Curdione ameliorates bleomycin-induced pulmonary fibrosis by repressing TGF-β-induced fibroblast to myofibroblast differentiation

Sixty male C57BL/6 mice (8 weeks old) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in a specific pathogen-free (SPF) animal facility at the Tongji Medical College with sterile acidified water and irradiated food. The Institutional Animal Care and Use Committee of Tongji Hospital (Wuhan, China) approved the protocols used for the animal experiments in the present study.

Curdione was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China), and Bleomycin was obtained from MedChemExpress LLC (NJ, USA). Antibodies against β-actin, Gapdh, α-SMA, fibronectin, collagen I, p-Jnk, Jnk, p-p38, p38, p-Erk1/2, Erk1/2, p-Smad2, p-Smad3, Smad2/3 were purchased from Cell Signal Technology Inc. (MA, USA). BCA Protein Assay Kit was supplied by Boster Biological Technology co. Ltd. (Wuhan, China). RT-PCR assay kit was obtained from Takara (Dalian, China). Hydroxyproline assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Cell Counting Kit-8 was available from DojindoChemical technology co. LTD (Shanghai, China).

All animals were divided randomly into four groups: (i) PBS group, (ii) DMSO group, (iii) BLM + DMSO group, (iiii) BLM + curdione (BLM + CUR) group. For BLM + DMSO group or BLM + CUR group, mice were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg) and then intratracheally administered 1.75 mg/kg BLM in sterile PBS using established techniques [10]. Next, mice were treated intraperitoneally with curdione (100 mg/kg, dissolved in 10%DMSO) or 10%DMSO every 2 days following BLM injection. Mice were sacrificed under anesthesia on the 21st day (Fig. 1 b).

HPFs were purchased from ScienCell Research Laboratories, Inc. (Nanjing, China). HPFs were cultured in fibroblast medium supplemented with 10% fetal calf serum,1% FGS and 1% solution of penicillin and streptomycin at 37 °C in a humid atmosphere containing 5% CO₂. HPFs were treated with stimulated with curdione (160 μM or 300 μM) or equal amount of DMSO 1 h before stimulation with human recombinant TGF-β1 (10 ng/ml, PeproTech, NJ, USA) for 48 h.

Cell viability was assayed by the Cell Counting Kit-8. Briefly, cells were seeded in 96-well plates at 4 × 10³ cells per well, then subjected to serum starvation for 24 h by reducing FBS to 2%. Next, the cells were treated different concentrations of curdione (100 μM, 200 μM, 300 μM, 400 μM, 500 μM) or 2‰ of DMSO for 1 h and 48 h, and then 5 mg/ml CCK8 was added to the wells and incubated for additional 2 h. After mix well, the optical density was measured at 450 nm.

Lung specimens were washed with PBS and hydrolyzed with 0.6% hydrochloric acid at 95 °C for 5 h. Hydrolysates were neutralized with sodium hydroxide and diluted with distilled water. Hydroxyproline level in hydrolysates was colorimetrically determined by absorbance at 550 nm with p-dimethylaminobenzaldehyde and expressed as μg/mg wet tissue.

The left lungs were fixed by intratracheal infusion of 4% aqueous buffered formalin for 24 h, then subjected to paraffin embedding and sliced into 4 μm sections. After dewaxing, the sections were subjected to hematoxylin and eosin (H&E), Sirius red and Masson staining using established techniques [10]. The severity of lung fibrosis was assessed by two pathologists using a brightfield (Olympus, Tokyo, Japan) in a blinded fashion via Ashcroft scoring system. For immunostaining, after dewaxing to water, the sections were co-incubated with primary antibodies against CD3 (1:100) or F4/80(1:100), followed by probing with Horseradish peroxidase labeled secondary antibodies.

Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA). RNA quantity and quality were measured using the NanoDrop 2000 spectrophotometer (Thermo scientific, MA, USA). Complementary DNA synthesis was performed using the M-MLV reverse transcriptase kit (Invitrogen, CA, USA) as previous reported [11]. RT-PCR was performed using a SYBR green-based PCR master mix kit (Takara, Dalian, China) on a Rotor Gene 3000 real-time PCR system from Corbett Research (Sydney, Australia). The following primers were used for each target gene: Fibronectin (5′-GAT GTC CGA ACA GCA GCT TAT TTA CCA-3′, 5′- CCT TGC GAC TTC AGC CAC T-3′); Collagen 1 (5′- TAA GGG TCC CCA ATG GTG AGA-3′, 5′- GGG TCC CTC GAC TCC TAC AT-3′); α-SMA (5′- GGA CGT ACA ACT GGT ATT GTG C -3′, 5′- TCG GCA GTA GTC ACG AAG GA-3′); and β-actin (5′- GCC ACA GCA CTC CAT CGA C-3′, 5′-GTC TCC GAT CTG GAA AAC GC-3′).

Lung tissues and cultured cells were homogenized in RIPA lysis buffer supplemented with a protease inhibitor cocktail, as previously described [12]. The proteins were subjected to western blotting with the indicated primary antibodies and detected by chemiluminescencec (Advansta, CA, USA).

For the Immunofluorescence assay, the slides were first probed with Fibronectin or Collagen 1 or α-SMA antibody at 4 °C overnight. After rinsed with PBS, cells were incubated with Alexa 488-conjugated anti-rabbit antibody (Invitrogen, CA, USA), as previously described [13]. The sections were immediately subjected to fluorescence analysis under a fluorescence microscope.

Liver function and renal function was assessed by measuring blood Alanine aminotransferase, Aspartate aminotransferase, urea and serum creatinine at the Department of Clinical Laboratories of Tongji Hospital.

We first sought to evaluate the effects of curdione on the development of pulmonary fibrosis. For this purpose, the mice were administered with curdione for every 2 days, followed by BLM injection as described [10]. First, the toxic effects of curdione to the mice and cells were detected. Notably, curdione did not seem to exist toxic effects on the mice, as we failed to detect perceptible differences of liver function and renal function (Fig. 2 a-d). Furthermore, curdione may not affect the cells viability, even if 500 μM of curdione lasted for 48 h, a very high dose and long enough stimulating time, was administrated to the cells (Fig. 2 e-f). Next, histopathological changes of the lungs were observed with HE, Masson and Sirius red staining to evaluate pulmonary morphology. Interestingly, compared with the BLM + DMSO groups, the BLM + CUR group showed significantly attenuated lung injury, as evidenced by H&E staining (Fig. 3 a). Significantly alleviated pulmonary fibrosis were noted in BLM + CUR group as shown by the Masson staining (Fig. 3 b) and Sirius red staining (Fig. 3 c). In particular, the Ashcroft scores of lung fibrosis was lower in BLM + CUR group compared with BLM + DMSO group (Fig. 3 d). In addition, both BLM-induced mice were undergoing significant weight loss on day 7 of BLM induction compared to mice from the control group. However, mice treated with curdione had a less weight loss than mice from the BLM + DMSO group at all examined time points (Fig. 3 e). Furthermore, from the immunohistochemical results, the infiltration level of inflammatory cells (lymphocytes and macrophages) in the curdione treatment group was also significantly lower than that in the BLM + DMSO group (Additional file 1: Figure S1).

To confirm the above data, levels of hydroxyproline, an amino acid formed upon hydrolysis of connective-tissue proteins such as collagen, were detected in all group. Indeed, compared with control group, significantly higher levels of hydroxyproline were detected in BLM-induced mice, while curdione administration attenuated BLM-induced hydroxyproline content by 1.6-fold (Fig. 3 f).

To further evaluate the effects of curdione on pulmonary fibrosis, western blot analysis of fibrotic markers was adopted in lung homogenates. Consisted with above data, the expression of fibronectin, collagen 1 and α-SMA was lower in BLM + CUR group (Fig. 4 a) than BLM + DMSO group. Similarly, RT-PCR analysis and immunofluorescence analysis of fibronectin, collagen 1 and α-SMA expression all showed consistent results (Fig. 4 b-e).

Given the important role that fibroblast to myofibroblast differentiation plays in the progression of pulmonary fibrosis [14], we examined differentiation of fibroblast in the lungs of the mice by western blot analysis, RT-PCR analysis and immunofluorescence. Interestingly, mice treatment with curdione displayed substantially lower α-SMA positive cells after BLM induction than the mice originating from BLM + DMSO group (Fig. 4 e), indicating that administration of curdione may reduce the differentiation of fibroblasts in vivo.

Based on the above observations, HPFs were used to validate the effects of curdione on fibroblast differentiation. Interestingly, administration of curdione significantly inhibited fibroblast differentiation to myofibroblast in dose dependent manner as evidenced by the significantly reduced levels of fibronectin, collagen 1 and α-SMA after TGF-β1 treatment for 48 h analyzed by western blot (Fig. 5 a), RT-PCR (Fig. 5 b - d) and immunofluorescence (Fig. 5 e).

TGF-β/Smad signaling has been suggested to be critical for an optimal and sustained fibroblast differentiation upon TGF-β stimulation [15]. Therefore, we examined the impact of curdione on TGF-β/Smad signaling in TGF-β1-stimulated HPFs. Interestingly, TGF-β1 treatment significantly induced Smad activation as manifested by increasing of p-Smad2 and p-Smad3 levels, while curdione administration significantly attenuated TGF-β1-induced p-Smad3 activation, but not p-Smad2 (Fig. 6 a). Furthermore, we also detected others Smad signal pathway proteins by RT-PCR (Fig. 6 b-d). Interestingly, the expression of Smad7, which could inhibit p-Smad3, was increased following curdione treatment. Therefore, we speculate that up-regulation of Smad7 is a potential intermediate mechanism for the down-phosphorylation of Smad3 by curdione. Of note, MAPK signaling are also implicated in fibroblast differentiation [16]. However, no differences were detected in terms of phosphorylated forms (p-P38, p-Jnk, p-Erk1/2) between all experimental groups after TGF-β1 induction for 1 h (Fig. 6 e).