QC@mmSiO_(2)-NIPAM对矽肺纤维化病变中M1型巨噬细胞极化的调节作用

The effect of QC@mmSiO_(2)-NIPAM on the polarization of M1 macrophages in silicosis

目的 探讨负载槲皮素的核壳温敏型药物载体(QC@mmSiO_(2)-NIPAM)通过Toll样受体4(TLR4)/核因子κB(NF-κB)信号通路对矽肺纤维化形成过程中M1型巨噬细胞极化及炎症反应的作用与机制。方法 C57BL/6小鼠随机分为正常对照组、mSiO_(2)-NIPAM低剂量组(50mg/kg)、中剂量组(100mg/kg)、高剂量组(200mg/kg),每组5只小鼠,苏木素-伊红(HE)染色法观察不同浓度mmSiO_(2)-NIPAM溶液对小鼠各脏器的影响。C57BL/6小鼠随机分为正常对照组、矽肺模型组(SiO_(2)悬液100mg/kg)、QC@mmSiO_(2)-NIPAM组(SiO_(2)悬液100mg/kg,QC@mmSiO_(2)-NIPAM溶液100mg/kg),每组5只小鼠,HE及Van Gieson(VG)染色法观察各组小鼠肺组织的形态学变化,Western blot法及免疫荧光染色法检测I型胶原蛋白、M1型巨噬细胞标记蛋白以及TLR4/NF-κB信号通路蛋白的表达。结果 HE及VG染色结果显示,各浓度mmSiO_(2)-NIPAM溶液对小鼠各脏器均无影响,矽肺模型组小鼠肺组织有明显的矽结节形成,并可见粗大的胶原纤维分布,给予QC@mmSiO_(2)-NIPAM溶液灌胃治疗后,矽结节明显减少,且胶原沉积显著下降。Western blot结果显示,矽肺模型组小鼠肺组织内I型胶原蛋白、M1型巨噬细胞特异性标记蛋白诱导型一氧化氮合酶(iNOS)及炎症因子白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)、TLR4/NF-κB信号通路蛋白TLR4、髓样分化因子88(MyD88)、p-核因子-κB抑制蛋白α(IκBα)、p-NF-κB表达水平明显上调;而QC@mmSiO_(2)-NIPAM组小鼠肺组织内I型胶原蛋白、M1型巨噬细胞标记蛋白、TLR4/NF-κB信号通路蛋白表达水平明显降低。免疫荧光染色法结果显示,矽肺模型组小鼠肺组织内的iNOS与TLR4蛋白共表达。结论 QC@mmSiO_(2)-NIPAM可能通过调控TLR4/NF-κB通路抑制M1型巨噬细胞极化与炎症因子表达,从而发挥抗矽肺纤维化的作用。

Objective To investigate the effect and mechanism of core-shell type intelligent drug slow-release carrier(QC@mSiO_(2)-NIPAM) on the polarization of M1 macrophages and inflammatory response during silicosis through toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway. Methods C57BL/6 mice were randomly divided into normal control group, mmSiO_(2)-NIPAM low dose group(50mg/kg), mmSiO_(2)-NIPAM medium dose group(100mg/kg) and mmSiO_(2)-NIPAM high dose group(200mg/kg)(n=5). HE staining was used to observe the effects of different concentrations of mmSiO_(2)-NIPAM solution on the organs of mice. C57BL/6 mice were randomly divided into normal control group, silicosis model group(mSiO_(2)100mg/kg) and QC@mmSiO_(2)-NIPAM group(mSiO_(2)100mg/kg, QC@mmSiO_(2)-NIPAM100mg/kg)(n=5). Hematoxylin-eosin(HE) and Van Gieson(VG) staining were observed the morphological changes in each group. The expressions of type I collagen, M1 macrophage marker proteins and TLR4/NF-κB signaling pathway were detected by western blot and immunofluorescence staining. Results The results of HE staining showed that mmSiO_(2)-NIPAM solution had no effect on the organs of mice. HE and VG staining showed that there are silicosis nodules and the distribution of collagen fibers in the lung tissue of mice in the silicosis model group. After intragastric administration of QC@mmSiO_(2)-NIPAM solution, silicosis nodules were significantly reduced and collagen deposition was significantly decreased. The results of western blot were showed that the levels of type I collagen, M1 macrophage marker protein inducible nitric oxide synthase(iNOS), inflammatory factors interleukin(IL)-1β, IL-6, tumor necrosis factor-α(TNF-α), TLR4/NF-KB signaling pathway proteins TLR4, myeloid differentiation factor 88(MyD88), p-nuclear factor-κB inhibitor protein α(IκBα), p-NF-κB were significantly up-regulated in silicosis model group, while the expression levels of type I collagen, M1 macrophage marker protein and TLR4/NF-κB signaling pathway were significantly decreased in QC@mmSiO_(2)-NIPAM group. Immunofluorescence staining showed that iNOS and TLR4 protein were co-expressed in the lung tissue of silicosis model group.Conclusion QC@mmSiO_(2)-NIPAM can inhibit the polarization of M1 macrophages and inflammatory factors by regulating TLR4/NF-κB pathway, thereby playing an anti-silicosis role.