Preferential suppression of fibroblast activity by eperisone
A library of drugs already in clinical use was screened to identify
drugs that are not toxic to alveolar epithelial cells but are
preferentially toxic to lung fibroblasts. Specifically, LL29 or A549
cells were treated with each drug, and 24 h later, the percentages of
viable cells were determined using the methylthiazole tetrazolium
reagent. Among the drugs that showed lower IC50 values in LL29 cells
than in A549 cells, idebenone and eperisone were selected based on the
difference in IC50 values between the two cell types, their clinical
safety, and other pharmacological activities. As described above, we
previously reported the preferential suppression of fibroblast activity
by idebenone and its efficacy against BLM-induced pulmonary fibrosis
(Sugizaki et al., 2019). Therefore, in this study, we focused on
eperisone, which is used in clinical practice as a central muscle
relaxant (Iwase, Mano, Saito, & Ishida, 1992), and examined its
efficacy against IPF using in vitro and in vivo systems.
As shown in Figure 1A, eperisone treatment (25–200 µM) decreased the
percentage of viable LL29 cells in a dose-dependent manner. In contrast,
the percentage of viable A549 cells treated with 200 µM of eperisone was
88.5 ± 3.0% (mean ± SEM, n= 4), revealing almost no decrease in viable A549 cells after eperisone
treatment. We next examined eperisone-induced cytotoxicity in LL29 cells
using CellTox™ Green Dye, which can detect cell membrane disruption. As
shown in Figure 1B, LL29 cells treated with eperisone exhibited
cytotoxic effects in a time- and concentration-dependent manner.
Furthermore, we compared the effect of eperisone on TGF-β1–induced
activation of lung fibroblasts. LL29 cells were pre-treated with
eperisone (10–30 µM), followed by the addition of TGF-β1 (5 µM), and
the expression of fibrosis-related factors was analyzed 72 h later by
real-time RT-PCR. As shown in Figure 1C, TGF-β1 increased the mRNA
expression of Collagen 1a1 (COL1A1) , α-SMA (ACTA2) ,
connective tissue growth factor (CTGF ), vascular endothelial
growth factor (VEGF ), basic fibroblast growth factor
(BFGF ), and platelet derived growth factor (PDGF-A ) in
LL29 cells, but this increase was suppressed by pre-treatment with
eperisone. These results suggest that eperisone preferentially
suppressed lung fibroblast activity in vitro .
Effects of other drugs on lung fibroblast viability
As described in the introduction, pirfenidone and nintedanib have been
used as anti-fibrotic agents in clinical practice to treat IPF patients.
Thus, to investigate the characteristic effect of eperisone on lung
fibroblasts, we measured the percentages of viable LL29 and A549 cells
after treatment with these existing drugs. After pirfenidone treatment
(up to 2 mM), almost no decrease was observed in the percentage of
viable cells of both cell types. In contrast, nintedanib decreased the
percentage of viable cells of both cell types, but there was no
difference in the degree of decrease between the cell types (Figure 2A).
Eperisone is a central muscle relaxant that has been used in clinical
practice to improve muscle tone in patients with lumbago and spastic
paralysis caused by cerebrovascular disease. Thus, we determined whether
other central muscle relaxants exert preferential effects on
fibroblasts. Among the six drugs examined, tolperisone, inaperisone, and
lanperisone preferentially reduced the viability of LL29 cells, similar
to eperisone. However, tizanidine, methocarbamol, and baclofen, at
concentrations up to 2 mM, did not reduce the viability of either cell
type (Figure 2B). As will be discussed in detail later, because
preferential suppression of fibroblasts was not observed for some
central muscle relaxants, we speculate that eperisone exerts its
preferential effects by a molecular mechanism other than its muscle
relaxant effect.