七甲川花菁类荧光小分子IR-61改善大鼠肺纤维化

Fluorescent small molecule IR-61 improves pulmonary fibrosis induced by intratracheal bleomycin in rats

目的探索七甲川花菁类荧光小分子IR-61在肺纤维化发生过程中的作用以及对肺成纤维细胞转分化、增殖和迁移的影响。方法采用气管滴注博来霉素(bleomycin,BLM,2.5 mg/kg)建立大鼠肺纤维化模型;腹腔注射PBS或IR-61(2.0 mg/kg,每周2次);BLM处理10 d后,尾静脉注射IR-61(2.0 mg/kg),于24 h后取各器官行近红外荧光成像(near infrared fluorescence imaging,NIR),观察IR-61在各器官的蓄积情况;治疗28 d后,取肺组织制作切片,行HE和Masson染色,显微镜下观察;采用RT-PCR、Western blot测定人肺成纤维细胞(human fetal lung fibroblast,HFL1)与原代大鼠肺成纤维细胞转分化及增殖相关基因Collagen-1、Collagen-3、α-SMA、Fibronectin、FOXM1、CCNB1、CDC25b和AURKB的mRNA及蛋白表达;用细胞划痕实验进行迁移检测。结果①NIR显示:IR61可靶向至博来霉素诱导的受损伤肺组织。②形态学观察显示:BLM+IR61组较博来霉素组形态改善,肺塌陷少,纤维结节少。经组织学鉴定,BLM+IR61处理组较BLM处理组肺纤维化程度低[(0.1033±0.01453)vs(0.4600±0.03786),P<0.05]。IR-61预处理HFL1与原代大鼠肺成纤维细胞后,用TGFβ1刺激,Western blot和RTPCR分析结果显示:TGF-β1+IR-61处理组较TGFβ1处理组转分化特异性基因Collagen-1、Collagen-3、α-SMA、Fibronectin的mRNA及蛋白表达降低(P<0.05),同时,增殖特异性FOXM1、CCNB1、CDC25b和AURKB基因的表达也降低(P<0.05),说明IR-61可以抑制TGF-β1诱导的肺成纤维细胞的转分化与增殖的作用。③细胞划痕实验结果显示:TGF-β1诱导肺成纤维细胞迁移活性增强,IR-61能有效抑制肺成纤维细胞的迁移能力。结论IR-61通过靶向到受损伤的肺组织并通过抑制肺成纤维细胞的转分化、增殖和迁移来减轻博来霉素诱导的肺纤维化。

Objective To evaluate the effects of IR61 on the development of pulmonary fibrosis and on the proliferation and migration of lung fibroblasts in rats.Methods Forty male SD rats(6 to 8 weeks old)were randomized equally into normal control group,IR-61 group,bleomycin group and bleomycin+IR61 group.In the latter 2 groups,rat models of pulmonary fibrosis were established by intratracheal infusion of bleomycin(2.5 mg/kg).In the 2 groups with IR-61 treatment,IR-61 was intraperitoneally injected at the dose of 2.0 mg/kg 30 min following intratracheal bleomycin infusion and was then administered twice a week.At 10 d after bleomycin treatment,3 rats from each group were selected for IR-61 injection(2 mg/kg)via the tail vein to observe the accumulation of IR-61 in each organ at 24 h using near infrared fluorescence imaging(NIR).After 28 d of treatment,the rats were euthanized and the lung tissue was collected for HE and Masson staining.RT-PCR and Western blotting were performed to detect the changes of the expression of the genes related with lung fibroblast transdifferentiation and proliferation(including Collagen-1,Collagen-3,α-SMA,fibronectin,FOXM1,CCNB1,CDC25b and AURKB)in human lung fibroblasts(HFL1)and primary rat lung fibroblasts in response to IR-61 and transforming growth factor-β1(TGF-β1)treatments;the migration of the cells was assessed using cell scratch test.Results imaging showed that IR-61 could target the injured lung tissue induced by bleomycin.Morphological observation showed that compared with bleomycintreated rats,the rats with IR-61 treatment presented with better structural integrity of the lungs with less obvious lung collapse,fewer fibrotic nodules and a significantly milder of pulmonary fibrosis(0.1033±0.01453 vs0.4600±0.03786,P<0.05).In HFL1 cells and primary rat lung fibroblasts,IR-61 pretreatment prior to TGF-β1 stimulation resulted in significantly lowered mRNA and protein expression of collagen-1,collagen-3,α-MA,and fibronectin as well as the proliferationspecific genes FOXM1,CCNB1,CDC25b and AURKB(P<0.05).Cell scratch test showed that TGF-β1 treatment significantly increased the cell migration activity,which was effectively suppressed by IR-61 treatment.Conclusion61 ameliorates bleomycininduced pulmonary fibrosis in rats by targeting the injured lung tissue and inhibiting the transdifferentiation,proliferation and migration of lung fibroblasts.