Antifibrotic Effect Of Pirfenidone In Connective Tissue Disease Associated Interstitial Lung Disease

BackgroundDiffuse connective tissue diseases(CTDs)are a group of diseases caused by autoimmune disorders which can affect almost every organ,especially the lung.Interstitial lung disease(ILD)is the most serious complication,which can lead to impaired lung function or even respiratory failure and seriously shorten the life expectancy.As so far,the pathogenesis of CTD-ILD has not been fully elucidated and a radical cure is in great need.Therefore,this paper intends to observe the anti-fibrotic effect of pirfenidone on vivo and vitro models and clinical treatment and explore the mechanism of ILD and therapeutic strategy to provide new evidence and ideas.Objective1.To observe the efficacy and side effects of pirfenidone in the treatment of CTD-ILD.2.To observe the antifibrotic effect of pirfenidone in bleomycin-induced pulmonary fibrosis in mice3.To investigate the effects and mechanism of pirfenidone on the proliferation,migration,invasion,secretion capacities and phenotype of HLFs.MethodsPatients diagnosed with CTD-ILD were administrated with a 12-months treatment with or without pirfenidone.At baseline and at months 3rd,6th,and 12th,laboratory tests,pulmonary function tests,six minute walk distance(6MWD)and HRCT scores were performed.Covariance analysis was used to calculate the difference between the PFD and control group.A repeated-measures ANOVA was conducted to assess the variations over time.Eight-week-old male C57BL/6 mice were induced to establish the pulmonary fibrosis model with bleomycin by intratracheal injection of bleomycin.Pirfenidone or the saline solution was orally administrated and then all mice were sacrificed at the day 28th.Pathological variation and expression of ?-SMA and ATF3 were observed by H&E staining,Masson staining and immunofluorescence staining.Primary human lung fibroblasts(HLFs)were isolated from pathologically confirmed normal lung tissues by surgeries.CTD-ILD lung specimens were obtained by CT-guided percutaneous transthoracic biopsy.The cell viability was detected by MTT assay.The migration and invasion ability of the cells were detected by Transwell assay.The protein levels of a-SMA,ATF3 and p-Smad3 were detected by western blot and immunofluorescence analysis.The levels of mRNA expression of?-SMA,ATF3 and collagen type ? and ? were detected by real-time quantitative PCR.HLFs were infected with ATF3 shRNA or scramble lentivirus.ResultsFVC,TLC and DLco were significantly improved(p=0.044,0.017 and 0.001,respectively)in the PFD group compared with the CTR group,and DLco was increased in the 6th month(p=0.033).During the follow-up period,the TLC of the PFD group improved markedly in the first 3 months,and then maintained at the same level(p=0.046,0.030 and 0.056,respectively).Moreover,FVC and DLco showed improvement in the 6th month(p<0.05).A lower alveolar score in HRCT in the PFD group approached statistically significance even after 3-month treatment with pirfenidone compared with control group(p=0.021,0.010,and 0.036,respectively)and decreased gradually during the follow-up period whereas no such changes were observed in the control group.The most common adverse reactions are fatigue,dyspnea,cough,rash and dyspepsia during pirfenidone treatment,which can be improved after clinical symptomatic therapy.Pretreatment of pirfenidone protected from lung structural damage and collagen deposition and ameliorated pulmonary fibrosis in bleomycin-induced pulmonary fibrosis model of mice.Alpha-SMA expression was obviously upregulated in lung tisses from bleomycin-treated mice compared with control group and pirfenidone-treated group.ATF3 expression was obviously increased in lung tissues from CTD-ILD patients and from bleomycin-induced mice compared with controls while pirfenidone enabled to decrease the expression of ATF3 in bleomycin-induced mice.The expression of p-Smad3 in protein level and the expression of ATF3 in protein and mRNA levels were increased in TGF-?1-induced HLFs than in the control group,as well as the proliferation,migration and invasion ability and secretion and mobility capacities.Pretreatment of the HLFs with pirfenidone not only markedly suppressed the changes of proliferation,migration,invasion and secretion abilities and mobility capacity but also down-regulated the expression of ?-SMA and ATF3 triggered by TGF-?1.ATF3 knockdown(KD)HLFs were less responsive to TGF-?1 stimulation than the wildtype(WT)fibroblasts:they had reduced proliferation,migration and invasion capacities and the expression of ?-SMA and p-Smad3 in HLFs of ATF3 KD was significantly decreased than that of WT HLFs even after stimulated by TGF-?1 ConclusionPirfenidone could improve the pulmonary function tests and HRCT scores of CTD-ILD patients and reduce the lung damage and collagen deposition and alleviate fibrosis in bleomycin-induced pulmonary fibrosis in mice via inhibiting fibroblast to myofibroblast transition,which depends on interfering with the ATF3/Smad3 signalling pathway.