Thalidomide has a therapeutic effect on interstitial lung fibrosis: evidence from in vitro and in vivo studies

Abstract

The objective of this study was to investigate the effects of thalidomide (THD) on interstitial lung fibrosis (ILF). In vitro, human fetal lung fibroblast (HFL-F) to myofibroblast (MF) trans-differentiation was induced by transforming growth factor (TGF)-beta1. The effects of THD on trans-differentiation process or differentiated MF were evaluated by measuring hydroxyproline (HYP) content by alkaline hydrolysis colorimetry, alpha-smooth muscle actin (alpha-SMA) protein by Western blot and alpha-SMA and pro-collagen III mRNA expressions by semi-quantitative reverse transcription-polymerase chain reaction; in vivo, a mouse model of ILF was generated by daily subcutaneous injection of bleomycin (BLM) in female C3H mice. Gastric perfusion of THD began 1 week prior to injection and lasted for 8 weeks. Lung specimens were harvested at different time-points (1, 4, 6 and 8 weeks) for pathology and immunohistochemistry examination. The HYP content, alpha-SMA and pro-collagen III mRNA expressions were also assessed. THD inhibited the up-regulation of HYP protein, pro-collagen III mRNA and alpha-SMA protein induced by TGF-beta1 in HFL-F cells, and additionally inhibited pro-collagen III mRNA expression on trans-differentiated MF. THD reduced HYP synthesis in the lung tissues of BLM-treated mice at week 4, and slightly reduced the numbers of alpha-SMA-positive cells. THD had an effect on ILF models both in vitro and in vivo.

Figures

Fig. 2

Hydroxyproline (HYP), α-smooth muscle actin (α-SMA) protein, α-SMA and pro-collagen III mRNA expressions in human fetal lung fibroblast (HFL-F) treated by transforming growth factor (TGF)-β1 with or without thalidomide (THD) or phosphate-buffered saline only (each experiment was repeated at least three times, data were shown as mean ± standard deviation). (a) Relative levels of HYP protein, α-SMA mRNA and pro-collagen III mRNA; (b) reverse transcription–polymerase chain reaction: α-SMA and pro-collagen III mRNA; (c) Western blot: α-SMA protein.

Fig. 1

Effects of thalidomide (THD) on morphological transformation of myofibroblast (MF) induced by transforming growth factor (TGF)-β1. Human fetal lung fibroblast (HFL-F) were incubated for 4 days with 0·1% fetal bovine serum (a), TGF-β1 5 µg/l (b), TGF-β1 5 µg/l and THD 50 µg/l (c). Fluorescein isothiocyanate stain (α-smooth muscle actin) ×200.

Fig. 3

Relative levels of hydroxyproline (HYP), α-smooth muscle actin (α-SMA) protein, α-SMA and pro-collagen III mRNA expressions in trans-differentiated myofibroblast (MF) treated by thalidomide (THD) or phosphate-buffered saline (PBS) (each experiment was repeated at least three times, data were shown as mean ± standard deviation). Group 1: human fetal lung fibroblast (HFL-F) was cultured with PBS for 144 h; group 2: HFL-F was cultured with 5 µg/l transforming growth factor (TGF)-β1 for 96 h, then TGF-β1 was removed and cells were incubated with PBS for additional 48 h; group 3: HFL-F was cultured with 5 µg/l TGF-β1 for 96 h, then TGF-β1 was removed and cells were incubated with 50 µg/l THD for another 48 h. (a) Relative levels of HYP protein, α-SMA mRNA and pro-collagen III mRNA; (b) reverse transcription–polymerase chain reaction: α-SMA and pro-collagen III mRNA; (c) Western blot: α-SMA protein.

Fig. 4

Haematoxylin and eosin staining and immunohistochemistry staining of the lung tissues in mice (×100). (a–c) Haematoxylin and eosin staining; (d–f) immunohistochemistry staining of α-smooth muscle actin (α-SMA). (a,d) Group n; (b,e) group bleomycin (BLM); (c,f) group thalidomide (THD). Group n: mice injected with phosphate-buffered saline subcutaneously and gastric perfused with carboxymethyl cellulose (CMC); group BLM: mice injected with BLM subcutaneously and gastric perfused with CMC; group THD: mice injected with BLM subcutaneously and gastric perfused with THD.