【摘要】 为探讨毛蕊花糖苷(verbascoside, VB)对类风湿关节炎(rheumatoid arthritis, RA)小鼠骨侵蚀的作用及其与JNK/细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)通路的调控关系，将DBA/1J雌性小鼠随机分为正常组、胶原诱导的关节炎(collagen-induced arthritis, CIA)组、VB组和甲氨蝶呤(methotrexate, MTX)组，测量小鼠关节炎评分、后足肿胀、体质量和缩足阈值，观察踝关节病理变化；用ELISA检测血清中IL-6、TNF-α和抗牛胶原蛋白Ⅱ(collagenⅡ, CⅡ)特异性抗体水平；抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色观察踝关节破骨细胞变化；Western blotting检测关节组织中磷酸化JNK(phosphorylated JNK, p-JNK)、JNK、磷酸化ERK(phosphorylated ERK, p-ERK)、ERK蛋白表达。从C57BL/6小鼠中分离骨髓源性巨噬细胞(bone marrow-derived macrophage, BMM),将其分为巨噬细胞集落刺激因子(macrophage colony stimulating factor, M-CSF)组、NF-κB配体受体激活因子(receptor activator of NF-κB ligand, RANKL)组、VB低剂量组(VB-L组)、VB中剂量组(VB-M组)、VB高剂量组(VB-H组), TRAP染色观察体外破骨细胞分化；qRT-PCR检测组织蛋白酶K(cathepsin K,CTSK)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)mRNA表达；Western blotting检测CTSK、MMP-9、原癌基因(c-Fos)、活化T细胞核因子1(activated T-cell nuclear factor 1, NFATc1)、p-JNK、JNK、p-ERK、ERK蛋白表达。结果显示，与CIA组比较，VB组和MTX组关节炎评分和后足肿胀，血清中IgG、IgG2a、IgG2b抗体及IL-6、TNF-α水平，关节组织中p-JNK/JNK、p-ERK/ERK比值显著降低，缩足阈值和体质量显著升高，骨侵蚀显著减轻，TRAP阳性细胞数显著减少(P<0.05)。与M-CSF组比较，RANKL组TRAP阳性破骨细胞数量显著增多，CTSK和MMP-9 mRNA水平，CTSK、MMP-9、c-Fos、NFATc1蛋白水平以及p-JNK/JNK、p-ERK/ERK比值显著升高(P<0.05);与RANKL组比较，VB-L组、VB-M组和VB-H组上述指标水平均显著逆转，且呈剂量依赖性(P<0.05)。该研究提示，VB可通过阻滞JNK/ERK通路激活，抑制RANKL诱导破骨细胞生成，并改善CIA小鼠的骨侵蚀，VB是治疗RA骨侵蚀的一种有前景的治疗方法。更多还原

【Abstract】 This study aims to evaluate the impact of verbascoside(VB) on bone erosion in rheumatoid arthritis(RA) mice and its regulation on the JNK/extracellular signal-regulated kinase(ERK) pathway. DBA/1J female mice were randomly divided into the normal group, collagen-induced arthritis(CIA) group, VB group, and methotrexate(MTX) group. Arthritis scores, hind paw swelling, body weight, and paw withdrawal threshold of the mice were measured, and the pathological changes of the ankle joint were observed; ELISA was performed to measure the levels of IL-6, TNF-α, and anti-bovine collagen Ⅱ(CⅡ)-specific antibodies in serum; tartrate-resistant acid phosphatase(TRAP) staining was performed to observe the changes of ankle osteoclasts; Western blotting was used to detect the protein expressions of phosphorylated JNK(p-JNK), JNK, phosphorylated ERK(p-ERK), and ERK in the joint tissue. Bone marrow-derived macrophages(BMMs) were isolated from C57BL/6 mice, and divided into macrophage colony stimulating factor(M-CSF) group, receptor activator of NK-κB ligand(RANKL) group, VB low-dose group(VB-L group), VB medium-dose group(VB-M group), and VB high-dose group(VB-H group). TRAP staining was performed to observe osteoclast differentiation in vitro; qRT-PCR was performed to measure the mRNA expressions of cathepsin K(CTSK), and matrix metalloproteinase 9(MMP-9); Western blotting analysis was performed to measure the protein levels of CTSK, MMP-9, protooncogene(c-Fos), activated T-cell nuclear factor 1(NFATc1), p-JNK, JNK, p-ERK, and ERK. The results showed that compared to those of in the CIA group, the arthritis score and hind paw swelling, the serum IgG, IgG2a, and IgG2b antibodies, IL-6 and TNF-α levels, the ratios of p-JNK/JNK and p-ERK/ERK in joint tissue in the VB group and the MTX group were markedly decreased while the paw withdrawal threshold and body weight significantly increased, the bone erosion significantly reduced, and the number of TRAP-positive cells significantly decreased(P<0.05). Compared to those in the M-CSF group, the number of TRAP-positive osteoclasts in the RANKL group was significantly increased, same as the mRNA levels of CTSK and MMP-9, the protein levels of CTSK, MMP-9, c-Fos, NFATc1, and the ratios of p-JNK/JNK and p-ERK/ERK(P<0.05). Compared to those in the RANKL group, the levels of the above indexes in the VB-L, VB-M, and VB-H groups were significantly reversed in a dose-dependent manner(P<0.05). The study suggests that VB blocks JNK/ERK pathway activation, resulting in inhibition of RANKL-induced osteoclastogenesis and improvement of bone erosion in CIA mice. Therefore, VB may be a promising treatment for RA bone erosion.更多还原