The calpain inhibitor calpeptin prevents bleomycin-induced pulmonary fibrosis in mice

Abstract

Pulmonary fibrosis is characterized by progressive worsening of pulmonary function leading to a high incidence of death. Currently, however, there has been little progress in therapeutic strategies for pulmonary fibrosis. There have been several reports on cytokines being associated with lung fibrosis, including interleukin (IL)-6 and transforming growth factor (TGF)-β1. We reported recently that two substances (ATRA and thalidomide) have preventive effects on pulmonary fibrosis by inhibiting IL-6-dependent proliferation and TGF-β1-dependent transdifferentiation of lung fibroblasts. Rheumatoid arthritis is a chronic autoimmune disorder, and its pathogenesis is also characterized by an association with several cytokines. It has been reported that calpain, a calcium-dependent intracellular cysteine protease, plays an important role in the progression of rheumatoid arthritis. In this study, we examined the preventive effect of Calpeptin, a calpain inhibitor, on bleomycin-induced pulmonary fibrosis. We performed histological examinations and quantitative measurements of IL-6, TGF-β1, collagen type Iα1 and angiopoietin-1 in bleomycin-treated mouse lung tissues with or without the administration of Calpeptin. Calpeptin histologically ameliorated bleomycin-induced pulmonary fibrosis in mice. Calpeptin decreased the expression of IL-6, TGF-β1, angiopoietin-1 and collagen type Iα1 mRNA in mouse lung tissues. In vitro studies disclosed that Calpeptin reduced (i) production of IL-6, TGF-β1, angiopoietin-1 and collagen synthesis from lung fibroblasts; and (ii) both IL-6-dependent proliferation and angiopoietin-1-dependent migration of the cells, which could be the mechanism underlying the preventive effect of Calpeptin on pulmonary fibrosis. These data suggest the clinical use of Calpeptin for the prevention of pulmonary fibrosis.

Fig. 1

Effect of calpain inhibitor Calpeptin on bleomycin-induced pulmonary fibrosis. Eight-week-old mice were injected intraperitoneally with or without [dimethylsulphoxide (DMSO) only] Bleo on days 1, 8 and 15. Calpeptin (0·04 mg/mouse/day) (Bleo+ Calpeptin) or distilled water alone (Bleo + DMSO) was administered intraperitoneally three times a week during the time–course. On day 28, the mice (n = 5 in each experiments) were killed. Histological changes were demonstrated by haematoxylin and eosin (H&E), Azan and calpain staining (original magnification: ×200) (a). Real-time reverse transcription–polymerase chain reaction was performed to determine the changes in the lung tissues of mice in the mRNA levels of Calpain1 (b) and Calpain2 (c). All results are indicated as the mean ± standard deviation of three separate experiments.

Fig. 2

Effect of calpain inhibitor Calpeptin on the interleukin (IL)-6/IL-6R system. WI38VA-13 and IMR90 cells were cultured with or without Calpeptin (1–100 nM) for 48 h and cell proliferation was assayed (a). IL-6 concentration in the culture supernatants for 24 h (b), and the changes in mRNA levels for 7 h for IL-6 (c) and IL-6R (d) of WI38VA-13 cells in the presence or absence of 100 nM Calpeptin were measured by enzyme-linked immunosorbent assay and real-time reverse transcription–polymerase chain reaction (RT–PCR), respectively. WI38VA-13 cells were cultured in the presence or absence of IL-6 (1000 pg/ml) with or without Calpeptin (100 nM) for 96 h and cell proliferation was assayed (e). Real-time RT–PCR was performed to determine the changes in mRNA levels of IL-6 in the lung tissues of mice (f). All results are indicated as the mean ± standard deviation of three separate experiments.

Fig. 3

Inhibitory effect of calpain inhibitor Calpeptin on the Ang-1/Tie-2 system. WI38 VA13 cells were cultured in the presence or absence of 100 nM Calpeptin for 7 h and real-time reverse transcription–polymerase chain reaction (RT–PCR) was performed to determine the changes in mRNA levels for Ang-1 (a) and Tie-2 (b). WI38 VA13 cells were precultured overnight with or without Calpeptin (100 nM) and cultured further in the presence or absence of Ang-1 (100 ng/ml) and a cell migration assay was performed (c). Real-time RT–PCR was performed to determine the changes in the lung tissues of mice in the mRNA levels of Ang-1 (d) and Tie-2 (e). All results are indicated as the mean ± standard deviation of three separate experiments.

Fig. 4

Effect of calpain inhibitor Calpeptin on collagen synthesis and production of transforming growth factor (TGF)-β1. WI38 VA13 cells were cultured in the presence or absence of 100 nM of Calpeptin for 24 h, and the supernatants of the cells were collected and examined for the amount of collagen (a) using a Sircol soluble collagen assay and TGF-β1 concentration (b) was measured by enzyme-linked immunosorbent assay. WI38 VA13 cells were cultured in the presence or absence of 100 nM Calpeptin for 7 h and real-time reverse transcription–polymerase chain reaction (RT–PCR) was performed to determine the changes in mRNA levels for COL1A1 (c) and TGF-β1 (d). Real-time RT–PCR was performed to determine the changes in the lung tissues of mice in the mRNA levels of COL1A1 (e) and TGF-β1 (f). All results are indicated as the mean ± standard deviation of three separate experiments.