Bleomycin was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, China). Artesunate was purchased from Guilin Pharmaceutical Co., Ltd. (Guilin, China). The Hydroxyproline assay kit (#A030-2) was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Primers were synthesized by Invitrogen Life Technologies (Shanghai, China). The cDNA synthesis kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The total RNA extraction kit and 2X Taq PCR Master mix were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). Western blot and immunoprecipitation tissue and cell lysis solution were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The bicinchoninic acid protein assay kit was obtained from Beyotime Institute of Biotechnology (Shanghai, China). The TGF-β1 antibody and horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibody were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Smad3 antibody was purchased from Abzoom Biolabs, Inc. (Dallas, TX, USA). α-SMA antibody and the Hsp47 antibody were purchased from Epitomics (Burlingame, CA, USA).

Male Sprague Dawley (SD) rats weighing 180–250 g (Guilin Medical University, Guilin, China) were used in all experiments. SD rats were housed in specific-pathogen-free conditions with free access to food and water.

All experimental protocols performed on all rats were approved by the Guilin Medical University Animal Experiment Ethics Committee. Animals were randomly assigned to one of the following four groups: i) Control group (n=24), which received intratracheal administration of 0.9% NaCl solution alone; ii) bleomycin group (n=39), which received intratracheal administration of bleomycin (5 mg/kg); iii) artesunate group (n=24), which received daily intraperitoneal injections of artesunate (100 mg/kg) and the iv) bleomycin + artesunate group (n=39), which received intratracheal administration of bleomycin (5 mg/kg) and daily intraperitoneal injection of artesunate (100 mg/kg). Bleomycin was injected into the trachea exposed through a midline anterior neck incision using a 24-gauge needle. At 7, 14, 21 or 28 days after bleomycin treatment, rats were sacrificed by decapitation under anesthesia by inhaling isofluorane (3.5% in oxygen; sc-363629Rx; Santa Cruz Animal Health, Paso Robles, CA, USA), and lung tissue was quickly removed and processed as described below.

Left lungs obtained from rats were instilled with 10% formalin. Tissues were embedded in paraffin and then cut into 4 μm thickness sections that were used for hematoxylin and eosin (H&E), Masson staining and immunohistochemistry analysis. The severity of interstitial fibrosis was observed and assessed by a blinded pathologist using the Ashcroft score (15). Masson staining was performed in the present study to observe collagen deposition. Right lungs obtained from rats were immediately frozen in liquid nitrogen and then stored at −80°C in a freezer until use for immunoblot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and hydroxyproline measurement.

The total collagen content of the right lung was determined by hydroxyproline measurement using a hydroxyproline determination kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. Data are expressed as milligrams of hydroxyproline per gram of protein in the lungs (mg/g).

The lung tissues were homogenized in WIP tissue and cell lysis solution with the protease inhibitor cocktail (#04693159001; Roche Diagnostics, Basel, Switzerland) using a tissue grinder. The concentration of protein was determined using a protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Approximately 30 μg protein from each sample was electrophoresed in a 10 or 12% polyacrylamide gel. Following transferring proteins onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA), immunoblotting was performed using monoclonal rabbit anti-rat HSP47 (1:1,000 dilution; #3198-1), TGF-β1 (1:1,000 dilution; #RS-0105R), α-SMA (1:500 dilution; #5264-1) and polyclonal rabbit anti-rat Smad3 (1:500 dilution; #AM4061) antibodies. The proteins were visualized by incubating the membrane with chemiluminescence reagent (NEN Life Science Products, Boston, MA, USA) and exposing the membrane to X-ray films.

RT-PCR was used in the present study to determine the mRNA expression of HSP47 and collagen type I mRNA in the lung. In brief, total RNA was isolated from the lung tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). PCR was performed using a DNA thermal cycler in a 25 μl reaction volume, containing the cDNA template (2 μl), forward and backward primers (1 μl each), 12.5 μl 2X Master mix and 8.5 μl ddH₂O for 30 cycles via GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The primer sequences were as follows: HSP47, forward 5′-AGA ACC AAG GCA GAC TTA TCGC-3′ and reverse 5′-CCG TAG ATG TCC TGG TCA AAG-3′; collagen type I, forward 5′-TGC CGT GAC CTC AAG ATG TG-3′ and reverse 5′-CAC AAG CGT GCT GTA GGT GA-3′; GAPDH, forward 5′-GTG CTG AGT ATG TCG TGG AG-3′ and reverse 5′-ACC AGT GGA TGC AGG GAT-3′. The rat GAPDH housekeeping gene was used as an internal control.

Slides were rinsed in 1X phosphate-buffered saline (PBS) and then incubated in 0.3% H₂O₂ dissolved in methanol for 30 min in order to quench endogenous peroxidase. Following being rinsed with 1X PBS several times, slides were incubated with 3% normal serum in 1X PBS for 1 h to block nonspecific binding. Slides were then incubated with HSP47 antibody (1:400) or α-SMA antibody (1:500) overnight at 4°C. Following being rinsed with 1X PBS several times, slides were incubated with biotinylated goat anti-rabbit polyclonal secondary antibody (1:200; #BA1000; Vector Laboratories, Burlingame, CA, USA) for 1 h and then avidinbiotin complex reagent (Vector Laboratories) for 30 min. Slides were rinsed in 1X PBS and covered with diaminobenzidine peroxidase substrate solution from the Impact DAB kit (Vector Laboratories) for 2 min and then rinsed in water. Counterstaining was performed with hematoxylin. Slides were then dehydrated using increasing concentrations of ethanol and xylenes and mounted. Finally, images of tissue sections were captured using an Olympus BX53 digital microscope (Olympus, Tokyo, Japan).

Statistical analyses were performed with Sigmaplot software (SPSS) version 17.0 software (IBM, Armonk, NY, USA). The data are presented as the mean ± standard deviation. Statistical differences were determined by two-way analysis of variance and P<0.05 was considered to indicate a statistically significant difference.

As shown in Table I, ~25.64, 8.70 and 11.11% of the bleomycin group rats died within 7, 14 and 28 days after the administration of bleomycin, however, administration of artesunate reduced mortality to 20.51, 4.00 and 0.00% within 7, 14 and 28 days, respectively. The data demonstrated that the mortality rate was highest during the first 7 days after the administration of bleomycin. Following that, the mortality rate remained at ~10.00% in the bleomycin group, while the mortality rate reduced to 4.00% at 14 days. No mortality was observed between 14 and 28 days in the bleomycin+artesunate group. No mortality in the saline and artesunate group was observed during the whole experimental period. Notably, between 14 and 21 days, no mortality was found in all groups.

In order to determine the pathological alterations in the lung, H&E staining was performed (Fig. 1) and Masson staining was used to visualize the collagen fibrils (blue color) in the tissue (Fig. 2). The histological evaluation of lung sections at 7, 14, 21 and 28 days after bleomycin treatment revealed evidence of marked infiltration of inflammatory cells (particularly at day 7), excessive deposition of mature collagen in the interstitium (particularly at 14, 21 and 28 days), diffuse consolidation of parenchyma with loss of alveolar architecture and increased cell number, clear alveolar wall thickening and finally lung tissue was severely damaged. However, following artesunate treatment, the pathological alterations in the lung tissues were attenuated. The rats in the control group and artesunate alone group demonstrated no histological alterations. Similar to Masson staining, the RT-PCR results demonstrated that the mRNA expression of collagen type I (at day 28) was higher in bleomycin-treated rats compared with that in the control rats, and that the addition of artesunate then reduced this effect of bleomycin (Fig. 3). As for pulmonary fibrosis evaluation, the hydroxyproline content was measured. On days 7 and 14, the hydroxyproline contents were similar between the four groups. Out of all time points, the highest hydroxyproline contents were observed at day 14 in all groups. On day 21, the rats receiving bleomycin demonstrated a higher hydroxyproline content than the control group. However, artesunate did not significantly reduce bleomycin-induced increases in hydroxyproline content on day 21. On day 28, the bleomycin group had a higher hydroxyproline content than the control group. However, artesunate significantly reduced increases in bleomycin-induced hydroxyproline content (Table II). Additionally, artesunate alone had no effect on hydroxyproline content. The severity of interstitial fibrosis among the four groups was compared using the Ashcroft score. As shown in Table III, on day 14, 21 and 28, the Ashcroft score was higher in the bleomycin group than that in the control group. Administration of artesunate could reduce bleomycin-induced increases in the Ashcroft score on day 21 and 28, but not on day 14. In addition, artesunate alone had no effect on the Ashcroft score.

As stated above, bleomycin induced collagen deposition in lung tissues and artesunate inhibited this. HSP47, a collagen-binding glycoprotein, is associated with collagen accumulation and disease progression in an experimental pulmonary fibrosis model (2). Therefore, in the present study, the effect of artesunate on HSP47 was investigated. The data demonstrated that bleomycin induced HSP47 upregulation and artesunate inhibited HSP47 upregulation, which is evidenced by the results of RT-PCR (Fig. 3), immunohistochemistry (Fig. 4) and western blotting (Fig. 5).

TGF-β1 and its downstream molecule α-SMA are known to be critical for pulmonary fibrosis (5,6). Western blotting confirmed enhanced protein expression of TGF-β1 on day 28 after bleomycin injection compared with the control group (Fig. 5). In addition, Fig. 5 also shows that bleomycin-induced increases in TGF-β1 were inhibited by artesunate. Western blotting demonstrated that bleomycin treatment led to increased Smad3 expression and artesunate administration attenuated this increase (Fig. 5). Similar to TGF-β1, α-SMA protein was enhanced by bleomycin treatment and this enhancement was also inhibited by artesunate administration, which was confirmed by immunohistochemistry (Fig. 4) and western blotting (Fig. 5).