养阴清肺汤抑制矽肺模型小鼠TLR4信号的机制

The inhibitory effect of Yang-Yin-Qing-Fei Decoction on TLR4 signaling in silicotic mice

目的探讨养阴清肺汤通过对Toll样受体4(TLR4)信号和核因子κB受体激活剂配体(RANKL)信号的调控,发挥抑制矽肺小鼠肺纤维化的作用机制。方法将72只SPF级C57BL/6J雄性小鼠随机分为对照组、矽肺组、吡非尼酮组和养阴清肺汤高、中、低剂量组,每组12只。采用气管一次性灌注5mg SiO_(2)建立矽肺小鼠模型,2周后予以200mg/kg/d吡非尼酮,1000、500、250mg/kg/d高、中、低剂量养阴清肺汤连续灌胃2周。HE染色、Masson染色观察肺组织病理形态和胶原沉积。微小计算机断层扫描(micro-CT)重建肺三维结构;全身体积描记系统(WBP)检测肺功能;免疫荧光染色标记Toll样受体4(TLR4);抗酒石酸酸性磷酸酶(TRAP)染色标记肺组织阳性细胞;免疫印迹法检测肺组织I型胶原蛋白(CoL I)、波形蛋白(Vimentin)、Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子κB(NF-κB)、白细胞介素6(IL-6)、核因子κB受体激活因子(RANK)、核因子κB受体激活因子配体(RANKL)的蛋白表达。结果与矽肺模型组比较,养阴清肺汤可减少矽结节面积和胶原沉积(P<0.05),改善矽肺小鼠肺功能(P<0.05),养阴清肺汤组ColI、vimentin、TLR4、MyD88、NF-κB、IL-6、RANK和RANKL蛋白水平均明显下调(P<0.05)。结论养阴清肺汤可通过抑制TLR4和RANKL信号的活化,改善肺功能和减轻矽肺小鼠肺纤维化进展。

Objective To explore the effects and mechanisms of Yang-Yin-Qing-Fei Decoction(YYQFD)on lung fibrosis in silicotic mice by regulating Toll-like receptors 4(TLR4)and Receptor activator of nuclear factor kappa-B ligand(RANKL)signal pathways.Methods Seventy-two SPF grade C57BL/6J male mice were randomly divided into six groups(n=12),as control group,silicosis group,pirfenidone treatment group,and YYQFD high-dose,medium-dose,low-dose treatment groups.Silicotic mice was made by intratracheal instillation of SiO_(2)(5mg/mice)for 2 weeks and treated with YYQFD(1000 mg,500 mg,250mg/kg/d)or pirfenidone(200mg/kg/d)for another 2 weeks.The pathological morphology and collagen deposition of lung tissue were observed HE staining and Masson staining.Three-dimensional lung structure was reconstructed by micro computed tomography(Micro-CT).Whole body plethysmography(WBP)was used to detect lung functions.The expression of TLR4 in lung tissue was was observed by immunofluorescence staining.Tartrate-resistant acid phosphatase(TRAP)staining was used to observe the expression of positive cells in lung tissue.The expression of Type I collagen(Col I),Vimentin,TLR4;Myeloid differentiation primary response gene 88(MyD88),Nuclear factor kappa-B(NF-κB),interleukin 6(IL-6),Receptor activator of nuclear factor kappa-B ligand(RANK),and RANKL in lung tissue was detected by Western blot.Results Compared with the silicosis group,YYQFD treatment groups attenuated silicotic area,collagen deposition and improved impair lung functions in silicotic mice(P<0.05).The expression of Col I,vimentin,TLR4,MyD88,NF-κB,IL-6,RANK and RANKL were all reduced in YYQFD groups(P<0.05).Conclusion YYQFD alleviated lung fibrosis and improved lung function in mice exposed to silica by regulating TLR4 and RANKL signal pathways.