基于“肾主骨”理论探讨加味六味地黄汤对肾间质纤维化Klotho-FGF23轴调控肾和骨Wnt/β-catenin信号的影响

It is considered that kinney could control bone in the Chinese traditional medcine, suggestting that there is closed connect between the kidney and bone.Renal osteodystrophy is an important complication in the Renal interstitial fibrosis（RIF）. The activation of Wnt/β-catenin pathway is associated with the regulation of the bone metabolic abnormalities in the RIF. Oue previons works have proved that JiaWeiLiuWeiDiHuang decoction play a role through anti-oxidization, anti-microinflammation station,blocking the activation of Notch signaling, restrainning the TGF-β1, CTGF, MMP-2. However,there is not reports at home and abroad about whether JiaWeiLiuWeiDiHuang decoction could adjust the Klotho-FGF23 axis, provide the bi-directional regulation of the Wnt/β-catenin pathway. In this study, both Klotho-FGF23 axis and the Wnt/β-catenin pathway are regard as the entry point .The effect of JiaWeiLiuWeiDiHuang decoction on.the serum Calcium phosphorus metabolism and the level of iPTH, the markers of bonemetabolism including FGF-23, RANKL and Osteocalcin, Klotho-FGF23 axis, the signal molecule in the Wnt/β-catenin pathway and its downstream l molecule about c-myc and FN through the way of Immunohistochemistry and western blot, discussing the protictive mechanism and target of JiaWeiLiuWeiDiHuang decoction on the anti-RIF and bone protective role, and the material basis of the kinney controlling bone.

中医学认为肾主骨，肾与骨关系密切。肾性骨营养不良是肾间质纤维化（RIF）的重要并发症。Wnt/β-catenin信号通路活化介导调控RIF的骨代谢异常。前期的研究证实加味六味地黄汤通过抗氧化、抗微炎症状态、阻断Notch信号通路活化及抑制TGF-β1、CTGF、MMP-2发挥抗RIF作用，但是否能调节Klotho-FGF23轴、双向调控肾和骨的Wnt/β-catenin信号发挥保护作用，国内外未见相关报道。本课题将Wnt/β-catenin信号及Klotho-FGF23为切入点，应用免疫组化、western blot等观察加味六味地黄汤对血清钙磷代谢和iPTH水平、骨代谢标记物（FGF-23、RANKL和Osteocalcin）、肾和骨组织Klotho、Wnt/β-catenin信号分子及下游的c-myc、FN的影响，探讨加味六味地黄汤抗RIF和骨保护作用机制和靶点以及肾主骨理论的物质基础

中医学认为，肾和骨关系密切，肾主骨藏精生髓，髓居骨中以养骨。慢性肾脏病矿物质和骨代谢异常（CKD-MBD）是CKD的主要并发症之一，还会引起心血管事件发生,可严重影响患者的生命及预后。因此，深入开展CKD-MBD的基础研究，对CKD患者的预后具有重要意义。本课题以腺嘌呤联合高磷的CKD大鼠模型为研究对象，我们的结果表明，CKD大鼠血清BUN、Scr、血磷、iPTH、RANKL、Osteocalcin及小管间质半定量评分均显著增高，血钙、Klotho及股骨骨密度降低，骨质疏松发生，提示CKD可诱发肾性骨病。深入研究发现，肾间质纤维化可导致大鼠血清FGF23升高、Klotho降低，且大鼠肾脏Wnt和β-catenin蛋白表达上调，而骨骼Wnt和β-catenin蛋白表达下调，提示肾间质纤维化可致Klotho-FGF23轴失调，进而通过Klotho-FGF23轴调控肾骨组织中Wnt/β-catenin信号通路的活化，参与了CKD诱发CKD-MBD的发展，这可能是CKD-MBD发生发展的重要分子机制；进一步研究发现，加味六味地黄汤可降低大鼠血清BUN、Scr、血磷、iPTH、FGF23、RANKL、Osteocalcin及小管间质半定量评分，血钙、Klotho及股骨骨密度上升，下调了肾组织FGF23、Wnt和β-catenin蛋白表达，上调了骨组织Wnt和β-catenin蛋白表达，这提示加味六味地黄汤可通过调节Klotho-FGF23轴平衡进而抑制肾Wnt/β-catenin信号通路，活化骨Wnt/β-catenin信号通路，通过双向调控肾骨组织Wnt/β-catenin信号通路，发挥肾和骨的保护作用，且Klotho-FGF23轴贯穿于肾脏与骨骼两个系统，Klotho-FGF23轴与Wnt/β-catenin信号通路可能是CKD-MBD病理生理过程中“肾主骨”的物质基础。