Recombinant human TGFβ1 was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Isor was purchased from Baomanbio (Shanghai, China). BLM was purchased from Hisun Company (Zhejiang, China). Antibodies targeting collagen I (dilution, 1:6,000; cat. no. ab138492), α-smooth muscle actin (α-SMA; dilution, 1:300; ab32575) and 78 kDa glucose-regulating protein (GRP78)/binding immunoglobulin protein (BiP; dilution, 1:1,200; cat. no. BM0134) were obtained from Abcam (Cambridge, UK). Antibodies targeting TGFβ1 (dilution, 1:1,000; cat. no. AM4195), protein kinase R-like endoplasmic reticulum kinase (PERK; dilution, 1:1,200; cat. no. BM0524) and E-cadherin (dilution, 1:1,200; cat no. BM0537) were obtained from Abzoom Biolabs, Inc. (Dallas, TX, USA). Antibodies targeting vimentin (dilution, 1:1,200; YT4880), phosphorylated (p)-PERK (dilution, 1:2,000; cat. no. YP1055), DNA damage-inducible transcript 3 (DDIT3; also known as CHOP; dilution, 1:1,200; cat. no. YT0911), eukaryotic translation initiation factor 2 subunit α (eIF2α; dilution, 1:1,200; cat. no. YT1507) and p-eIF2α (dilution, 1:1,000; cat. no. YP0093) were obtained from ImmunoWay Biotechnology, Plano, TX, USA. Horseradish peroxidase (HRP)-coupled sheep anti-rat (dilution, 1:15,000; cat. no. SA001) or sheep anti-rabbit (dilution, 1:15,000; cat. no. SA009) secondary antibodies were obtained from Auragene Technology, Co., Inc. (Changsha, China).

A total of 15 male 4-week old C57 mice (20-25 g in weight; SLRC Laboratory Animal Company, Changsha, China) were housed in rooms with a 12-h light/dark cycle at 25°C and 40-70% humidity for 1 week prior to the experiment. Mice were fasted for 12 h and had ad libitum access to fresh tap water up until the beginning of the experiment. During the experiment, the mice had ad libitum access to food and water. The mice were then randomly assigned to the Isor treatment group, the BLM group or the control group. Mice in the Isor and BLM groups were intraperitoneally injected with BLM (3.5 U/kg; Hisun Company, Zhejiang, China), while mice in the control group were injected with normal saline. The Isor treatment group was divided into two subgroups: High dose (30 mg/kg) and low dose (10 mg/kg). Each subgroup was treated with Isor by intragastric administration once a day. Mice in the control group and BLM group were administered the same volume of distilled water by gavage. After 28 days, the mice were euthanized by pentobarbitone overdose. Lung tissues were collected and used for hematoxylin and eosin (H&E) and Sirius red staining and western blot analyses. All experiments involving animals were approved by the Ethics Committee of Hunan Normal University Medical College (Changsha, China).

Human A549 cells and human bronchial epithelial cells (HBECs) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO₂. These cells were divided into five groups: The control group, the TGFβ1 group, and three TGFβ1 + Isor groups. Cells in the TGFβ1 group were stimulated with TGFβ1 alone (5 ng/ml) for 48 h. Cells in the TGFβ1 + Isor groups were treated with 5 ng/ml TGFβ1 and 25, 50 or 100 µM Isor for 48 h.

The lung specimens were fixed in 4% paraformaldehyde at room temperature for 48 h, dehydrated in a graded alcohol series and embedded in paraffin blocks. Sections were blocked with 3% hydrogen peroxide for 15 min at room temperature. Five-micron-thick sections were then stained with H&E for 10 min for routine examination, or with Sirius red for 8 min to visualize collagen deposition at room temperature. For immunohistochemistry staining, sections were stained with the aforementioned antibodies, developed with 3,3′-diaminobenzidine, and counterstained with hematoxylin. Samples were viewed using a light microscope (magnification for H&E staining, ×100 and ×400; magnification for immunohistochemistry staining, ×200 and ×400).

Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Inc.). A total of 2 µg RNA was subjected to reverse transcription using the PrimeScript Reverse Transcription Reagent kit (Thermo Fisher Scientific, Inc.). Quantitative analysis of the change in expression levels was performed using SYBR Premix Ex Taq (Dongsheng Biotech, Guangzhou, China) on an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: An initial incubation at 95°C for 10 sec, followed by 40 cycles of denaturation at 95°C for 10 sec and annealing at 60°C for 30 sec. The sequences of the primers used were: GRP78, sense, 5′-GAAGGTTACCCATGCAGTT-3′ and antisense, 5′-AGCAATAGTTCCAGCGTCT-3′; CHOP, sense, 5′-CCTGAAAGCAGCACCAAA-3′ and anti-sense, 5′-ACAAGCTCCATGTAGCAAAC-3′; and β-actin, sense, 5′-AGGGGCCGGACTCGTCATACT-3′ and antisense, 5′-GGCGGCACCACCATGTACCCT-3′. Data analysis was performed by the comparative C_q method using ABI software (25). mRNA expression was normalized to that of β-actin.

Protein was extracted from lung tissues and cells with RIPA lysis buffer (Auragene Technology, Co., Inc., Shenzen, China). Protein concentrations were determined with bicinchoninic acid protein assay reagents (Auragene Technology, Co., Inc.) according to the manufacturer’s instructions. Equal amounts of protein (10 µg) from each sample were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 3% BSA-TBST at room temperature for 60 min. The membranes were probed with primary antibodies overnight at 4°C and then incubated with a HRP-coupled sheep anti-rat (dilution, 1:15,000; cat. no. SA001) or sheep anti-rabbit (dilution, 1:15,000; cat. no. SA009; both from Auragene Technology, Co., Inc.) secondary antibody at room temperature for 40 min. Signals were visualized with an enhanced chemiluminescence detection kit (Auragene Technology, Co., Inc.). Densitometric analysis was performed using Image-Pro Plus v. 6.0 (Media Cybernetics, Silver Spring, MD, USA).

Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After three 5-min washes with PBS, the cells were blocked in PBS containing 3% goat serum (OriGene Technologies, Inc., Beijing, China) and 0.5% Triton X-100 for 1 h at room temperature. Afterwards, the cells were incubated with the indicated primary antibodies at 4°C overnight and then incubated with Cy3-conjugated goat anti-rabbit IgG(H+L) secondary antibody (1:500) for 1 h at room temperature (catalog no. SA012; Auragene Technology, Co., Inc.). The coverslips were counter-stained with DAPI and imaged with a confocal laser scanning microscope (AE31; Motic Group, Co., Ltd., Xiamen, China).

Data from three independent experiments were expressed as the mean ± standard deviation and processed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Differences between groups were evaluated by one-way analysis of variance followed by Tukey’s test. P<0.05 was considered to indicate a statistically significant difference.

In accordance with previous studies (26,27), histological examination of lung tissues demonstrated that four weeks of exposure to bleomycin in mice led to significant pulmonary fibrotic changes, including disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts (Fig. 1A). However, Isor treatment (10 and 30 mg/kg) significantly attenuated the fibrotic changes induced by BLM (Fig. 1A), and the higher concentration exhibited better inhibitory effect on fibrosis, with the histological morphology being similar to the control mice. The hallmark characteristic of BLM-induced pulmonary fibrosis is the excessive deposition of extracellular matrix proteins, such as collagen. Collagen deposition was clearly observed in the lungs of BLM-treated mice by Sirius red staining (Fig. 1B). As expected, Isor treatment significantly attenuated matrix protein deposition in the lungs (Fig. 1B). In addition, α-SMA is a hallmark of myofibroblasts and a marker of pulmonary fibrosis. As illustrated in Fig. 1C, the protein expression levels of α-SMA and collagen I were markedly upregulated in the lungs of BLM-treated mice. Isor treatment significantly reduced the production of collagen I and α-SMA in BLM-treated lungs (Fig. 1C). The expression of α-SMA was also monitored by immunohistochemical analysis. BLM treatment resulted in markedly increased expression of α-SMA in the area of pulmonary fibrosis, while this effect was largely reversed in the Isor-treated groups (Fig. 1D).

Recent studies have demonstrated that pulmonary ERS is involved in the pathogenesis of BLM-induced pulmonary fibrosis (14-16). Therefore, the effects of Isor on BLM-induced pulmonary stress were analyzed. The protein expression levels of GRP78 and CHOP were examined, as both proteins are widely used as markers for ERS. As illustrated in Fig. 2A and B, the expression of GRP78 and CHOP was upregulated in the BLM-treated mice compared with the control mice, at both the protein and mRNA level. Isor treatment significantly attenuated the BLM-induced increase in GRP78 and CHOP expression, at both the transcriptional and translational level (Fig. 2A and B). These findings suggested that Isor treatment inhibited ERS, which was in accordance with a previous study (28). Immunohistochemistry staining for CHOP was performed in lung tissues for further confirmation. CHOP expression was decreased in Isor-treated BLM-induced lung tissues (10 and 30 mg/kg) compared with BLM-induced lung tissues (Fig. 2C).

ERS-induced EMT contributes to fibrotic remodeling in the lungs (15,29). Isor obviously inhibited ERS-induced EMT of myofibroblasts in the lungs, as evidenced by repression of pulmonary α-SMA expression described above. For further confirmation, expression of the EMT-related proteins E-cadherin, Vimentin and TGFβ1 was evaluated by western blotting. The results demonstrated a significant upregulation of Vimentin and TGFβ1 expression and downregulation of E-cadherin expression in the BLM-treated group compared with the control group (Fig. 3A-E). However, Isor treatment alleviated the increase in Vimentin and TGFβ1 expression and the decrease in E-cadherin expression, compared with the BLM-treated group (Fig. 3A-E). These findings indicated that Isor treatment alleviated ERS-mediated EMT in BLM-induced pulmonary fibrosis in vivo.

To investigate the possible pathways by which Isor inhibits ERS, the expression levels of total and phosphorylated PERK were determined by western blotting. PERK phosphorylation levels significantly increased in the BLM-induced group compared with the control group, while a dramatic decrease in p-PERK was observed in the Isor-treated BLM groups (Fig. 4A). Immunohistochemistry staining results for p-PERK in lung tissue sections supported the western blot analysis results (Fig. 4B). eIF2α is a downstream target of the PERK pathway. As illustrated in Fig. 4A, consistent with PERK phosphorylation, the levels of phosphorylated eIF2α in the lungs of BLM-treated mice were elevated dramatically. Isor treatment significantly attenuated BLM-induced eIF2α phosphorylation in the lungs (Fig. 4A). Thus, Isor exhibited an antifibrotic effect on BLM-induced pulmonary fibrosis in vivo.

To further validate the antifibrotic effect of Isor, in vitro studies were conducted in A549 cells and HBECs simulated with TGFβ1. Results obtained in vitro were similar to those observed in vivo. Treatment of cells with TGFβ1 increased the expression of fibrosis-related factors (collagen I and α-SMA; Fig. 5) and ER markers (GRP78 and CHOP; Fig. 6). However, Isor suppressed the upregulated expression of collagen I, α-SMA, GRP78 and CHOP in the presence of TGFβ1 in a dose-dependent manner (Figs. 5 and 6).

Exposure to TGFβ1 caused A549 cells and HBECs to undergo EMT, during which the morphology of A549 cells changed from polygonal or cobblestone-like to a spindle-like shape, and that of HBECs changed from a short fusiform shape to a long fusiform shape (Fig. 7A). Aside from the morphological changes, the expression levels of E-cadherin were decreased, while the expression levels of Vimentin were increased (Fig. 7B); these results were also confirmed by immunostaining analysis (Fig. 7C). By contrast, treating cells with Isor reversed the TGFβ1-induced EMT in a dose-dependent manner, as illustrated by phenotypic cellular alterations (Fig. 7C) and the expression profiles of the EMT markers E-cadherin and Vimentin (Fig. 7B). Taken together, the in vivo and in vitro data suggest that Isor protects against BLM-induced pulmonary fibrosis by inhibiting ERS-mediated EMT in the lungs.