Therapeutic effects of eperisone on pulmonary fibrosis via preferential suppression of fibroblast activity

Abstract

Although the exact pathogenesis of idiopathic pulmonary fibrosis (IPF) is still unknown, the transdifferentiation of fibroblasts into myofibroblasts and the production of extracellular matrix components such as collagen, triggered by alveolar epithelial cell injury, are important mechanisms of IPF development. In the lungs of IPF patients, apoptosis is less likely to be induced in fibroblasts than in alveolar epithelial cells, and this process is involved in the pathogenesis of IPF. We used a library containing approved drugs to screen for drugs that preferentially reduce cell viability in LL29 cells (lung fibroblasts from an IPF patient) compared with A549 cells (human alveolar epithelial cell line). After screening, we selected eperisone, a central muscle relaxant used in clinical practice. Eperisone showed little toxicity in A549 cells and preferentially reduced the percentage of viable LL29 cells, while pirfenidone and nintedanib did not have this effect. Eperisone also significantly inhibited transforming growth factor-β1-dependent transdifferentiation of LL29 cells into myofibroblasts. In an in vivo study using ICR mice, eperisone inhibited bleomycin (BLM)-induced pulmonary fibrosis, respiratory dysfunction, and fibroblast activation. In contrast, pirfenidone and nintedanib were less effective than eperisone in inhibiting BLM-induced pulmonary fibrosis under this experimental condition. Finally, we showed that eperisone did not induce adverse effects in the liver and gastrointestinal tract in the BLM-induced pulmonary fibrosis model. Considering these results, we propose that eperisone may be safer and more therapeutically beneficial for IPF patients than current therapies.

Fig. 1. Preferential suppression of fibroblast activity by eperisone.

LL29 or A549 cells were incubated with the indicated concentrations (µM) of eperisone for 24 h. The percentage of viable cells was determined using a CellTiter-Glo® 2.0 assay (A). LL29 cells were incubated with the indicated concentrations (µM) of eperisone for 18 h. The cytotoxicity was measured every hour using CellTox™ Green Dye and a microplate reader (excitation: 485 nm, emission: 530 nm) (B). LL29 cells were incubated with transforming growth factor (TGF)-β1 (5 ng/ml) for 72 h in the presence of the indicated concentrations of eperisone. Total RNA was extracted and subjected to real-time RT-PCR using a specific primer set for each gene. The values were normalized to GAPDH gene expression and expressed relative to the control sample (C). Values represent the mean ± SEM ** or ^##P < 0.01; * or ^#P < 0.05. (*, vs A549 cells (A) or Control (B, C); ^#, vs TGF-ß (C)).

Fig. 2. Effect of other drugs on the percentage of viable LL29 and A549 cells.

LL29 or A549 cells were incubated with the indicated concentrations of pirfenidone, nintedanib (A), tolperisone, inaperisone, lanperisone, tizanidine, methocarbamol, or baclofen (B) for 24 h. The percentage of viable cells was determined using a CellTiter-Glo® 2.0 assay. Values represent the mean ± SEM **P < 0.01; *P < 0.05. (*, vs A549 cells).

Fig. 3. Effect of eperisone on pre-developed pulmonary fibrosis.

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on day 0. The mice were then orally administered the indicated dose of eperisone (Epe) once daily for 9 days (from day 10 to day 18). Pulmonary tissue sections were prepared on day 20 and subjected to a histopathological examination (Masson’s trichrome staining; scale bar = 500 µm) (A). The collagen-positive area was determined based on Masson’s trichrome staining images (B). The pulmonary hydroxyproline level was determined on day 20 (C). The total respiratory system elastance, tissue elastance, and forced vital capacity (FVC) were measured on day 20 (D). Values represent the mean ± SEM **P < 0.01; ^#P < 0.05; NS not significant. (*, vs vehicle; ^#, vs BLM alone).

Fig. 4. Effect of eperisone on bleomycin-induced increases in myofibroblasts.

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on day 0. The mice were then orally administered the indicated dose of eperisone (Epe) once daily for 9 days (from day 10 to day 18). Pulmonary tissue sections were prepared on day 20 and subjected to immunohistochemical analysis with an antibody against α-smooth muscle actin (SMA) (scale bar = 100 µm) (A). The α-SMA-positive area was determined using ImageJ software (B). Total RNA was extracted from lung tissue and subjected to real-time RT-PCR using a specific primer set for each gene. The values were normalized to Hprt1 gene expression and expressed relative to the control sample (C). Values represent the mean ± SEM ** or ^##P < 0.01; ^#P < 0.05. (*, vs vehicle; ^#, vs BLM alone).

Fig. 5. Effects of eperisone administration on gastric and colonic mucosa.

Mice were treated with bleomycin (BLM, 1 mg/kg) or vehicle once only on day 0. The mice were then orally administered 250 mg/kg of eperisone (Epe) once at day 10. After 24 h, the stomach and colon were collected from the mice. Gastric (A) and colonic (B) tissue sections were prepared and subjected to histopathological examination (hematotoxin and eosin staining; scale bar = 200 µm).