with the effect of TAK-242 injection. Compared with the control group, the levels of TREM-1 mRNA and TLR4 mRNA in LRAW264.7 cells induced by LPS were increased(P<0.05), the levels of inflammatory factors TNF-α, IL-6, IL-1β, IFN-γ were increased(P<0.05), cells migration were increased(P<0.01), and the levels of TLR4, MyD88, NF-κBp65, and p-IκBα proteins were increased in cells(P<0.05).Pretreatment of LP17 could reverse these changes in RAW264.7 cells induced by LPS. Taken together, TREM-1 inhibitor can effectively alleviate LPS-induced myocardial injury, and inhibit cell migration of RAW264.7 cells, in which the TLR4/NF-κB pathway may play roles.