Effect of eperisone on BLM-induced pulmonary fibrosis
Pulmonary fibrosis was induced by intratracheal administration of BLM to male ICR mice. Specifically, 10 days after BLM administration, mice were divided into three groups based on the rate of change in body weight (excluding the vehicle group), and the effect of oral eperisone administration on lung fibrosis was examined. At 20 days after BLM administration, lung tissue sections were prepared and stained for collagen using Masson’s trichrome stain. Collagen deposition in the lungs was observed in a BLM administration-dependent manner. In contrast, oral eperisone administration suppressed the BLM-dependent collagen deposition in a dose-dependent manner (Figure 3A and 3B). Next, we performed quantitative analysis of hydroxyproline, a collagen-specific amino acid, in lung tissue. As shown in Figure 3C, BLM treatment significantly increased the amount of hydroxyproline in lung tissue, while eperisone treatment suppressed this increase. When considering the clinical application of eperisone for the treatment of lung fibrosis, it is important to improve respiratory function as well as histological and biochemical indices. Moreover, our previous analysis showed that lung elastance is increased and FVC is decreased in BLM-induced pulmonary fibrosis (Sugizaki et al., 2019; K. I. Tanaka et al., 2017). Thus, we measured the respiratory function of mice using a computer-controlled ventilator and negative pressure reservoir. As shown in Figure 3D, BLM treatment increased the total elastance (elastance of the entire lung including the bronchi, bronchioles, and alveoli) and tissue elastance (elastance of the alveoli) and decreased the FVC. In contrast, eperisone significantly improved the deterioration of respiratory function induced by BLM administration. These results indicate that eperisone has an ameliorating effect on BLM-dependent pulmonary fibrosis.
Fibroblasts are known to differentiate into myofibroblasts upon activation and play an important role in the development and exacerbation of lung fibrosis (Hinz et al., 2007; Scotton & Chambers, 2007). Thus, we performed an immunohistochemical analysis of α-SMA, a myofibroblast marker. As shown in Figure 4A, BLM treatment increased the number of α-SMA-positive cells in the lung, i.e., myofibroblasts increased in a BLM-dependent manner. In contrast, administration of 50 mg/kg eperisone decreased the BLM-dependent increase in α-SMA-positive cells in the lung to the same level as observed in the vehicle group. These results suggest that eperisone inhibits fibroblast activation not only in vitro but also in vivo and that it suppresses BLM-dependent pulmonary fibrosis through its inhibitory effect on fibroblast activation.