Li Yiqun, Yang Chao, Jia Kexin, Wang Jinxia, Wang Jingxia, Ming Ruirui, Xu Tengteng, Su Xiaohui, Jing Yu, Miao Yandong, Liu Chunfang, Lin Na
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Science and Technology Innovation Center, Guangzhou University of Chinese Medicine, Guangzhou, 510405, China.
J Ethnopharmacol. 2022 Jan 10;282:114602. doi: 10.1016/j.jep.2021.114602. Epub 2021 Sep 4.
Bone destruction plays a key role in damaging the joint function of rheumatoid arthritis (RA). Fengshi Qutong capsule (FSQTC) consisting of 19 traditional Chinese medicines has been used for treating RA in China for many years. Preliminary studies show that FSQTC has analgesic activity and inhibits synovial angiogenesis of collagen-induced arthritis (CIA), but its role on bone destruction of RA is still unclear.
To explore the effect of FSQTC on bone destruction of RA and the possible mechanism of osteoclastogenesis in vivo and in vitro.
LC-MS system was used to detect the quality control components of FSQTC. The anti-arthritic effect of FSQTC on CIA rats was evaluated by arthritis score, arthritis incidence and histopathology evaluation of inflamed joints. The effect of treatment with FSQTC on bone destruction of joint tissues was determined with X-ray and micro-CT quantification, and on bone resorption marker CTX-I and formation marker osteocalcin in sera were detected by ELISA. Then, osteoclast differentiation and mature were evaluated by TRAP staining, actin ring immunofluorescence and bone resorption assay both in joints and RANKL-induced RAW264.7 cells. In addition, RANKL, OPG, IL-1β and TNFα in sera were evaluated by ELISA. The molecular mechanisms of the inhibitions were elucidated by analyzing the protein and gene expression of osteoclastic markers CTSK, MMP-9 and β3-Integrin, transcriptional factors c-Fos and NFATc1, as well as phosphorylation of ERK1/2, JNK and P38 in joints and in RANKL-induced RAW264.7 cells using western blot and/or qPCR.
In this study, 12 major quality control components were identified. Our data showed that FSQTC significantly increased bone mineral density, volume fraction, trabecular thickness, and decreased trabecular separation of inflamed joints both at periarticular and extra-articular locations in CIA rats. FSQTC also diminished the level of CTX-I and simultaneously increased osteocalcin in sera of CIA rats. The effects were accompanied by reductions of osteoclast differentiation, bone resorption, and expression of osteoclastic markers (CTSK, MMP-9 and β3-Integrin) in joints. Interestingly, FSQTC treatment could reduce the protein level of RANKL, increase the expression of OPG, and decrease the ratio of RANKL to OPG in inflamed joints and sera of CIA rats. In addition, FSQTC inhibited the levels of pro-inflammatory cytokines implicated in bone resorption, such as IL-1β and TNFα in sera. When RAW264.7 cells were treated with RANKL, FSQTC inhibited the formation of TRAP + multinucleated cells, actin ring and the bone-resorbing activity in dose-dependent manners. Furthermore, FSQTC reduced the RANKL-induced expression of osteoclastic genes and proteins and transcriptional factors (c-Fos and NFATc1), as well as phosphorylation of mitogen-activated protein kinases (MAPKs).
FSQTC may inhibit bone destruction of RA by its anti-osteoclastogenic activity both in vivo and in vitro.
骨破坏在类风湿关节炎（RA）关节功能损害中起关键作用。由19味中药组成的风湿祛痛胶囊（FSQTC）在中国用于治疗RA已有多年。初步研究表明，FSQTC具有镇痛活性，并能抑制胶原诱导性关节炎（CIA）的滑膜血管生成，但其对RA骨破坏的作用仍不清楚。
探讨FSQTC对RA骨破坏的影响以及体内外破骨细胞生成的可能机制。
采用液相色谱 - 质谱联用（LC - MS）系统检测FSQTC的质量控制成分。通过关节炎评分、关节炎发病率及炎症关节的组织病理学评估来评价FSQTC对CIA大鼠的抗关节炎作用。用X射线和显微CT定量测定FSQTC治疗对关节组织骨破坏的影响，并用酶联免疫吸附测定（ELISA）法检测血清中骨吸收标志物I型胶原交联C末端肽（CTX - I）和骨形成标志物骨钙素。然后，通过抗酒石酸酸性磷酸酶（TRAP）染色、肌动蛋白环免疫荧光和骨吸收试验在关节及RANKL诱导的RAW264.7细胞中评估破骨细胞的分化和成熟情况。此外，用ELISA法评估血清中的RANKL、骨保护素（OPG）、白细胞介素 - 1β（IL - 1β）和肿瘤坏死因子α（TNFα）。通过蛋白质印迹法和/或实时定量聚合酶链反应（qPCR）分析关节及RANKL诱导的RAW264.7细胞中破骨细胞标志物组织蛋白酶K（CTSK）、基质金属蛋白酶 - 9（MMP - 9）和β3整合素、转录因子c - Fos和活化T细胞核因子c1（NFATc1）的蛋白和基因表达，以及细胞外信号调节激酶1/2（ERK1/2）、应激活化蛋白激酶（JNK）和p38的磷酸化，以阐明抑制作用的分子机制。
本研究鉴定出12种主要质量控制成分。我们的数据表明，FSQTC显著增加了CIA大鼠炎症关节在关节周围和关节外部位的骨矿物质密度、体积分数、小梁厚度，并减小了小梁间距。FSQTC还降低了CIA大鼠血清中CTX - I的水平，同时增加了骨钙素水平。这些作用伴随着关节中破骨细胞分化、骨吸收及破骨细胞标志物（CTSK、MMP - 9和β3整合素）表达的减少。有趣的是，FSQTC治疗可降低CIA大鼠炎症关节和血清中RANKL的蛋白水平，增加OPG的表达，并降低RANKL与OPG的比值。此外，FSQTC抑制了血清中参与骨吸收的促炎细胞因子如IL - 1β和TNFα的水平。当用RANKL处理RAW264.7细胞时，FSQTC以剂量依赖性方式抑制TRAP + 多核细胞的形成、肌动蛋白环和骨吸收活性。此外，FSQTC降低了RANKL诱导的破骨细胞基因和蛋白以及转录因子（c - Fos和NFATc1）的表达，以及丝裂原活化蛋白激酶（MAPKs）的磷酸化。
FSQTC可能通过其体内外抗破骨细胞生成活性抑制RA的骨破坏。
