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.
ABSTRACT
Objective:To investigate the clinical features, risk factors, treatment and prognosis of dermatomyositis (DM) patients with positive anti-melanoma differentiation associated gene 5(MDA5) antibody with rapidly progressive interstitial lung disease (RPILD).Methods:The clinical data of 88 DM patients from June 2019 to June 2020, at the rheumatology department of Guangdong Provincial People's Hospital were collected and retrospectively analyzed. T-test, non-parametric Mann-Whitney U test, Chi-squared test, Fisher exact probability and Logistics regression analysis were used for data analysis. Results:① 37%(36/88) DM patients were positive for anti-MDA5 antibody. The frequency of ulcerative rash, Gottron's sign, arthritis, clinically amyopathic dermatomyositis (CADM), and erythrocyte sedimentation rate (ESR) was significantly higher in patients with anti-MDA5 antibody ( P<0.05). The cell count of white blood cell, neutrophil, lymphocyte, and serum creatine kinase (CK) level were significantly lower in the anti-MDA5 antibody positive group than those in the negative group ( P<0.05). Of anti-MDA5 antibody positive DM patients, 100% developed ILD, 34% (11/32)developed RP-ILD, 16%(5/32) died, which were significantly higher than those of anti-MDA5 antibody negative patients ( P<0.05). ② Of anti-MDA5 antibody positive DM patients, the C reactive protein (CRP) level, positive rate of anti-Ro-52 antibody and mortality rate were significantly higher RPILD group than those in the non-RPILD group [15.70(4.49, 29.00) vs 3.22 (1.66, 7.15), Z=-2.440, P=0.014; 91% vs 43%, P=0.011; 46% vs 0, P=0.002]. Logistics regression analysis indicated that positive anti-Ro-52 antibody [ OR=4.561, 95% CI (1.797, 11.580), P=0.001] might be a risk factor for anti-MDA5 antibody positive DM-RPILD. ③ Among patients with anti-MDA5 antibody with RPILD, serum ferritin and D-dimer level was significantly higher and oxygenation index was significantly lower in the non-survival group than those in the survival group [1 931 (1 377, 7 379) vs 638(196, 876), Z=-2.556, P=0.009; 2 760(1 995, 4 854) vs 985(533, 1 588), Z=-2.379, P=0.017; 230(140, 256) vs 309(262, 382), Z=2.191, P=0.030]. In addition, the delayed intensive treatment time was significantly longer in the non-survival group than those in the survival group [(14.0±2.6) vs (4.5±1.4), t=7.899, P<0.01]. Furthermore, the proportion of combined therapy with two disease modifying antirheumatic drug (DMARDs) was significantly lower in the non-survival group than those in the survival group (0 vs 83%, P=0.015). Conclusion:Anti-MDA5 antibody may be associ-ated with characteristic clinical manifestations of DM, ILD, RPILD and high mortality rate. Positive anti-Ro-52 antibody may be a risk factor for anti-MDA5 antibody positive DM-RPILD. High serum ferritin and D-dimer level and low oxygenation index in RPILD patients may be associated with poor prognosis. Early treatment with two DMARDs may improve the prognosis of RPILD.