STAT-6/KLF-4/PPAR-γ激活在二氧化硅致肺纤维化中肺泡巨噬细胞极化改变中的作用

The role of STAT-6/KLF-4/PPAR-γactivation in alveolar macrophage polarization changes in silica-induced pulmonary fibrosis

目的:观察二氧化硅(SiO_(2))对小鼠肺泡巨噬细胞(AMs)极化的影响,探讨信号转导及转录激活因子-6(STAT-6)/Krüppel样因子-4(KLF-4)/过氧化物酶体增殖激活受体-γ(PPAR-γ)信号分子在AMs中的表达情况及意义。方法:于2020年11月,将C57BL/6小鼠随机分为结晶型SiO_(2)组和生理盐水(NS)组,每组12只。通过气管滴注100μl SiO_(2)混悬液(20 mg/ml)或NS,28 d后处死小鼠,Masson染色观察小鼠肺组织纤维化情况,并检测羟脯氨酸(HYP)水平;流式细胞术检测支气管肺泡灌洗液(BALF)中M1和M2型AMs比例,用实时荧光定量PCR检测AMs中诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)、白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、IL-6、IL-10、转化生长因子-β(TGF-β)、STAT-6、KLF-4、PPAR-γ等mRNA相对表达水平;酶联免疫吸附试验检测BALF中iNOS、Arg-1活力和IL-1β、TNF-α、IL-6、IL-10、TGF-β含量,免疫荧光检测AMs中磷酸化信号转导及转录激活因子-6(p-STAT-6)、KLF-4、PPAR-γ蛋白相对表达水平。结果:处理28 d后,结晶型SiO_(2)组小鼠肺组织结构被破坏,胶原蛋白沉积明显增多;与NS组比较,结晶型SiO_(2)组小鼠肺组织HYP水平明显升高,M2型AMs比例明显增加,M1型AMs比例明显下降;Arg-1、IL-10和TGF-βmRNA相对表达水平和含量升高,而iNOS、IL-1β、TNF-α、IL-6 mRNA相对表达水平和含量降低,STAT-6、KLF-4、PPAR-γmRNA和p-STAT-6、KLF-4、PPAR-γ蛋白相对表达水平上调,差异均有统计学意义(P<0.05)。结论:结晶型SiO_(2)可能通过激活STAT-6/KLF-4/PPAR-γ信号通路,促进AMs向M2型极化,从而介导肺纤维化过程。

Objective To observe the effect of silicon dioxide(SiO_(2))on the polarization of alveolar macrophages(AMs),and to explore the expressions and the significance of signal transducer and activator of transcription-6(STAT-6)/Krüppel-like factor-4(KLF-4)/peroxisome proliferators-activated receptors-γ(PPAR-γ)signaling molecules in AMs.Methods In November 2020,C57BL/6 mice were randomly divided into crystalline SiO_(2) group and normal saline(NS)group,and 12 mice in each group.Mice were intratracheally instillated with 100μl crystalline SiO_(2) suspension(20 mg/ml)or 100μl NS,and were sacrificed after 28 days.Masson staining was used to observe the degree of pulmonary fibrosis of mice and hydroxyproline(HYP)level were assessed.The proportions of M1-typed and M2-typed AMs in bronchoalveolar lavage fluid(BLAF)were analyzed by flow cytometry.The mRNA relative expression levels of inducible nitric oxide synthase(iNOS),arginidase-1(Arg-1),interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,IL-10,transforming growth factor-β(TGF-β),STAT-6,KLF-4 and PPAR-γwere detected by real-time fluorescence quantitative PCR.Activities of iNOS and Arg-1,as well as contents of IL-1β,TNF-α,IL-6,IL-10 and TGF-βwere assessed by the enzyme-linked immunosorbent.The protein relative expression levels of phosphorylation-signal transducer and activator of transcription-6(p-STAT-6),KLF-4 and PPAR-γwere evaluated by immunofluorescence.Results After 28 days of treatment,the structure of the lung tissue of the mice was destroyed,and the deposition of collagen was significantly increased in the crystalline SiO_(2) group.Compared with NS group,HYP level of lung tissue in crystalline SiO_(2) group were increased,the proportion of M2-typed AMs in crystalline SiO_(2) group was increased,the proportion of M1-typed AMs in crystalline SiO_(2) group was decreased,the mRNA relative expressions and contents of Arg-1,IL-10,TGF-βin crystalline SiO_(2) group were significantly increased,the mRNA relative expressions and contents of iNOS,IL-1β,TNF-α,IL-6 in crystalline SiO_(2) group were significantly decreased,the mRNA of STAT-6,KLF-4,PPAR-γand the protein relative expression levels of p-STAT-6,KLF-4,PPAR-γwere significantly increased in crystalline SiO_(2) group,and the the differences were statistically significant(P<0.05).Conclusion Crystalline SiO_(2) may mediate the process of pulmonary fibrosis through promote AMs polarization toward M2-typed by activating the STAT-6/KLF-4/PPAR-γsignaling pathway.