7-Hydroxycoumarin mitigates the severity of collagen-induced arthritis in rats by inhibiting proliferation and inducing apoptosis of fibroblast-like synoviocytes via suppression of Wnt/β-catenin signaling pathway

Abstract

Background: 7-Hydroxycoumarin (7-HC) as a coumarin compound is widely found in Chinese herbs and exhibits diverse biological activities. Promoting cell apoptosis of fibroblast-like synoviocytes (FLS) is a meaningful strategy for rheumatoid arthritis (RA). Though the protective effect of 7-HC on RA experimental models has been reported, the specific mechanisms, especially the possible relationships of this effect to regulating FLS proliferation and apoptosis, still need clarification.

Purpose: This study clarified the therapeutic effects of 7-HC on collagen-induced arthritis (CIA) in rats and explored the underlying mechanisms.

Methods: In vivo, 7-HC (15, 30 or 60 mg/kg) was intraperitoneally given to CIA rats, and its therapeutic effect and anti-inflammatory activity were evaluated. Ki67 immunohistochemistry, TUNEL assay and synovial proteins detection were conducted. In vitro, after treating with 7-HC (20, 40 or 80 μM) in TNF-α-stimulated RA FLS (MH7A cell line), cell proliferation and apoptosis were examined. The involvement of Wnt/β-catenin pathway was checked in vivo and in vitro.

Results: 7-HC attenuated the severity of rat CIA, evidenced by the reduction of paw swelling, arthritis index, joint damage, collagen type II antibody serum level, and IL-1β, IL-6, TNF-α production in serum and synovium. Particularly, 7-HC in vivo had anti-proliferative and pro-apoptotic effects on CIA rat synovial cells, indicated by reduced synovial Ki67 expression, raised synovial apoptosis index, decreased Bcl-2 protein level and increased level of Bax and cleaved caspase 3 protein. Further, 7-HC in vitro suppressed proliferation and promoted apoptosis of TNF-α-stimulated MH7A cells by regulating the mitochondrial pathway. Mechanistically, 7-HC treatment inhibited Wnt/β-catenin pathway, suggested by the reduction of pathway-related proteins (e.g. Wnt1, LRP6, p-GSK-3β (Ser9), β-catenin, cyclin D1 and c-Myc), the recovery of GSK-3β activity and the inhibition of β-catenin nuclear translocation. As expected, combined use of lithium chloride, an activator of Wnt/β-catenin signaling, reversed the anti-proliferative and pro-apoptotic effects of 7-HC in vitro.

Conclusion: 7-HC relieved the severity of rat CIA by inhibiting cell proliferation and inducing apoptosis of rheumatoid FLS via inhibition of Wnt/β-catenin pathway.

Keywords: 7-Hydroxycoumarin; Apoptosis; Fibroblast-like synoviocytes; Proliferation; Rheumatoid arthritis; Wnt/β-catenin signaling pathway.