Chrysophanol administration alleviates bleomycin-induced pulmonary fibrosis by inhibiting lung fibroblast proliferation and Wnt/β-catenin signaling

Purpose: To determine the functional effect of chrysophanol (CH) on bleomycin (BLM)-induced pulmonary fibrosis (PF) and reveal its mechanism of action.Methods: A mouse model of PF was established by intratracheal instillation of BLM (5 mg/kg), prior to CH administration. Masson’s trichrome staining was used to analyze interstitial fibrosis and collagen deposition. Hydroxyproline (HYP) content was measured, and lung fibroblast viability determined by MTT assay. Bronchoalveolar lavage fluid (BALF) was collected, and levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and interferon-γ (IFN-γ) were evaluated using enzyme-linked immunosorbent assays (ELISA). Expression of cell signaling, adhesion, and apoptotic proteins were determined by western blotting.Results: Administration of CH reduced collagen deposition and HYP content, downregulated α-smooth muscle actin, upregulated E-cadherin, and decreased the levels of TNF-α, IL-1β, IL-6, and IFN-γ in BLM-treated mice. The viability of lung fibroblasts was also reduced, and Bcl-2-associated X protein and cleaved caspase-3 were upregulated after CH treatment in BLM-treated mice. In addition, CH treatment in BLM-treated mice significantly increased levels of cytoplasmic β-catenin but decreased its expression in the nucleus.Conclusion: Administration of CH alleviated BLM-induced PF by inhibiting lung fibroblast proliferation and nuclear translocation of β-catenin. Thus, this study provides a potential therapeutic strategy for PF. Keywords: Chrysophanol, Bleomycin, Pulmonary fibrosis, Hydroxyproline, E-cadheri

Regulatory volume decrease is actively modulated during the cell cycle

Nasopharyngeal carcinoma cells, CNE-2Z, when swollen by 47% hypotonic solution, exhibited a regulatory volume decrease (RVD). The RVD was inhibited by extracellular applications of the chloride channel blockers tamoxifen (30 μM; 61% inhibition), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μM; 60% inhibition), and ATP (10 mM; 91% inhibition). The level and time constant of RVD varied greatly between cells. Most cells conducted an incomplete RVD, but a few had the ability to recover their volume completely. There was no obvious correlation between cell volume and RVD capacity. Flow cytometric analysis showed that highly synchronous cells were obtained by the mitotic shake-off technique and that the cells progressed through the cell cycle synchronously when incubated in culture medium. Combined application of DNA synthesis inhibitors, thymidine and hydroxyurea arrested cells at the G1/S boundary and 87% of the cells reached S phase 4 h after being released. RVD capacity changed significantly during the cell cycle progression in cells synchronized by shake-off technique. RVD capacity being at its highest in G1 phase and lowest in S phase. The RVD capacity in G1 (shake-off cells sampled after 4 h of incubation), S (obtained by chemical arrest), and M cells (selected under microscope) was 73, 33, and 58%, respectively, and the time constants were 435, 769, and 2,000 sec, respectively. We conclude that RVD capacity is actively modulated in the cell cycle and RVD may play an important role in cell cycle progress