从EphB4/ephrinB2逆向信号调控滑膜及骨髓中破骨细胞分化探讨肾虚在绝经后RA发展中的作用

Rheumatoid arthritis (RA) incidence and prevalence were higher in women than in men, and RA incidence and severity in postmenopausal women were significantly higher than in premenopausal. We have found that incidence and severity were significantly higher in collagen induced arthritis (CIA) rats of castration- induced kidney deficiency than in CIA rats, and Yishen Juanbi pill can reduce the incidence and severity of ovariectomized CIA rats. Many studies have shown that osteoclasts in synovium and bone marrow played an important role in articular cartilage and bone erosion of RA, and the latest reseach confirmed that EphB4/ephrinB2 reverse signaling pathway plays an important regulatory role on osteoclasts differentiation. Estrogen had an important impact on this pathway, and the significant reduction of estrogen after menopause was one of the important causes of kidney deficiency. Presumably, long-term decline of estrogen in postmenopausal rheumatoid arthritis can cause kidney deficiency state of rheumatoid arthritis. Kidney deficiency could attenuate the inhibition to osteoclast differentiation by this pathway, and enhance the osteoclast differentiation excessively, further lead to the destruction of articular cartilage and bone. However, tonifying kidney could enhance this pathway to achieve the therapeutic effect. This project intends to deeply explore the role of kidney deficiency in the development of postmenopausal rheumatoid arthritis from EphB4/ephrinB2 reverse signaling by the method of combining in vitro and in vivo experiment, and partly reveals the scientific connotation of kidney deficiency, and further provide experimental basis for treatment of postmenopausal rheumatoid arthritis from kidney of Chinese medicine.

类风湿关节炎(RA)发病率女性明显高于男性，而绝经后妇女RA发病率及病变程度又明显高于未绝经妇女。我们发现大鼠去卵巢使机体处于肾虚状态，其RA发病率及病变程度明显高于未去卵巢大鼠；而具有补肾作用的“益肾蠲痹丸”能明显减轻去卵巢大鼠RA发病率及病变程度。研究表明滑膜和骨髓中破骨细胞(OC)在RA软骨及骨破坏中起关键作用；而最新证实EphB4/ephrinB2逆向信号通路对OC分化具有重要调控作用。雌激素对该通路具有重要的影响，而绝经后雌激素大幅度降低又是导致肾虚的重要原因之一。据此推测：患有RA的绝经后妇女雌激素长期大幅度降低，当机体处于“肾虚”时，可使这一通路对OC的抑制作用减弱，导致OC分化过度增强，从而造成软骨及骨质受到进一步破坏；而补肾可使这一通路增强而达到治疗作用。对此本项目采用体内与体外实验相结合的方法，从这一通路深入探讨肾虚在绝经后RA发展中的作用，以部分揭示其科学内涵。

临床研究表明绝经后妇女RA发病率及病变程度明显高于未绝经妇女，动物实验发现去卵巢（肾虚）大鼠RA发病率及病变程度明显高于未去卵巢大鼠；补肾可以改善肾虚证RA骨质破坏。据此，本项目通过体内实验和体外实验相结合的方法，探讨肾虚在绝经后RA发展中的作用及机制。体内实验结果表明：去卵巢（肾虚）CIA大鼠不仅滑膜组织中OC形成增加，骨髓组织中OC数量也明显增加。去卵巢CIA大鼠滑膜组织和骨髓组织中均有EphB4和ephrinB2的表达，但二者在滑膜和骨髓中表达趋势不同。益肾蠲痹丸能改善去卵巢CIA大鼠踝关节的肿胀程度及跗骨骨表面骨微观结构的破坏，减轻关节局部炎细胞浸润、滑膜增生及软骨与骨的破坏。其机制一方面是通过上调骨髓中EphrinB2的表达，减少骨髓中OC分化转录因子NFATc1、c-FOS、c-JUN、RANK的表达和OC的形成，从而减少关节外的骨吸收；另一方面通过降低滑膜组织中OC分化转录因子的表达，抑制滑膜组织中OC的形成，减少滑膜组织对关节内软骨和骨组织的侵蚀。体外实验结果表明：与空白组血清和CIA血清组相比，去卵巢CIA大鼠血清组骨髓和脾脏来源的破骨前体细胞诱导的OC形成、分化及活性均增强，而OC分化负调控因子ephrinB2的表达明显下降。益肾蠲痹丸均可抑制OC的分化及活性，并上调ephrinB2的表达。ephrinB2基因敲减可促进OC形成及其转录因子的表达，并减弱益肾蠲痹丸对OC分化的调控，说明益肾蠲痹丸对OC分化的调控作用与ephrinB2信号相关。以上结果表明肾虚可以从骨组织内部引起骨的侵蚀，从而加重RA引起的骨破坏，具有补肾作用的益肾蠲痹丸可以同时调控骨髓与滑膜组织中OC数量而减轻关节内外软骨及骨质的破坏，ephB4/ephrinB2信号通路对绝经后（肾虚）RA骨破坏及益肾蠲痹丸作用的发挥均具有调控作用。本研究为“肾主骨”理论提供了进一步的实验依据，同时也为绝经后RA“从肾论治”提供进一步的实验依据。