巨噬细胞分泌转化生长因子β对百草枯中毒致肺纤维化的影响

Effect of transforming growth factor-β secreted by macrophages on pulmonary fibrosis induced by paraquat poisoning

目的研究巨噬细胞参与百草枯(PQ)致肺纤维化可能的作用机制。方法用佛波酯(PMA)320 nmol·L^(-1)诱导THP-1细胞24 h获得巨噬细胞。检测PQ 20,40,60,80,100,120,140,160和180μmol·L^(-1)染毒该巨噬细胞48 h,CCK-8法检测细胞存活率。用Transwell技术构建该巨噬细胞和人胚肺成纤维细胞MRC-5共培养体系,PQ 100μmol·L^(-1)染毒48 h建立肺纤维化细胞模型;染毒同时加入转化生长因子β(TGF-β)受体抑制剂GW78838810μmol·L^(-1)抑制TGF-β受体,实时荧光定量PCR检测巨噬细胞和MRC-5细胞TGF-βmRNA表达,Western印迹法检测巨噬细胞TGF-β蛋白、MRC-5细胞纤维化相关蛋白α平滑肌肌动蛋白(α-SMA)和纤连蛋白及TGF-β/Smads通路相关蛋白Smad2和基质金属蛋白酶9(MMP-9)蛋白表达,免疫荧光法检测MRC-5细胞纤维状肌动蛋白表达。结果PQ在20~180μmol·L^(-1)浓度范围内可抑制巨噬细胞存活,IC_(50)为57.13μmol·L^(-1)。与细胞对照组相比,PQ染毒组巨噬细胞TGF-βmRNA和蛋白表达显著增加(P<0.05);与MRC-5细胞相比,巨噬细胞在PQ染毒前后TGF-βmRNA均显著增加(P<0.05)。PQ染毒共培养体系48 h,MRC-5细胞Smad2和MMP-9蛋白表达显著增加(P<0.05,P<0.01),α-SMA和纤连蛋白显著增加(P<0.01);与PQ染毒组相比,GW788388处理可显著降低MRC-5细胞Smad2和MMP-9蛋白表达(P<0.05)及α-SMA和纤连蛋白表达(P<0.05,P<0.01)。免疫荧光染色结果表明,与PQ染毒组相比,GW788388处理后MRC-5细胞纤维状肌动蛋白表达显著降低(P<0.01)。结论PQ染毒共培养体系后,巨噬细胞可能通过旁分泌TGF-β激活肺成纤维细胞TGF-β/Smads通路而促进肺纤维化。

OBJECTIVE To study the mechanism by which macrophages are involved in pulmonary fibrosis induced by paraquat(PQ).METHODS Phorbol 12-myristate-13-acetate(PMA)320 nmol·L^(-1) for24 h was used to induce THP-1 cells to differentiate into macrophages.CCK-8 was used to detect the survival rate of macrophages after 48 h exposure to different concentrations of PQ(20,40,60,80,100,120,140,160 and 180μmol·L^(-1)).Transwell technology was used to establish a co-culture system that was exposed to PQ 100μmol·L^(-1) for 48 h for a pulmonary fibrosis cell model.The transforming growth factor-β(TGF-β)receptor inhibitor GW78838810μmol·L^(-1) was added while the co-culture system was exposed to PQ to inhibit TGF-βreceptors.qRT-PCR was used to detect TGF-βmRNA expression in macrophages and MRC-5 cells.Western blotting was used to detect the expression levels of TGF-βprotein,fibrosis-related proteins such asα-smooth muscle actin(α-SMA)and fibronectin,and TGF-β/Smads pathway related proteins such as Smad2 and matrix metalloproteinase 9-(MMP-9),while immunofluorescence assay was adopted to detect F-actin protein in MRC-5 cells.RESULTS PQ reduced cell viability of macrophages in the concentration range of 20-180μmol·L^(-1),and the IC_(50) was 57.13μmol·L^(-1).Compared with the cell control group,the expressions of TGF-βmRNA and protein of macrophages in the PQ group were significantly increased(P<0.05).Compared with MRC-5 cells,TGF-βmRNA was increased significantly before and after PQ exposure in macrophages(P<0.05).After the co-culture exposure to PQ,the protein expressions of Smad2 and MMP-9 in MRC-5 cells were increased significantly(P<0.05,P<0.01),so didα-SMA and fibronectin protein expression(P<0.01).Compared with the PQ group,GW788388 treatment significantly reduced the protein expressions of Smad2(P<0.05),MMP-9(P<0.05),fibronectin andα-SMA in MRC-5 cells(P<0.01).Compared with the PQ group,phalloidin fluorescently labeled cell fibrinoid actin also showed that F-actin protein expression was significantly reduced in MRC-5 cells after GW788388 intervention(P<0.01).CONCLUSION After the exposure of the co-culture systerm to PQ,TGF-βsecreted by macrophages activates the TGF-β/Smads pathway of lung fibroblasts to promote pulmonary fibrosis.