基于OPG/RANK/RANKL信号通路探讨铁筷子对CIA大鼠骨破坏的防护作用

Exploring the protective effect of helleborus thibetanus franch alcohol extract on bone destruction in CIA rats based on the OPG/RANK/RANKL signaling pathway

目的 探讨铁筷子对胶原诱导性关节炎(collagen induced arthritis, CIA)模型大鼠的抗炎作用及对OPG/RANK/RANKL信号通路的影响。方法 雌性Wistar大鼠60只分为:正常组、模型组、阳性药物组(甲氨蝶呤,MTX)、低剂量组、中剂量组和高剂量组。采用胶原抗体诱导法于大鼠尾根部注射牛Ⅱ型胶原蛋白建立CIA大鼠模型,模型建立成功后进行灌胃给药,正常组:给予10 mL/(kg·d)生理盐水;模型组:给予10 mL/(kg·d)生理盐水;阳性药物组每次给予2.0 mL/(kg·d) MTX,每周3次;铁筷子低、中、高剂量组每次分别给予0.25 g/(kg·d)、 0.5 g/(kg·d)、1.0 g/(kg·d);连续灌胃治疗25 d。通过记录大鼠体重;观察大鼠足肿胀程度;大鼠踝关节炎指数评分;micro-CT观察踝关节骨组织病理改变;苏木素-伊红(HE)染色对大鼠踝关节骨组织和滑膜病理变化;抗酒石酸酸性磷酸酶(TRAP)染色法检测观察破骨细胞数量的改变;PCR检测骨保护素(OPG)、核因子-κB受体激活剂(RANK)、RANK配体(RANKL)、肿瘤坏死因子(TNF-α)和骨形成蛋白2(BMP-2)的mRNA水平,OPG、RANK、RANKL、TNF-α和BMP-2蛋白相对表达量检测用蛋白免疫印迹法(Western Blot),以确定铁筷子对类风湿关节炎大鼠的治疗作用及其对骨关节保护的潜在药理机制。结果 数据统计结果表明,与正常组相比,CIA大鼠体重增长减慢(P<0.05),足底厚度增加(P<0.05),踝关节出现关节腔变窄,骨组织啃噬样骨质破坏、滑膜的病理性变化,如炎性细胞增生和滑膜异常增生浸润;Micro-CT结果显示:与正常组相比,模型组、低剂量组、中剂量组,踝关节表面凹凸不平、外形不完整,踝关节表面出现啃噬样骨质破坏,高剂量组与正常组相比,各项指标变化都不明显;HE染色发现模型组大鼠踝关节出现关节腔变窄,骨组织啃噬样骨质破坏、滑膜组织增生和炎性细胞浸润等病理改变;与模型组相比,低、中、高剂量组大鼠踝关节骨组织增生和炎性浸润显著改善;TRAP染色观察与正常组比,模型组破骨细胞数量最多,低、中、高剂量组干预下TRAP染色阳性破骨细胞数量逐渐减少;qRT-PCR的结果,与模型组相比,低、中、高剂量组OPG、BMP-2 mRNA相对表达量升高(P<0.05),RANK、RANKL、TNF-α mRNA相对表达量降低(P<0.05);Western Blot结果,与模型组比,低、中、高剂量组OPG、BMP-2蛋白相对表达量升高(P<0.05),RANK、RANKL、TNF-α蛋白相对表达量降低(P<0.05)。结论 铁筷子可能通过调节OPG/RANK/RANKL信号通路缓解RA大鼠体内炎症反应,发挥其抗类风湿关节炎机制的作用。

Objective To investigate the anti-inflammatory effect of helleborus thibetanus franch on collagen-induced arthritis(CIA)model rats and its effect on the OPG/RANK/RANKL signaling pathway.Methods Sixty female Wistar rats were divided into normal group,model group,positive drug methotrexate(MTX)group,low dose group,medium dose group,and high dose group.The CIA model was established by injecting bovine typeⅡcollagen into the tail root of rats using the collagen antibody induction method,and drugs were administered by gavage after the model was successfully established.In the normal group,10 mL/(kg·d)saline was administered.In the model group,10 mL/(kg·d)saline was administered.In the positive drug group,2 mg/(kg·d)MTX was administered three times a week.In low,medium,and high dose group,025,05,and 10 g/(kg·d)were administered,respectively.Continuous gavage treatment was applied for 25 days.Body mass of rats was recorded to observe the degree of foot swelling.The ankle arthritis index score was calculated.Micro-CT was used to observe histopathological changes in the ankle joint bone.Hematoxylin-eosin(HE)staining was performed to observe pathological changes in ankle joint bone tissue and synovial membrane.Changes in the number of osteoclasts were determined by tartaric acid phosphatase(TRAP)staining.PCR was used to measure mRNA levels of osteoprotegerin(OPG),nuclear factor-κB receptor activator(RANK),RANK ligand(RANKL),tumor necrosis factor(TNF)-α,and bone morphogenetic protein 2(BMP-2).Relative protein expression of OPG,RANK,RANKL,TNF-α,and BMP-2 was measured by Western Blot.helleborus thibetanus franch on rats with rheumatoid arthritis and its potential pharmacological mechanism for osteoarthritic protection.Results Compared with the normal group,CIA rats had a lower body mass(P<005),increased thickness of the plantar foot(P<005),narrowing of the joint cavity in the ankle joint,gnawing-like bone destruction,and pathological changes in the synovium,such as inflammatory cell proliferation and abnormal synovial hyperplasia infiltration.Micro-CT showed that,compared with the normal group,model,low and medium-dose groups showed an uneven ankle joint surface with an incomplete shape and gnawing-like bone destruction,and the high-dose group showed no significant changes in all indexes.HE staining revealed that the ankle joint of the model group showed joint cavity narrowing,gnawing-like bone destruction,synovial tissue proliferation,and inflammatory cell infiltration.Pathological changes,such as destruction of bone tissue,synovial tissue hyperplasia,and inflammatory cell infiltration,were found in the ankle joint of rats in the model group.Compared with the model group,bone tissue hyperplasia and inflammatory cell infiltration in the ankle joint of rats in low,medium,and high dose groups were significantly improved.TRAP staining showed that the model group had the largest number of osteoclasts,and the number of TRAP-positive osteoclasts was reduced in low,medium,and high dose groups compared with the normal group.qRT-PCR showed that,compared with the model group,relative mRNA expression of OPG and BMP-2 was increased in low,medium,and high dose groups(P<005),and relative mRNA expression of RANK,RANKL,and TNF-αwas decreased in low,medium,and high dose groups(P<005).Western Blot showed that,compared with the model group,relative protein expression of OPG and BMP-2 was increased in low,medium,and high dose groups(P<005),and relative protein expression of RANK,RANKL,and TNF-αwas decreased(P<005).Conclusions Helleborus thibetanus franch may alleviate the inflammatory response in RA rats through an anti-rheumatoid arthritis mechanism involving the OPG/RANK/RANKL signaling pathway.