Iguratimod inhibits transforming growth factor-β 1 induced human lung fibroblast activation and collagen secretion via the Smad3/p300 pathway / 中华风湿病学杂志

ABSTRACT

Objective:To investigate the effect of iguratimod (IGU) on transforming growth factor-β 1 (TGF-β 1)-induced primary human lung fibroblasts (pHLFs) activation and collagen secretion. Methods:Mice pulmonary fibrosis (PF) models were established in vivo and were divided into three groups: the control group (CTR group), the Bleomycin (BLM) group and the BLM+IGU group, hematoxylin-eosin (HE) staining was used to observe lung morphology, and Masson staining was used to observe the degree of collagen accumulation in lung. Fibronectin and smooth muscle 22 (SM22) were detected by immunofluorescence, and the content of hydroxyproline in lung tissue was detected by chloramine-T method. In vitro, pHLFs were used to assess the effect of IGU on TGF-β 1 stimulation in four groups: CTR group, IGU group, TGF-β 1 group and TGF-β 1+IGU group, the apoptosis of cells was detected by flow cytometry, and the mRNA expression of collagen type Ⅰ (COL-Ⅰ) and collagen type Ⅲ (COL-Ⅲ) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of α-smooth muscle actin (α-SMA), fibronectin, p-Smad2, p-Smad3 and transcription coactivator p300 were detected by Western blot and immunofluorescence. One-way ANOVA was used for all data, and LSD- t test or Kruskal-Wallis test was used for pair comparison. Results:The content of hydroxyproline in CTR group, the BLM group and the BLM+IGU group was (0.552±0.075) μg/mg, (1.293±0.081) μg/mg and (0.833±0.053) μg/mg ( F=169.672, P<0.01) respectively. IGU reduced the content of hydroxyproline in the lung tissue of mice, reduced the accumulation of collagen in the lung, and thus reduced the degree of BLM-induced pulmonary fibrosis, and improved the pathological changes in the lung of mice. In cell experiments, IGU had no significant effect on apoptosis ( F=0.83, P=0.54). The relative expression levels of COL-Ⅰ mRNA in the CTR group, TGF-β 1 group and TGF-β 1+IGU group were (100.4±1.2), (299.0± 13.0) and (202.5±7.0) respectively ( F=468.7, P<0.01). The relative expression levels of COL-Ⅲ mRNA in the CTR group, TGF-β 1 group and TGF-β 1+IGU group were (99.8±1.9), (350.6±8.0) and (220.3±9.9) respectively ( F=468.7, P<0.01). The relative expression levels of α-SMA protein were (0.193±0.038) in CTR group, (0.530±0.061) in TGF-β 1 group, and (0.410±0.065) in TGF-β 1+IGU group ( F=35.620, P<0.01); The relative expression levels of fibronectin in CTR group, TGF-β 1 group, and TGF-β 1+IGU group were (0.200±0.020), (0.700±0.020) and (0.410±0.066) respectively ( F=123.326, P<0.01). The relative expression levels of p-Smad3 protein in CTR group, TGF-β 1 group, and TGF-β 1+IGU group were (0.120±0.020), (0.573±0.586) and (0.327±0.252) respectively( F=92.987, P<0.01); The relative expression levels of p300 in CTR group, TGF-β 1 group and TGF-β 1+IGU group were (0.180±0.055), (0.923±0.025) and (0.650±0.050) respectively ( F=207.676, P<0.01). IGU significantly decreased the mRNA expression levels of COL-Ⅰ and COL-Ⅲ induced by TGF-β 1, inhibited the protein expression levels of α-SMA, fibronectin, p300, and phosphorylation of Smad2/3. Conclusion:Our results revealed the beneficial effect of IGU on the inhibition of TGF-β 1-mediated pHLFs activation and collagen secretion via the Smad3/p300 pathway, thus suggest that it might act as an effective anti-fibrotic agent in preventing the progression of PF.