从DANCR调控NF-κB骨免疫通路探讨PMOP肾阳虚证的分子机制

Osteoimmunology has been well-known introduced in postmenopausl osteoporosis (PMOP). As one of the best important pathway of PMOP, the exact mechanism of NF-κB signaling remains unclear. DANCR, as a long non-coding RNA, plays a critical role in regulation of NF-κB pathway, osteoblastdifferentiation and osteoclast differentiation. Our previous results of gene chip have shown upregulation of DANCR in Kidney-Yang Dificiency of Postmenopausal osteoporosis. Thus we hypothesize that regulation of DANCR on NF-κB osteoimmunology pathway mediates Kidney-Yang Dificiency of Postmenopausal osteoporosis. To verify this hypothesis, three main experiments will be performed through implying CRISPR/Cas9, Adenovirus silence and overexpression,Bio-Plex Assays,etc.Firstly, osteoimmunology mechanism of Kidney-Yang Dificiency of Postmenopausal osteoporosis in mouse will be clarified based on regulation of DANCR on NF-κB pathway. Secondly, the effect of DANCR regulating NF-κB osteoimmunology pathway on osteoblastic/osteoclastic potentials of BMSCs and BMMs, which are from mouse with Kidney-Yang Dificiency of Postmenopausal osteoporosis, will be investigated. Lastly, the correlation between regulation of DANCR on NF-κBosteoimmunology pathway and Kidney-Yang Dificiency of Postmenopausal osteoporosis were demonstrated from human specimen of serum and vertebrae. This study will provide experimental evidence for new therapeutic target of PMOP and theories of “Kidney Dominating Bone”and “Pattern Identification Differentiation and Treatment”.

近期骨免疫学被用于解释绝经后骨质疏松症（PMOP）的发病机理。NF-κB骨免疫通路是PMOP发病的重要途径，但其确切调节机制仍然未知。DANCR是近年来新发现的长链非编码RNA，具有调节NF-κB通路和成、破骨的作用。我们前期芯片检测发现DANCR在PMOP肾阳虚证中显著上调，因此我们假设DANCR调控NF-κB骨免疫通路是PMOP肾阳虚证的重要机制。为证实这一假说，我们应用CRISPR/Cas9、腺病毒沉默和过表达、蛋白悬液芯片等技术来探讨：①DANCR调控NF-κB通路在PMOP肾阳虚证小鼠模型中的骨免疫机制；②在体外，DANCR调控NF-κB骨免疫通路对PMOP肾阳虚证小鼠BMSCs/BMMs成、破骨分化的调节作用；③人血清和椎体骨中，DANCR调控NF-κB骨免疫通路与PMOP肾阳虚证的关联。为挖掘中医药防治PMOP的新靶点及丰富“肾主骨”、“辨证论治”理论的科学内涵提供实验依据。

NF-κB骨免疫通路是沟通免疫与骨代谢系统的重要通路，近期被用于解释PMOP的发病机理，而其确切调节机制仍未知。DANCR是近来新发现的LncRNA，具有调节NF-κB通路和成、破骨的作用，且我们前期芯片检测发现DANCR在PMOP肾阳虚证中显著上调。为了明确DANCR是否通过介导NF-κB通路从而导致PMOP肾阳虚证的发生，我们应用CRISPR/Cas9、RNA干扰技术等进行了以下的研究：1.体内研究：①采用去卵巢法成功构建了PMOP肾阳虚病证结合模型（micro-CT检测骨微细结构，行为学评定肾阳虚证候）；②验证NF-κB上调导致的骨免疫失衡是PMOP的病理基础，而肾气丸通过抑制NF-κB通路的激活从而改善PMOP骨损害及肾阳虚证候；③应用NF-κB抑制剂对小鼠骨量的变化进行初步探索，发现NF-κB抑制剂能对抗PMOP的骨量丢失；④由于DANCR在PMOP肾阳虚证中显著上调，我们应用siRNA将WT组小鼠DANCR沉默后，小鼠腰椎骨量显著上升，而 NF-κB通路上的标记性基因P50显著下降，表明沉默DANCR可抑制NF-κB通路，而将NF-κB过表达后可解除siDANCR对腰椎骨量的保护作用；⑤基于PMOP肾阳虚模型的进一步研究中，我们发现siDANCR可逆转骨量丢失，将NF-κB过表达后该保护作用被解除。2.体外研究：①肾气丸促进小鼠BMSCs增殖并通过DANCR抑制NF-κB通路促进成骨分化；②由于DANCR是骨量的负性调控因子且骨免疫与破骨形成密切相关，因此我们专注于破骨的研究。我们应用CRISPR/Cas9 技术，构建了小鼠单核巨噬细胞系RAW264.7的DANCR敲除细胞系（敲除G1、F5片段），并验证了DANCR片段的敲除效果。③研究发现，RAW264.7的DANCR敲除细胞系破骨分化显著被抑制，且NF-kB通路关键基因P50表达下降，破骨关键因子NFatc1亦显著下降，而应用NF-κB通路激活剂LPS干预则能逆转该效应，重新激活破骨分化。3.临床研究：大样本研究验证发现，血清的维生素D、微量元素等与PMOP骨量无相关性，而骨免疫标志物与骨量成负相关。综上，我们从动物-细胞-人三方面证实DANCR促进NF-κB骨免疫通路的激活是介导PMOP肾阳虚证的分子机制，从骨免疫角度探索了防治PMOP肾阳虚证的新思路，为中医辨证论治PMOP提供了分子基础。