Contributed equally
Pulmonary fibrosis is an aggressive end-stage disease. Transforming growth factor-β1 (TGF-β1) mediates lung fibroblast activation and is essential for the progress of pulmonary fibrosis. BML-111, a lipoxinA4 (LXA4) receptor (ALX) agonist, has been reported to possess anti-fibrotic properties. The present study aimed to elucidate whether BML-111 inhibits TGF-β1-induced mouse embryo lung fibroblast (NIH3T3 cell line) activation
Pulmonary fibrosis is a common occurrence in the final stages of various lung diseases, including acute lung injury, drug reactions, sarcoidosis and autoimmune disease. Due to the lack of timely and effective intervention, the majority of patients who succumb to the disease exhibit respiratory failure 3-5 years following diagnosis (
Although the underlying mechanisms of pulmonary fibrosis are complex, previous studies have demonstrated that lung fibrosis develops from the maladaptive regulation of repair processes following lung injury and inflammation, where various profibrotic factors precipitate the formation of α smooth muscle actin (α-SMA)-expressing myofibroblasts, which in turn synthetize and secrete immoderate extracellular matrix (ECM) components, replacing normal lung tissue and driving lung fibrosis (
Previous studies have revealed a strong association between inflammation, fibroblast activation and lung fibrosis (
In the present study, it was demonstrated that BML-111 reduces the expression of α-SMA, fibronectin and total collagen induced by TGF-β1 in NIH3T3 cells, and that it interferes with TGF-β1 associated signaling pathways. The results of the current study indicated that BML-111 inhibits the activation of fibroblasts and exerts direct anti-fibrotic affects. In addition, BML-111 treatment markedly improved murine survival rates in the BLM intratracheal mouse model, while BOC-2 (N-tertbutyloxy-carbonyl-phenyalanine-le-ucyl- phenyalanine-leucyl-phenyalanine) partially weakened the effects of BML-111. Furthermore, it was concluded that BML-111 alleviates BLM-induced pulmonary fibrosis by binding to ALX, and that these mechanisms may be involved in the anti-inflammatory response and in the inhibition of fibroblast activation.
NIH3T3 cells were obtained from China Center for Type Culture Collection (Wuhan, China) and were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) to 75% confluence. The cells were then serum-starved for 12 h prior to each experiment. To select an optimal concentration of BML-111 (Cayman Chemical, Ann Arbor, MI, USA), cells were treated with varying concentrations (1, 10, 100, 200 and 500 nM) of BML-111 or vehicle (0.035% methanol) for 30 min at 37°C prior to the addition of 5 ng/ml TGF-β1 (PeproTech Inc., Rocky Hill, NJ, USA) for 24 h at 37°C. Although BML-111 at concentrations of 1 and 10 nM did not appear to affect a-SMA protein levels, the other concentrations of BML-111 substantially suppressed TGF-β1-induced a-SMA expression, with 200 and 500 nM concentrations producing the most notable effects. Notably, there was no substantial difference between these two concentrations. Therefore 200 nM BML-111 was selected for subsequent experiments. To assess whether the action of BML-111 is associated with ALX, 10
Total RNA was isolated from cultured cells using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA reverse transcription was performed using an ReverTra Ace kit (Toyobo Life Science, Osaka, Japan). Briefly, the reaction was incubated in steps of 65°C for 5 min, 37°C for 15 min, 95°C for 5 min and held at −20°C. The amplified products of PCR were resolved using 2% agarose gel electrophoresis. The primers utilized were as follows: 5′-GGC AAC TCT GTT GAG GAA AG-3′ and 5′-GGCTCTCGGTAGACGAGA-3′ for ALX homeobox 1 (ALX1)/formyl peptide receptor related sequence 1 (FPR-rs1); and 5′-GTC AA-G ATC AAC AGA AGA AAC C-3′ and 5′-GGG CTC TCT CAA GAC TAT AAG G-3′ for ALX homeobox 2 (ALX2)/formyl peptide receptor 2 (FP-R2); and 5′-CTG AGA GGG AAA TCG TGC GT-3′ and 5′-CCA CAG GAT TCC ATA CCC AAG A-3′ for actin (
For the detection of the expression of FPR2, NIH3T3 cells were cultured in DMEM at 37°C for 24 h on sterile glass cover slips in 6-well plates and treated as aforementioned. Cells were then fixed with 4% paraformaldehyde. Following permeabilization, washing and blocking, the cells were incubated with rabbit anti-FPR2 anti-bodies (1:100; cat. no. AFR-002; Alomone Labs, Jerusalem, Israel) at 4°C overnight and then washed and incubated with a fluorescent-labeled secondary antibody (fluorescein isothiocyanate-labelled goat anti-rabbit immunoglobulin G; 1:50; cat. no. AS1110; Aspen Biological, Wuhan, China) for 45 min at 37°C. After washing again, over slips were mounted with anti-fade mounting medium (Beyotime Institute of Biotechnology, Haimen, China) on slides, and observed using a confocal microscope (Olympus FluoviewFV500).
Total protein was extracted using a Total Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentration was determined using a BCA Protein Assay kit (cat. no. KGP902; KeyGen Biotech Co., Ltd). A total of 30
A total of 100
A total of 100
C57BL/6 male mice (age, 6-8 weeks; weight, 20-25 g; n=76) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and housed in a specific pathogen-free animal facility. The mice were maintained under pathogen-free conditions, constant temperature, 22±2°C; humidity, 40-50%; 12 h light/dark cycle and were given food and water
The use of mice within the present study was reviewed and approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (Huazhong, China). All animal studies (including the mice euthanasia procedure) were completed in compliance with the regulations and guidelines of Huazhong University institutional animal care and conducted according to the AAALAC and the IACUC guidelines.
Lung samples were fixed in 10% formalin for 24 h at room temperature, embedded in paraffin and sectioned onto slides at a thickness of 4-5
Lung tissues were minced and hydrolyzed in 0.5 ml of 6 mol/l HCl for 6 h at 100°C. After adjusting the pH to 6.0-6.8, activated carbon was added to hydrolyzation products (25 mg activated carbon with 4 ml diluted hydrolysate) diluted with distilled water. Samples were centrifuged at 1,445.5 × g for 10 min at room temperature and the supernatant was used to measure the hydroxyproline content with a Hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; cat. no. A030-3) according to the manufacturer’s protocol.
BALF was acquired according to a previously described method (
Survival rates were evaluated using the log-rank (Mantel-Cox) test. Results were expressed as mean ± standard deviation and analyzed using one-way analysis of variance analysis followed by a Bonferoni post hoc test. Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analysis. P<0.05 was considered to indicate a statistically significant result.
BML-111 functions by binding to its corresponding receptor. As detected by PCR, NIH3T3 cells expressed rs1 and FPR2 (
Smad-dependent and -independent signaling pathways mediate the pro-fibrotic effects of TGF-β1. To assess whether BML-111 mediated fibrosis is regulated by these pathways, the effect of BML-111 on TGF-β1-induced Smad2, Smad3, ERK and Akt phosphorylation in NIH3T3 cells were analyzed using western blotting. The results demonstrated that BML-111 significantly reduces pSmad2, pSmad3, pERK and pAkt levels in cells stimulated by TGF-β1 (
The survival rate of mice was monitored for 21 days following BLM injection. As presented in
BML-111 demonstrated a high efficiency in protecting lungs from fibrosis. As observed on H&E and Massion-stained slides (
Lung fibrosis can be divided into two stages: The inflammatory and fibrotic stage. During the inflammatory stage, inflammatory cells infiltrate into the area of injury, attempt to clear tissue debris, and replace damaged cells (
BML-111 is a commercially stable ALX agonist, which possesses excellent anti-inflammatory and pro-resolving action, similar to that of LXA4. BML-111 suppresses pulmonary inflammatory reactions in ventilator/hemorrhagic shock/LPS-induced lung injury (
TGF-β1 mediated fibroblast activation occurs primarily via Smad-dependent and independent pathways. To further investigate the mechanisms by which BML-111 inhibits TGF-β1-mediated NIH3T3 cell activation, the downstream components of TGF-β1 signaling were determined. The results indicated that BML-111 inhibits TGF-β1-induced phosphorylation of Smad2 and Smad3. Previous studies have also demonstrated that phosphorylated Smad2 and Smad3 integrate with Smad4 and translocate into the nucleus from the cytoplasm, where they ultimately activate the transcription of pro-fibrotic genes, including collagen I, fibronectin and α-SMA through reaction cascades (
BLM is widely used to induce pulmonary fibrosis in mice. Following BLM instillation, mice are infected with acute alveolitis within 2-3 days, where upon profibrosis media is released, initiating ECM synthesis and the progression to fibrosis (
To conclude, the present study demonstrated that BML-111 treatment suppresses TGF-β1-induced fibroblast α-SMA protein synthesis and total collagen and fibronectin expression, by suppressing Smad-dependent and Smad-independent signaling pathways. Furthermore, BML-111 inhibited TGF-β1 levels and the synthesis of inflammatory mediators in BLM-induced pulmonary fibrosis. These results indicate that BML-111 may be used as a potential agent for the treatment of pulmonary fibrosis.
The present study was supported by grants obtained from the National Natural Science Foundation of China (grant nos. 30930089, 81372036, 81671890, 81500064, 81601669 and 81500436) and the Key Clinical Project of Ministry of Health of China (grant. No. 2010-47).
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.
YDJ, SLY and YS produced substantial contributions to the conception and design of the present study. YDJ, ZLL, CXC, BL and JG performed the experiments. ZLL, YXW and LC analyzed the data. YDJ and ZLL drafted the paper. YS edited and revised the manuscript. All authors read and approved the final manuscript.
The use of mice within the present study was reviewed and approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (Huazhong, China).
Not applicable.
The authors declare that they have no competing interests.
Not applicable.
BML-111 decreased TGF-β1-induced NIH3T3 cell α-SMA expression in a dose-dependent manner. (A) NIH3T3 cells express rs1 and (B) FPR2. Cells were pretreated with a vehicle (0.035% ethanol) or BML-111 (1, 10, 100, 200 and 500 nM) for 30 min and then treated with TGF-β1 (5 ng/ml) for 24 h. (C) The expression of α-SMA was assessed using western blotting and (D) quantified. Similar results were obtained from at least 3 sections. Data are expressed as the mean ± standard deviation. #P<0.05 and ##P<0.01 vs. the vehicle group. *P<0.05 and **P<0.01 vs. the TGF-β1 group in the absence of BML-111. Magnification, ×200. TGF-β1, Transforming growth factor-β1; α-SMA, smooth muscle α actin; rs1, related sequence 1; FPR2, formyl peptide receptor; marker 1, Trans DNA ladder (Tiangen Biotech, Co., Ltd., Beijing, China).
BML-111 suppressed TGF-β1-induced NIH3T3 cell activation. Cells were pretreated with a vehicle (0.035% ethanol) or BML-111 (200 nM) for 30 min in the absence or presence of BOC-2 (10
BML-111 inhibited TGF-β1-induced NIH3T3 cell Smad-dependent and Smad-independent signaling. NIH3T3 cells were stimulated using 5 ng/ml TGF-β1 in the absence or presence of 200 nM BML-111 (added 30 min prior to experimentation). Levels of (A) Smad2/3 and phosphorylated (B) Smad2/(C) Smad3 were assessed. (D) ERK and (E) phosphorylated ERK, (F) Aktand (G) phosphorylated Aktwere also analyzed using western blotting, 24 h following cell stimulation. The figures are representative results of three independent experiments. Data are expressed as mean ± standard deviation. ##P<0.01 vs. the vehicle group. **P<0.01 vs. the TGF-β1 group in the absence of BML-111. TGF-β1, Transforming growth factor-β1; Smad2/3, mothers against decapentaplegic homolog 2/3; ERK, extracellular signal-regulated kinase.
BML-111 treatment improved mortality after BLM instillation. Mice received intratracheal injection of 50
BML-111 treatment mitigated the destruction of lung architecture and production of TGF-β1, TNF-α and IL-1β in BALF following BLM iniection. Mice were treated with 50
BML-111 treatment inhibited α-SMA expression and ECM deposition in lungs following BLM injection. Mice treated with 50