Periploca forrestii saponin ameliorates CIA via suppressing proinflammatory cytokines and nuclear factor kappa-B pathways

ObjectivePeriploca forrestii Schltr has been used as a Chinese folk medicine for the treatment of rheumatism, arthralgia and fractures. However, the anti-arthritic activity of Periploca forrestii saponin (PFS) and the active compound has still not been revealed. This study aimed to investigate the protective effects and mechanisms of PFS on collagen type II (CII) collagen-induced arthritis (CIA) mice. We sought to investigate whether PFS and Periplocin could regulate osteoclastogenesis, and if so, further investigation on its mechanism of action.MethodsArthritis was induced in female BALB/c mice by CIA method. PFS was administered at a dose of 50 mg/kg body weight once daily for five weeks. The effects of treatment in mice were assessed by histological and biochemical evaluation in sera and paws. Anti-osteoclastogenic action of PFS and Periplocin was identified using an osteoclast formation model induced by RANKL.ResultsPFS ameliorated paw erythema and swelling, inhibited bone erosion in ankle joint histopathological examination. PFS treatment resulted in decreased IgG2a, and increased IgG1 levels in the serum of CIA mice. Decreased TNF-α, and increased interleukin (IL)-4 and IL-22 levels were also found in PFS-treated mice. PFS inhibited the I-κBα phosphorylation, blocked nuclear factor (NF)-κB/p65 phosphorylation and abrogated AP-1/c-Fos activity. PFS downregulated toll-like receptor (TLR) 4, STAT3 and MMP-9 expression in CIA mice and RANKL-induced osteoclastogenesis. PFS and Periplocin inhibited RANKL-induced osteoclast formation in a dose dependent manner within nongrowth inhibitory concentration, and PFS decreased osteoclastogenesis-related marker expression, including cathepsin K and MMP-9.ConclusionThis study revealed that the protective mechanism of PFS on CIA was associated with regulatory effects on proinflammatory factors and further on the crosstalk between NF-κB and c-Fos/AP-1 in vivo and in vitro. Therefore, PFS is a promising therapeutic alternative for the treatment of RA, evidencing the need to conduct further studies that can identify their active components in treating and preventing RA.杠柳（Periploca forrestii Schltr）作为中国民间传统药材，历来用于治疗风湿病、关节痹痛与骨折。然而，杠柳皂苷（Periploca forrestii saponin, PFS）的抗关节炎活性及其活性物质基础仍未被阐明。本研究旨在探讨PFS对Ⅱ型胶原（collagen type II, CII）诱导的关节炎（collagen-induced arthritis, CIA）模型小鼠的保护作用及其作用机制，并探究PFS与杠柳素（Periplocin）是否可调控破骨细胞生成，若可行则进一步解析其具体作用通路。方法 采用Ⅱ型胶原诱导法构建雌性BALB/c小鼠关节炎模型。以50 mg/kg体质量的剂量每日单次灌胃给予PFS，连续给药5周。通过血清与足部组织的组织学检测及生化指标分析，评价PFS对模型小鼠的治疗效果。采用核因子κB受体活化因子配体（receptor activator of nuclear factor-κB ligand, RANKL）诱导的破骨细胞生成模型，验证PFS与杠柳素的抗破骨细胞生成活性。结果 PFS可显著改善模型小鼠足部红斑与肿胀症状，踝关节组织病理学检查显示其可有效抑制骨侵蚀。PFS给药可降低CIA模型小鼠血清中IgG2a水平，同时升高IgG1抗体滴度。此外，PFS处理组小鼠血清中促炎因子TNF-α水平显著降低，抗炎因子白细胞介素（interleukin, IL）-4与IL-22水平明显升高。分子机制层面，PFS可抑制I-κBα磷酸化，阻断核因子（nuclear factor, NF）-κB/p65的磷酸化过程，并抑制AP-1/c-Fos的转录活性。在CIA模型小鼠体内及RANKL诱导的破骨细胞生成模型中，PFS均可下调Toll样受体（toll-like receptor, TLR）4、STAT3及基质金属蛋白酶9（matrix metalloproteinase 9, MMP-9）的表达。在非细胞毒性浓度范围内，PFS与杠柳素均可呈剂量依赖性抑制RANKL诱导的破骨细胞生成，且PFS可降低破骨细胞生成相关标志物的转录与蛋白表达，包括组织蛋白酶K与MMP-9。结论 本研究揭示，PFS对CIA的保护作用机制与调控促炎因子表达、介导体内外NF-κB与c-Fos/AP-1信号通路的交叉对话密切相关。综上，PFS是一种极具潜力的类风湿关节炎（rheumatoid arthritis, RA）治疗候选药物，本研究结果也提示需开展进一步研究以明确其治疗与预防RA的活性物质成分。