基于11.7T核磁共振波谱的骨髓脂肪酸代谢组学对骨质疏松发生机制的研究

The pathogenesis of osteoporosis(OP) is not clear. There is a increasing recognition that the deterioration of quantity and quality of OP is accompanied by the accumulation of bone marrow adipose tissue(MAT).However, very limited studies in the literature that have investigated the association between the metablism of fatty acid(FA)and BMD, have shown mixed results. High-resolution magic angle spinning nuclear magnetic resonance spectroscopy (NMRS) has been suggested as a non-invasive powerful tool for quantifying lipid composition. Seventy-two 3-month-old female Sprague-Dawley rats are studied. NMRS（11.7T, quantifying FA composition）, Micro CT (measuring BMD and microstructure parameters), gas chromatography and mass spectrometry(GC-MS, metabolomics of FA), RT-PCR（PPARγ and Runx2 mRNA expression）as well as serological examination(OCN/CTX-Ⅰ、Estrogen) and bone marrow pathology examination(quantifing osteoclast/osteoblast and bone mineralization),were performed at baseline and 2, 4, 8and 14 weeks after ovariectomy (OVX group) or sham surgery (Sham group).The longitudinal relationship between FA composition and BMD, as well as their causal relationship in OP are dynamically evaluated by comparing the parameters between two groups in order to illustrate the metabolic biomarkers with the potentially predictive diagnosis values in osteoporosis research. Meanwhile, the alteration of FAs between NMRS and GC-MS is compared to identify what actually happened in FA compositions which affect the bone marrow microenvironment thus lead to OP. Furthermore, the damaging impact of FA metabolic disorder on bone homeostasis and its mechanism leading to OP is revealed, from the aspect of expression of PPARγ/Runx2 mRNA and NOS activity in bone metabolism, serological osteogenic/osteoclastic markers and bone marrow pathological examination, which potentially provide new targets for the prevention and control of OP.

骨质疏松（OP）发病机制不清，骨质量衰变伴随骨髓脂肪堆积已成共识，但骨髓脂肪酸（FA）代谢与骨密度（BMD）的关系报道很少，且结果不一。11.7T核磁共振波谱（NMRS）是定量FA成分的有效技术。本研究拟对绝经后OP模型大鼠OVX术后不同时间点行胫骨NMRS（测定FA指数）和Micro CT（测定BMD）检查，同时，行骨髓液的气相色谱-质谱（GC-MS，FA绝对量）和 RT-PCR、血清学和骨髓病理检查，1）观察OP发生过程中，骨髓FA的时序变化规律，动态分析其与BMD相关性，寻找早期诊断OP的生物学标志物。2）分析NMRS与GC-MS的FA代谢组学结果一致性，明确影响骨质量的具体FA成分，为揭示骨髓FA影响骨髓微环境、导致OP提供依据。3）从骨代谢特异性转录因子表达、血清学成骨/破骨标志物、骨髓病理等方面，揭示骨髓FA代谢紊乱损害骨质量、导致OP可能机制，为OP防治提供新靶点。

骨质疏松（OP）发病机制不清，骨质量衰败伴随骨髓脂肪堆积已成共识，骨髓脂肪酸（FAs）代谢紊乱与成骨、破骨的关系多为体外实验结果，采用影像学技术在体研究骨髓FAs动态时序变化规律及其与OP形成关系、可能的作用机制，未见报道。11.7T核磁共振波谱（NMRS）是在体定量骨髓FAs成分的有效技术。本研究将96只雌性SD大鼠（12周）随机分为绝经后OP组和Sham组（n=48只/组），分别接受双侧卵巢切除术（OVX）或假手术。二组大鼠在术后不同时间点（3、5、7、14、21和28天）过量麻醉药处死，右侧股骨和肱骨，使用氘代氯仿和磷酸缓冲盐溶液（PBS）反复冲洗后获取骨髓冲洗液（BMSF）分别行11.7T NMRS（测定FAs指数）、气相色谱-质谱（GC-MS，FAs绝对定量）检测，左侧股骨远端用于Micro CT扫描（测定骨密度，BMD）和骨病理检查（VEGF/PPARγ），左侧肱骨BMSF采用化学比色法测定一氧化氮（NO）以及一氧化氮合成酶（NOS），同时，心脏穿刺采血测量血清成骨/破骨标志物。.结果发现：实验组大鼠OVX术后14天骨微结构显著受损，OP形成。每个时间点NMRS对FAs半定量测量结果与GC-MS定量结果显著相关。OVX组和Sham组在术后3天主要是棕榈油酸、肉豆蔻酸和花生四烯酸出现显著差异，表明骨髓FAs在PMOP形成早期即出现紊乱。术后第14天二组实验大鼠的棕榈油酸、肉豆蔻酸、α-亚麻酸、硬脂酸和二十碳烯酸（VIP＞1，p＜0.05）出现显著性差异，OVX组大鼠较对照组的棕榈油酸、肉豆蔻酸、α-亚麻酸和二十碳烯酸显著降低，而花生四烯酸和硬脂酸水平显著升高（p＜0.05）。术后3天，棕榈油酸与1型胶原C端端肽（CTX-1，骨吸收标志物）呈负相关，而花生四烯酸与骨钙素（OCN，骨形成标志物）呈负相关（p＜0.05）。术后14天，肉豆蔻酸、α-亚麻酸与二十碳烯酸均与CTX-1呈负相关（p＜0.05），而二十碳烯酸和NOS表达之间也存在显著正相关性（p＜0.05）。骨病理免疫组化结果显示术后14天OVX组VEGF表达降低，PPARγ表达增加。以上结果表明PMOP早期骨髓FAs发生了显著性变化，它们可能通过干扰骨髓间充质干细胞成脂分化和NOS/NO通路影响骨髓微环境，从而导致OP形成。该研究结果揭示了骨髓FAs早期代谢紊乱是损害骨质量重要因素，并探讨了其导致OP