MS80, a novel sulfated oligosaccharide, inhibits pulmonary fibrosis by targeting TGF-beta1 both in vitro and in vivo

Abstract

Aim: The pro-fibrogenic cytokine transforming growth factor-beta 1 (TGF-beta1) has attracted much attention for its potential role in the etiology of idiopathic pulmonary fibrosis (IPF). Here, we demonstrate that MS80, a novel sulfated oligosaccharide extracted from seaweed, can bind TGF-beta1. The aim of the present study was to determine whether MS80 is capable of combating TGF-beta1-mediated pulmonary fibrotic events both in vitro and in vivo, and to investigate the possible underlying mechanisms.

Methods: Surface plasmon resonance was used to uncover the binding profiles between the compound and TGF-beta. MTT assay, flow cytometry, Western blot analysis, BCA protein assay and SDS-PAGE gelatin zymography were used to probe the antifibrotic mechanisms of MS80. The in vivo fibrotic efficacy was evaluated in a bleomycin instillation-induced rat model.

Results: We report that MS80, a new kind of sulfated oligosaccharide extracted from seaweed, inhibits TGF-beta1-induced pulmonary fibrosis in vitro and bleomycin-induced pulmonary fibrosis in vivo. Our results indicated that MS80 competitively inhibited heparin/HS-TGF-beta1 interaction through its high binding affinity for TGF-beta1. Moreover, MS80 arrested TGF-beta1-induced human embryo pulmonary fibroblast (HEPF) cell proliferation, collagen deposition and matrix metalloproteinase (MMP) activity. Intriguingly, MS80 deactivated both the ERK and p38 signaling pathways. MS80 was also a potent suppressor of bleomycin-induced rat pulmonary fibrosis in vivo, as evidenced by improved pathological settings and decreased lung collagen contents.

Conclusion: MS80 in particular, and perhaps oligosaccharide in general, offer better pharmacological profiles with appreciably few side effects and represent a promising class of drug candidates for IPF therapy.Acta Pharmacologica Sinica (2009) 30: 973-979; doi: 10.1038/aps.2009.86; published online 22 June 2009.

Figure 1

Chemical structure of MS80.

Figure 2

MS80 competitively inhibits the interaction between TGF-β1 and heparin. (A) The interaction between MS80 and TGF-β1 occurred in a concentration-dependent manner and the concentrations of TGF-β1, from bottom to top, were 22.5, 45, 90, 180, and 360 nmol/L. (B) Inhibitory effect of MS80 (from top to bottom: 0, 0.05, 0.1, 0.2, 0.4, and 0.8 μg/mL) on the binding of TGF-β1 (200 nmol/L) to heparin immobilized onto the biosensor chip surface. The experiment was carried out at 25°C with a flow rate of 15 μL/min. The data are a representative of three independent experiments.

Figure 3

Effects of MS80 on TGF-β1-induced fibrogenic activities. (A) Concentration-dependent effects of TGF-β1 on HEPF proliferation. (B) Effects of MS80 on TGF-β1 (5 ng/mL) induced cell proliferation were examined with the MTT assay; (C) Effects of MS80 on cell cycle of TGF-β1-treated HEPF cells. Serum-starved cells were seeded in 6-well plates and treated with TGF-β1 (5 ng/mL). After harvesting, the cells were incubated with PI for 30 min and analyzed with a flow cytometer. (D) Effect of MS80 on TGF-β1-induced collagen synthesis. Serum-starved cells were treated with TGF-β1 (5 ng/mL) and indicated concentrations of MS80 for 48 h. Collagen levels were measured with the collagen assay kit and are presented as collagen/total protein (%). (E) Effects of MS80 on TGF-β1-induced MMP inhibition. The effect of MS80 on 5 ng/mL TGF-β1- induced MMP decrement was determined using SDS-PAGE gelatin zymography. (F) Effects of MS80 on the phosphorylation of ERK and p38 MAP kinase. The HEPF cells were seeded in 6-well plates (9×10⁴ cells/well). Serum-starved cells were exposed for 12 h to MS80 (0, 25, 50, and 100 μg/mL) then treated with or without TGF-β1. Cells were then harvested, washed twice with cold PBS (pH 7.4) and lysed in 0.4 μL sample buffer on ice for 30 min. Subsequently, cell lysates were subjected to Western blotting using phospho-specific, control ERK1/2, p38 and β-actin antibodies. Four parallel samples were prepared in each group. Data are shown as means±SD (n=4). ^cP<0.01 vs control; ^fP<0.01 vs TGF-β1.

Figure 4

Effects of MS80 on BALF-induced HEPF proliferation and collagen synthesis. (A) Concentration-dependent effects of BALF on HEPF proliferation. (B) Effects of MS80 on BALF-induced cell proliferation were detected with the MTT assay. (C) Effect of MS80 on BALF-induced collagen synthesis. Serum-starved cells were treated with 100 μL/well BALF and indicated concentrations of MS80 for 48 h. The collagen was measured with the collagen assay kit and calculated using collagen/total protein (%). Four parallel samples were prepared in each group. Data are shown as means±SD (n=4). ^cP<0.01 vs control; ^fP<0.01 vs BALF.

Figure 5

Effects of MS80 on pulmonary fibrosis in vivo. (A) Morphological evaluations of lungs were made by immunohistochemistry. a, control; b, bleomycin; c, d, e, MS80 of 25, 50, and 100 mg/kg, respectively. (B) Hydroxyproline level of lungs were tested with a hydroxyproline assay kit. Eight parallel samples were prepared in each group and the data shown are representative of three independent experiments with similar results. Data are shown as means±SD (n=8). ^cP<0.01 vs control; ^eP<0.05, ^fP<0.01 vs bleomycin.