Bleomycin was purchased from Nippon Kayaku Co. (Chiyoda-ku, Tokyo, Japan). Anti-GAPDH, anti-H3, Alex a FluorH 800 goat anti-rabbit IgG or anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ERK and anti-pERK antibody were bought from Cell Signaling Technology (Boston, USA). Anti-nuclear factor-(NF-) κB p65 were bought from Bioss (Bioss, Beijing, China). TRIzol Reagent was obtained from Invitrogen (Carlsbad, CA, USA). Reverse transcriptase kit was from Promega (Madison, Wisconsin, USA). Hydroxyproline detection kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China). Rat TNF-α and TGF-β1 ELISA kit were purchased from BOSTER (Wuhan, Hunan, China). Methyl thiazolyl tetrazolium (MTT) and darifenacin were from Sigma (St Louis, MO, USA). BCA Protein Assay kit was brought from Pierce (Rockford, IL, USA). The mirVana^TM qRT-PCR Detection miRNA Kit was from Ambion (Austin, TX, USA). Primers of miR-21 were from Gene Pharma (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) were purchased from Life Technologies, Inc (Carlsbad, CA, USA).

Wistar rats (6 - 8 w) weighing 200 - 250 g and neonatal rats (1 - 3 d) were obtained from the experimental animal center of the Second Affiliated hospital of Harbin Medical University. All experimental protocols were pre-approved by the Experimental Animal Ethic Committee of Harbin Medical University, China. Use of animals followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Briefly, at day 0, rats were anesthetized with intraperitoneal injection of 2% pentobarbital (0.5 ml/ 100g ) and intratracheally instilled bleomycin (5 mg/kg) to establish the model of rat pulmonary fibrosis. And the sham-operated control group rats (n = 7) were instilled saline instead of bleomycin in the same way. Rats of the bleomycin-induced pulmonary fibrosis model were randomly divided into bleomycin group (n = 7) and darifenacin group (n = 7). Darifenacin (5 mg/kg/day) was administered intraperitoneally in darifenacin group. bleomycin group with saline (0.9 percent sodium chloride) in the same manner. Seven days after treatment, the rat blood samples were collected via a tail vein from each group. Three weeks after bleomycin or saline administration, rats in each group were sacrificed and the lung tissues were harvested for the following experiment.

Pulmonary fibroblasts were isolated from neonatal rats. Briefly, whole lung was isolated and collected as quickly as possible. After washed out the residue blood with PBS, the lung tissues were chopped into suitable small pieces into and rinsed again in PBS. All of operations were performed on ice. Then the tissue fragments were digested with repetitive 0.125% trypsinization and shaken gently for suitable time in 37˚C water-bath. The supernatant was collected in DMEM containing 10% (vol/vol) fetal bovine serum (supernatant/DMEM = 1/1) and then kept at 4˚C provisionally. After filtering, the filtrate was collected and centrifuged for 5 min 1500 round/min. Finally, the cells were resuspended in the new culture medium DMEM, which contained 10% fetal bovine serum and 1% double-antibiotic (penicillin and etiolation). Resuspended cells were then plated in one culture bottle and incubated in a humidified incubator (95% O₂, 5% CO₂, 37˚C) for 1.5 h to remove non-fibroblast contamination.

Masson’s Trichrome and hematoxylin and eosin (HE) staining were used to observe the changes of lung histological. The middle lobes of left lung were fixed in 10% buffered paraformaldehyde (pH 7.4) for a week and then embedded in paraffin blocks. The lung lobes fixed were cut longitudinally into 5-μm-thick sections and then stained with Masson’s Trichrome and HE via Ashcroft scoring method, respectively.

Hydroxyproline level of lung tissues (40 - 60 mg per lung) or cell-culture medium of pulmonary fibroblasts was determined by hydroxyproline detection kit following the manufacturer’s direction and described using micrograms of hydroxyproline per gram of wet weight (mg/g).

ELISA for the measurement of cytokine TNF-α and TGF-β1 level in serum and cell culture supernatants were performed according to the manufacturer’s instructions.

Pulmonary fibroblasts (2 × 10⁴ cells/well) were seeded in 96-well plates with 200 μl of medium per well. After 24 h, the cells were treated with darifenacin. After incubation, 10 μl of MTT reagents (5 mg/ml) was added and incubated for 4 h under the same conditions. Then, the medium was removed from the plate, and the formazan was dissolved with 150 μl DMSO. The absorbance (OD) at 490 nm was measured by a microplate reader.

Total and nuclear proteins were extracted from lung tissues and primary culture pulmonary fibroblasts. The content of protein was then measured by BCA protein assay kit. The equal amounts of proteins samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk for 2 h at room temperature and then incubated with corresponding antibodies at 4˚C over-night. After that, the nitrocellulose membrane was washed 3 times with PBS containing 0.05% Tween-20 (PBS-T) followed by incubated with secondary antibodies (1:10,000) for 1h at room temperature. The images were captured on the Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA) and band intensity (area × optical density) was quantified using Odyssey v1.2 software. GAPDH was served as an internal control.

Total RNA was extracted using TRIzol Reagent. First-strand complementary DNA (cDNA) was synthetized using a reverse transcriptase kit according to the manufacturer’s instructions. Those cDNA were used as the template for quantitative RT-PCR (qRT-PCR) analysis on an ABI 7500 fast Real Time system (Applied Biosystems, Foster City, CA, USA), with U6 as an internal control. The sequences of the primers were as follows: U6, 5’-CTCCGATAGATCTGCCCTCTTGAA-3’ (forward), 5’-CGCTTCACGAATTTGCGTGTCAT-3’ (reverse); miR-21, 5’-GGGGTAGCTTATCAGACTGATG-3’ (forward), 5’-TGTCGTGGAGCGGCAATTG-3’ (reverse); collagen I, 5’-GCTCCTCTTAGGGGCCACT-3’ (forward), 5’-CCACGTCTCACCATTGGGG-3’ (reverse); α-SMA,5’-AGGGAGTAATGGTTGGAATGG-3’ (forward), 5’-GGTGATGATGCCGTGTTCTA-3’ (reverse); GAPDH, 5’-TGTGGGCATCAATGGATTTGG-3’ (forward), 5’-ACACCATGTATTCCGGGTCAAT-3’ (reverse).

Average data were expressed as mean ± standard error of the mean (SEM). Comparisons among multiple groups were analyzed by one-way analysis of variance (one-way ANOVA) followed by Bonferroni-corrected post-hoc tests were carried out using SPSS 13.0. A two-tailed P value < 0.05 indicated a statistically significant difference.

The changes of lung histological were observed by HE staining and Masson’s Trichrome staining. The signs of inflammatory cell infiltration and fibrotic changes were notable in the bleomycin group. Meanwhile, the alveolar septa became significant thickened compared with the control group in HE staining (Figure 1(a) and Figure 1(c)). Moreover, alveolar spaces became smaller even dent, accompanied by deposition of extensive blue collagen in Masson’s Trichrome staining (Figure 1(b) and Figure 1(d)). However, in the darifenacin group, the inflammatory infiltration, fibrotic and alveolar septa changed, as well as deposition of blue collagen all were remarkably alleviated compared with the bleomycin group.

The hydroxyproline levels in lung tissues among three groups were assessed using hydroxyproline detection kit (Figure 2(a)). In bleomycin group, the hydroxyproline level was significantly increased compared with the sham-operated control group. However, the increase of hydroxyproline induced by bleomycin was attenuated by administration with darifenacin. The expression level of cytokine

TGF-β1 and TNF-α in serum were assessed by ELISA. The levels of cytokines in the two groups were notable higher than control group (Figure 2(b) and Figure 2(c)). As we expected, after darifenacin treatment, all the cytokines level was decreased noticeably compared with bleomycin group.

Then, we examined the therapeutic effect of the darifenacin in the bleomycin-induced fibrotic process in human pulmonary fibroblasts (MRC-5 cells) by qPCR. Administration of darifenacin significantly decreased the expression of collagen I and α-SMA in bleomycin-treated MRC-5 cells (Figure 2(d) and Figure 2(e)).

Pulmonary fibroblasts derived from neonatal rats were used for proliferation assay. Pulmonary fibroblasts derived from neonatal rats were used for proliferation assay. Using MTT assay to measure the viability of pulmonary fibroblasts and nintedanib as a positive control, the results shown that cell viability of darifenacin and nintedanib groups decreased significantly compared with the control group (Figure 3(a)). The proliferation of cells was reduced to 53% by 1 μM darifenacin and that to 70% by 1 μM nintedanib, respectively. Besides, both darifenacin and nintedanib caused a significant decrease of the Hydroxyproline

level in pulmonary fibroblasts (Figure 3(b)). In the present study, both nintedanib and pirfenidone inhibited the proliferation of fibroblastic cells. At the same concentration, the effect of darifenacin was better than nintedanib.

To investigate whether ERK, NF-κB and miR-21 were involved in the antifibrotic effect of darifenacin on pulmonary fibrosis, we identified the ERK activation, NF-κB nuclear translocation, and miR-21 expression in pulmonary fibroblasts. The total protein level of ERK (t-ERK) remained unchanged between the two groups. However, the protein expression of p-ERK was markedly decreased in darifenacin group (Figure 4(a)). Additionally, further data showed that administration of darifenacin to pulmonary fibroblasts markedly reduced the nuclear NF-κB (N-NF-κB) and enhanced the cytoplasmic NF-κB (C-NF-κB) expression (Figure 4(b)). Besides miR-21 level was also decreased in darifenacin group (Figure 4(c)).