基于肠道菌群及肠道靶点的开环异落叶松脂酚抗骨质疏松作用机制研究

In recent years, gut microbiome becomes a research hotspot that is involved in bone metabolism. Serotonin synthesized by tyrosine hydroxylase I (Tph-1) in gut enterochromaffin cells plays a key role in modulating bone resorption. Our previous studies demonstrated that Secoisolariciresinol (SECO) exerted significantly protective effects on ovariectomy-induced bone loss. It was converted to enterodiol and enterolactone by incubation with gut microbiome in vitro. Surflex-docking showed a higher score of SECO than LP533401, a well-known Tph-1 inhibitor, binding to tryptophan hydroxylase I (Tph-1) protein, a rate-limiting enzyme of serotonin synthesis. It also inhibited the protein expression of Tph-1 on RBL-2H3 cells (Tph-1 high-expressing cells).In addition, it′s reported that SECO and its metabolites distributed to bone at trace amount. Therefore, in the current study, we hypothesis that SECO may exert its beneficial effects on bone by modulating gut microbiome and serotonin synthesis. The antibiotic-treated mice model, the fecal microbiota transplantation approach, LC-MS technique, micro-ct scanning, molecular biological method, cell biological approach et.al will be performed in this study. The underlying mechanism of SECO on treatment of osteoporosis through gut microbiota and serotonin will be clarified. The current study will fully support the development of SECO to be an anti-osteoporotic agent, and provide new idea and new targets for the development of anti-osteoporotic new drugs.

肠道菌群调控骨代谢是当前研究的热点，肠道色氨酸羟化酶（Tph-1）转化的5羟色胺是影响骨吸收的关键因素。我们前期研究证实，开环异落叶松脂酚（SECO）具有很强的抗骨质疏松作用，其可在肠道菌群作用下发生代谢转化，分子对接表明SECO作用强于现有Tph-1抑制剂，体外实验证实SECO能抑制肠道Tph-1蛋白的表达，此外，有研究表明SECO及其代谢物在骨组织中分布量极其低微。因此，我们提出了SECO通过调控肠道菌群及肠道靶点发挥抗骨质疏松作用的假说。本课题拟采用肠菌失调模型、肠菌转移技术、液质连用技术、医学影像学、分子生物学、细胞生物学等多种研究技术和方法，深入研究SECO调控肠道菌群和肠道靶点发挥抗骨质疏松作用的机制，为SECO进一步的新药开发提供科学依据，也为抗骨质疏松药物开发提供新思路和新靶点。

开环异落叶松脂酚（SECO）是亚麻籽中含量最丰富的亚麻木酚素（SDG）脱掉两个糖的苷元，SECO具有很好的抗骨质疏松效果，现有机制认为其主要通过肠道菌群转化为肠内脂，随后进入靶组织发挥生物学活性，然而，有报道SDG及其代谢产物主要分布在结肠和盲肠，仅有极少量的约0.1%分散在股骨、胫骨和椎骨中。因此，我们提出假设：SECO及其肠道转化产物可能作用于肠道菌群或肠道相关靶点发挥抗骨质疏松作用。本课题从肠道菌群结构和肠道靶点两个维度阐释SECO在肠道而非靶器官发挥抗骨质疏松作用的机制。通过去卵巢模型、抗生素干预模型、肠菌转移模型等动物实验，结合肠道菌群分析方法及骨参数，明确了SECO给药影响肠道菌群结构，SECO改变的肠道菌群与骨密度具有直接相关性，SECO给药后的肠菌转移可以显著改善去卵巢引起的骨流失；通过去卵巢模型和抗生素干预模型，发现了SECO具有除调控肠道菌群之外的肠道直接作用靶点；通过LC-MS含量测定、分子对接、表面等离子共振(SPR)、蛋白表达、蛋白活力、免疫组化等技术方法，发现并验证了SECO是TPH-1蛋白的抑制剂，其可以通过与TPH-1蛋白结合、抑制TPH-1蛋白活力及表达，从而降低5-HT的合成，进而间接影响骨代谢；通过蛋白组学测定，发现抗生素干预后，SECO仍然发挥骨保护作用可能与矿物质吸收相关。研究结果拓展了SECO调控骨代谢的作用机制，丰富了TPH-1抑制剂的化合物类型，为抗骨质疏松新药和TPH-1抑制剂的研发提供新的思路和新的化合物来源。