橙皮苷调控Jagged1/Notch1通路对巨噬细胞极化及细支气管炎小鼠肺损伤的影响

Hesperidin Regulates Jagged1/Notch1 Pathway to Promote Macrophage Polarization and Alleviate Lung Injury in Mice with Bronchiolitis

目的探究橙皮苷对呼吸道合胞病毒(RSV)细支气管炎模型小鼠肺损伤的影响及可能的作用机制。方法构建RSV细支气管炎小鼠模型,采用随机数字表法将60只BALB/c小鼠分为对照组、模型组、低剂量橙皮苷组(18 mg/kg)、高剂量橙皮苷组(36 mg/kg)、高剂量橙皮苷(36 mg/kg)+Jagged1(1 mg/kg)组,每组12只,给予相应剂量的药物进行干预,对照组和模型组给予等量生理盐水。收集小鼠支气管肺泡灌洗液(BALF),并分离肺泡巨噬细胞;ELISA检测BALF中白细胞介素(IL)-4、IL-6、肿瘤坏死因子α(TNF-α)、IL-10的水平;流式细胞术检测巨噬细胞M1/M2型极化;qRT-PCR、Western blot检测肺组织诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg-1)、Jagged1和Notch1的mRNA和蛋白表达水平。结果与对照组相比,模型组小鼠BALF中IL-4、IL-6、TNF-α、M1型巨噬细胞比例、肺组织炎症评分和黏液分泌评分以及iNOS、Jagged1、Notch1 mRNA和蛋白表达水平均显著升高(P均<0.001),而IL-10、M2型巨噬细胞比例、Arg-1 mRNA和蛋白表达水平显著降低(P均<0.001);与模型组相比,低、高剂量橙皮苷组小鼠BALF中IL-4、IL-6、TNF-α、M1型巨噬细胞比例、肺组织炎症评分和黏液分泌评分以及iNOS、Jagged1、Notch1 mRNA和蛋白表达水平显著降低(P均<0.05),而IL-10、M2型巨噬细胞比例、Arg-1 mRNA和蛋白表达水平显著升高(P均<0.001);使用重组Jagged1蛋白激活Notch1信号通路可显著减弱高剂量橙皮苷对M2型巨噬细胞极化的促进作用和对肺部炎症损伤的改善作用(P均<0.01)。结论橙皮苷可减轻RSV诱导的细支气管炎小鼠肺部炎症损伤,其作用机制可能与抑制Jagged1/Notch1通路,促进巨噬细胞M2型极化有关。

Objective To explore the effect and mechanism of hesperidin in treating the lung injury in the mouse model of respiratory syncytial virus(RSV)-induced bronchiolitis.Methods A mouse model of RSV-induced bronchiolitis was established,and 60 BALB/c mice were assigned into a control group,a model group,a low-dose hesperidin(18 mg/kg)group,a high-dose hesperidin(36 mg/kg)group,and a high-dose hesperidin(36 mg/kg)+Jagged1(1 mg/kg)group by random number table method,with 12 mice in each group.Corresponding doses of drugs were administrated for intervention,and the control group and model group were administrated with the same amount of saline.The bronchoalveolar lavage fluid(BALF)samples were collected and alveolar macrophages were isolated.ELISA was employed to detect the levels of interleukin(IL)-4,IL-6,tumor necrosis factor-α(TNF-α),and IL-10 in BALF,and flow cytometry to detect the M1/M2 polarization of macrophages.qRT-PCR and Western blotting were respectively conducted to detect the mRNA and protein levels of inducible nitric oxide synthase(iNOS),arginase 1(Arg-1),Jagged1,and Notch1 in the lung tissue.Results Compared with the control group,the modeling of RSV-induced bronchiolitis elevated the IL-4,IL-6,and TNF-αlevels,increased the proportion of M1-type macrophages and the lung inflammation and mucus secretion scores,and up-regulated the mRNA and protein levels of iNOS,Jagged1,and Notch1 in BALF(all P<0.001).Meanwhile,the modeling lowered the IL-10 level,decreased the proportion of M2-type macrophages,and down-regulated the mRNA and protein levels of Arg-1(all P<0.001).Compared with the model group,low-and high-dose hesperidin lowered the IL-4,IL-6,TNF-αlevels,decreased the proportion of M1-type macrophages and the lung inflammation and mucus secretion scores,and down-regulated the mRNA and protein levels of iNOS,Jagged1,and Notch1 in BALF(all P<0.05).Moreover,hesperidin elevated the IL-10 level,increased the proportion of M2-type macrophages,and up-regulated the mRNA and protein levels of Arg-1(all P<0.001).Using recombinant Jagged1 protein to activate Notch1 signaling pathway can significantly attenuate the promotion of high-dose hesperidin on M2 macrophage polarization and amelioration of lung inflammation damage(all P<0.01).Conclusion Hesperidin may alleviate the lung inflammation damage in mice with RSV-induced bronchiolitis by inhibiting the Jagged1/Notch1 signaling pathway and promoting the M2-type polarization of macrophages.