EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-β signaling in lung fibroblasts

Seven-week-old male ICR mice (Nippon CLEA; Tokyo, Japan) weighing 30 g each on average were randomly assigned to groups. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin (Nippon Kayaku; Tokyo, Japan) was dissolved in normal saline solution (NS) and administered intravenously to ICR mice at a dosageof 100 mg/kg body weight (0.3 ml per mouse). EM703 (Kitasato Institute for Life Sciences, Tokyo, Japan) at 75 mg/kg body weight was suspended in 5% gum arabic (AG) (Wako Pure Chemical Industries; Tokyo, Japan) at 0.3 ml per mouse and orally administered by force with a microtube daily to ICR mice.

NS was administered intravenously to the mice treated with NS alone (day 0). Bleomycin was administered intravenously to mice treated with bleomycin alone and bleomycin plus treatment with EM703 (day 0). AG was orally administered daily to mice of the NS-alone and bleomycin-alone groups from day -3 until the time of death. EM703 was administered daily to the EM703-treated groups from day -3 until the time of death. The mice in all groups were sacrificed under etheranesthesia on day 7 after bleomycin or NS injection. All groups were examined for cell populations in the BAL fluid and for induction of mRNA of Smad3 and Smad4 in the lung tissues by RT-PCR on day 7 after bleomycin or NS injection.

The bleomycin-untreated groups included the NS-treated group [group 1] and the EM703-alone group [group 2]. The bleomycin-treated groups included bleomycin alone [group 3], bleomycin plus pretreatment with EM703 (day -3 to day 13) [group 4], and bleomycin plus post-treatment with EM703 (day 3 to day 20) [group 5]. NS was administered intravenously to the bleomycin-untreated mice (day 0). Bleomycin was administered intravenously to the bleomycin-treated mice (day0). AG was orally administered daily to the mice of groups 1 and 3 (day -3 to day 27). EM703 was orally administered dailyto the mice of group 2 (day -3 to day 27) and those of groups 4 (day -3 to day 13) and 5 (day 3 to day 20). The mice in all groups were sacrificed under etheranesthesia on day 28 after bleomycin or NS injection. Fibroticfoci were assessed histologically in the left lung tissues, and the hydroxyproline content in the right lung tissues was chemically determined.

For histological examination, 10% formalin-fixed lung tissues were embedded in paraffin. The paraffin sections were stained with either hematoxylin and eosin (HE) or Masson Trichrome (MT), and systematically scanned with a light microscope (OLYMPUS AX80, Tokyo, Japan). We compared the severity of interstitial fibrosis among the groups using the Ashcroft score [33].

The total collagen content of the right lung was determined by hydroxyproline (HOP) assay [34]. After acid hydrolysis of the right lung with 12N HCL at 100°C for 20 hours in a sealed glass tube (Iwaki Tokyo, Japan), the HOP content was determined by high-performance liquid chromatography (HPLC).

BAL fluid was obtained by the injection of 1 ml saline (three times, total 3 ml) followed by gentle aspiration of the fluid from the right and left lungs after securing an intratracheal catheter within a trachea. With this catheter, the ratios of the recovery of lavage fluid ranged from 70% to 80% and did not significantly differ among the groups. The total numbers of cells in the BAL fluid were counted with a hemocytometer. For differential counts of leukocytes in the BAL fluid, cytospin smear slides were prepared (Labsystems Japan) and stained with Giemsa solution (Merck Japan). Differential cell counts were performed on 200 cells per smear.

A murine lung fibroblast cell line, MLg2908 (ATCC, CCL-206), originating from ddY mice was maintained in Roswell Park Memorial Institute (RPMI1640, Immuno-Biological Laboratories Gunma, Japan) with 10% fetal calf serum (FCS, Eguitech-BIO, INC. Kerrville, TX). Cultures of it were grown in a 5% CO₂ humidified atmosphere at 37°C. The cell groups tested included those of group 1 (control), group 2 [presence of TGF-β (TGF-β₁, BD Biosciences, Bedford, MA) alone], and group 3 (presence of TGF-β and treatment with EM703).

Lung fibroblast cells were suspended at 5 × 10⁴/ml in RPMI1640 with 10% FCS and plated in 96-well plates at 100 μl per well in a 5% CO₂ humidified atmosphere at 37°C for incubation for 24 hr. The medium was changed to serum-free Dulbecco's Modified Eagle's Medium (DMEM, GIBCO™, Grand Island, NY, USA) in all groups, EM703 was added at various final concentrations for group 3 incubation for 24 hr. Thereafter, TGF-β was added at various final concentrations for group 2 and 3 incubation for 24 hr again. Each group's cells were incubated with a Cell Counting Kit-8 (DOJINDO; Tokyo, Japan) at 37°C for 3 hr. OD (450 nm) values were measured on a microplate reader (BIO-RAD Model 3550, Tokyo, Japan).

Lung fibroblast cells were suspended at 5 × 10⁴/ml in RPMI1640 with 10% FCS and plated in 24-well plates at 1 ml per well and incubated in a 5% CO₂ humidified atmosphere at 37°C. After 24 hr of incubation, the medium was changed to serum-free DMEM in all groups, and EM703 (5 μg/ml) was added for group 3 incubation for 24 hr. Thereafter, TGF-β (5 ng/ml) was added for group 2 and 3, followed by incubation for 24 hr again. The cells of each group were then washed once and resuspended to 1 × 10⁵ cells/ml in serum-free DMEM and plated in 24-well plates at 1 ml per well for incubation. After 24 hr of incubation, the supernatants were collected and measured for collagen concentration with a soluble collagen assay kit (Biocolor Ltd., UK).

The cells of group 3 were divided into three subgroups as follows: group 3a: presence of TGF-β and pre-treatment with EM703; group 3b: presence of TGF-βand syn-treatment with EM703; and group 3c: presence of TGF-β and post-treatment with EM703. Lung fibroblast cells were suspended at 2 × 10⁴/ml in RPMI1640 with 10% FCS and plated in 24-well plates at 1 ml per well for incubation in a 5% CO₂ humidified atmosphere at 37°C. After 48-hr of incubation, the medium was changed to serum-free DMEM, and EM703 (5 μg/ml) was added for group 3a, with incubation continued for 24 hr. Thereafter, TGF-β (5 ng/ml) was added to the cells of groups 2 and 3 (a, b, c). EM703 (5 μg/ml) was simultaneously added to the cells of group 3b. After 24 hr of incubation, EM703 (5 μg/ml) was added for group 3c, followed by incubation for an additional 24 hr. Each cell culture was examined for the expression of mRNA of Smad3 and Smad4 by RT-PCR and for expression of Smad3 and Smad4 protein assay by western blotting.

The cell groups tested included those of the control, the presence of TGF-β alone, and the presence of TGF-β and pre-treatment with EM703. Conducting the cell cultures and treatment with EM703 before the presence of TGF-β used the same method as the Smad3 and Smad4 protein assay in group 3a. The cells were cultured in the presence of TGF-β (5 ng/ml) for 15 min and 12 hr. Followed by the presence of TGF-β, the cells were collected and the expression of p-Smad2/3 protein was examined by western blotting.

Total RNA was extracted from each specimen of lung tissue (in vivo) and lung fibroblast cells (in vitro) using ISOGEN (Nippon GENE; Tokyo, Japan). The methods of RNA extraction and RT-PCR used have been previously described [6, 35]. For the amplification of the desired cDNA, the following gene-specific primers were used[36, 37]. Glucose-6-phosphate dehydrogenase(G6PD) was measured as an internal control [38].

Smad3: sense 5'-CTGGCTACCTGAGTGAAGATGGAGA-3',

antisense 5'-AAAGACCTCCCCTCCGATGTAGTAG-3'

Smad4: sense 5'-GTATAATGCCACCAGTACCACCAAC-3',

antisense 5'-TGACCCAAGCAAAAGCGATCTCCTC -3'

G6PD: sense 5'-TAGGAATTCATCATCATGGGTGCATCG-3'

antisense 5'-TAGAAGCTTGTTTGCGGATGTCAGCCACTGT-3'

The PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, and observed with ultraviolet transillumination. Intensity analysis of the bands was performed with Adobe Photoshop 6.0 (Adobe Systems; Tokyo, Japan), with the expression of Smad3 mRNA indicated by the number of white pixel areas.

Western blotting was used for the measurement of Smad3, Smad4 and p-Smad2/3 protein assay protein in lung fibroblast cell line MLg2908. Total protein 100 μg was separated by 10% SDS-PAGE. Transfer to polyvinylidene difluoride membranes by a blot appliance (AE-6677P, ATTO CORPORATION, Tokyo, Japan) was performed according to the manufacturer's instructions. After having been blocked with 5% skim milk (Snow Brand Milk Production, Japan), the membrane was incubated with anti-Smad3 antibody (sc-8332, Santa Cruz Biotechnology, Inc. USA; rabbit polyclonal antibody, dilution 1/200) for 1 hr. The membrane was washed, and primary antibody was detected using alkaline phosphatase-conjugated affinipure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc. USA; dilution 1/10000) incubated for 1 hr. After having washed the membrane, the Smad3 protein band was visualized using an alkaline phosphatase substrate. Smad4 protein was detected by anti-Smad4 antibody (sc-7966, Santa Cruz Biotechnology, Inc. USA; mouse monoclonal antibody, dilution 1/100), and primary antibody was detected using alkaline phosphatase-conjugated affinipure goat anti-mouse IgG+IgM (Jackson ImmunoResearch Laboratories, Inc. USA; dilution 1/10000). Smad4 protein was detected using the same method as Smad3. p-Smad2/3 protein was detected by anti-p-Smad2/3 (sc-11769, Santa Cruz Biotechnology, Inc. USA; goat polyclonal antibody, dilution 1/100), and primary antibody was detected using donkey anti-goat IgG horseradish peroxidase (HRP) (sc-2056, Santa Cruz Biotechnology, Inc. USA; dilution 1/5000). P-Smad2/3 protein was detected by the chemiluminescence system (Amersham Biosciences ECL plus Western Blotting Detection System).

Numbersof macrophages and neutrophils in BAL fluid were significantly increased on day 7 after bleomycin injection. The increases in number of macrophages and neutrophils in BAL fluid were significantly attenuated by EM703 (Figure 2).

Bleomycin-induced pulmonary fibrosis was significantly inhibited by treatment with EM703 on day 28 after bleomycin injection in ICRmice. A typical picture of the attenuation of fibrosis is shown in Figure 3. Of the groups treated with EM703, the Ashcroft scores were significantly reduced compared to those in the bleomycin-alone group (Figure 4a). The administration of EM703 alone resulted in no remarkable changes in the results of histopathologic assessment of lung tissue.

The concentration of hydroxyproline on day 28 after bleomycin injection was significantly higher in the bleomycin-alone group than in the NS-alone group. Of the groups treated with EM703, the hydroxyproline content was significantly reduced compared to that in the bleomycin-alone group. The administration of EM703 alone resulted in no remarkable changes in the hydroxyproline content of the lung tissue (Figure 4b).

TGF-β significantly increased MLg2908 proliferation (Figure 5a). The proliferation of MLg2908 induced by TGF-β was significantly inhibited by EM703 (Figure 5b).

TGF-β significantly increased the production of soluble collagen by MLg2908 cells. The increase in the measured concentration of soluble collagen induced by TGF-β was significantly inhibited by EM703 (Figure 6).

The expression of Smad3 mRNA was eliminated by bleomycin, but recovered to control level by treatment with EM703 on day 7 after bleomycin injection. The expression of Smad4 mRNA was attenuated by bleomycin, but recovered to a higher control level by treatment with EM703 on day 7 after bleomycin injection (Figure 7a).

The expression of Smad3 and Smad4 mRNA was completely eliminated by the addition of TGF-β. The elimination of the expression of Smad3 and Smad4 mRNA by TGF-β was reversed to higher than the control level by pre-treatment with EM703, but was not recovered by syn-treatment or post-treatment with EM703 (Figure 7b).

The expression of Smad3 protein in murine lung fibroblasts was not changed by TGF-β. The expression of p-Smad2/3 protein was increased by TGF-β. The increased expression of p-Smad2/3 protein by TGF-β exposure for 15 min was remarkably inhibited by pre-treatment with EM703, but the increased expression of p-Smad2/3 protein by TGF-β exposure for 12 hr was not inhibited by pre-treatment with EM703. The expression of Smad4 protein was increased by TGF-β. The increased expression of Smad4 protein by TGF-β was inhibited by pre-treatment with EM703, but not inhibited by syn-treatment or post-treatment with EM703 (Figure 8).