胰高血糖素样肽-1受体介导骨髓巨噬细胞极化调控骨重建功能及分子机制研究

Osteoporosis (OP) is a growing public problem currently seriously affecting more than 200 million people's health worldwide. Currently, studying the pathogenesis of osteoporosis and developing new therapy have attracted more attentions. We previously found GLP-1 agonist exendin-4 could effectively treat osteoporosis. So far the mechanism is almost entirely unclear. Inflammatory cytokines in bone marrow microenvironment, including IL-1、IL-6 and TNF-α, are important for keeping bone remodeling balance. Many cells, such as macrophages, can produce inflammatory cytokines. The results of our previous study and reference showed the production and expression of IL-1, IL-6 and TNF-α were significantly increased in serum and bone tissue of osteoporotic rats. Meanwhile M1 macrophage-specific molecule MCP-1 was increased and M2 macrophage-specific molecule Arg-1 was reduced, indicating M1 polarization of macrophages was enhanced in osteoporosis. Exendin-4 could effectively ameliorate osteoporotic symptoms and decrease the production and expressions of IL-1, IL-6, TNF-α, MCP-1 and increased the level of Arg-1 in osteoporotic rats. As the same time, GLP-1 receptor is expressed on macrophages but not on osteoblasts and osteoclasts. So in this project we hypothesize that GLP-1 could improve bone remodeling balance and treat osteoporosis by regulating bone marrow macrophage polarization via activating GLP-1 receptor expressed on macrophages. In our research, we plan to investigate the correlation between bone marrow macrophage polarization and osteoporosis, and then to explore the protective effects and molecular machanisms of GLP-1 on osteoporosis through regulation of macrophage polarization. This study will reveal the new pathogenesis of osteoporosis, elucidate the molecular mechanism of GLP-1 regulating bone metabolism and bone remodeling, as well as explore new target and new drug for osteoporosis treatment.

骨质疏松(OP)是严重危害人类健康的全球公共卫生问题，其发病机制及防治研究是当前热点。我们前期发现GLP-1受体激动剂能有效治疗OP，但机制不清。骨髓微环境中IL-1、IL-6、TNF-α是维持骨重建平衡的重要细胞因子，巨噬细胞极化是其主要来源之一。文献和我们前期研究表明OP大鼠IL-1等分泌增多，巨噬细胞向M1极化；而GLP-1受体激动剂能有效治疗OP，降低IL-1等表达，抑制巨噬细胞的M1极化。成骨和破骨细胞上均不存在GLP-1受体，但巨噬细胞膜表面表达GLP-1受体，因此我们提出假说：GLP-1通过激活其受体调控骨髓巨噬细胞极化，改善骨重建平衡，治疗骨质疏松。本课题拟在多水平、多层次证实GLP-1调控骨髓巨噬细胞极化、治疗骨质疏松的作用及其机制。旨在揭示OP发病新机制，完善骨重建平衡的分子调控网络；阐明GLP-1受体调控骨代谢、改善骨重建平衡的分子机制，为防治OP提供新靶标和新药物。

骨质疏松(OP)是严重危害人类健康的全球公共卫生问题，其发病机制及防治研究是当前热点。我们前期发现GLP-1受体激动剂Exendin-4能有效治疗OP，但机制尚未阐明。骨髓微环境中IL-1、TGF-β、VEGF等细胞因子在维持骨重建平衡中发挥重要作用，而极化的巨噬细胞是其主要来源之一。有研究提示成骨和破骨细胞上均不存在GLP-1受体，巨噬细胞膜表面则表达该受体，因此我们提出假说：Exendin-4通过激活其受体调控骨髓巨噬细胞(BMDMs)极化，改善骨重建平衡，治疗骨质疏松。为了证明此假说，本课题在动物、细胞和分子水平上进行研究。OVX小鼠经Exendin-4治疗后骨小梁微结构显著改善，Exendin-4治疗组小鼠骨表面成骨细胞数量显著增加，Runx-2和Osterix的表达也显著增加，而破骨细胞活性明显抑制。免疫组化结果显示，Exendin-4显著增加了骨髓间充质干细胞(BMSCs)的数量，提高了TGF-β1表达，并且增加骨组织内TGF-β1蛋白的表达。同时OVX小鼠BMDMs数量与对照组相比显著增多，给予氯膦酸脂质体清除BMDMs后，Exendin-4改善骨形成的作用显著减弱，骨组织内TGF-β1的含量明显降低。表明BMDMs参与了Exendin-4改善骨质疏松的作用。同时我们在动物和细胞水平证明Exendin-4能显著增加M2型巨噬细胞标志物的表达，减少M1型巨噬细胞标志物的表达，提示Exendin-4能够促进BMDMs向M2型极化。Transwell结果显示，Exendin-4处理BMDMs后能够促进BMSCs迁移，而预先给予TGF-β1抗体处理，Exendin-4诱导BMSCs迁移的作用则显著减弱。上述结果提示Exendin-4通过促进BMDMs向M2极化，分泌TGF-β1，继而诱导BMSCs发生迁移。免疫组化及细胞实验也证明Exendin-4能促进BMDMs产生血管形成因子VEGF和MMP-2，增加CD31阳性血管内皮细胞在骨组织内的数量，促进骨组织新生血管生成，改善骨形成。最后Western blot和免疫共沉淀实验证明Exendin-4通过激活cAMP/PKA/STAT3信号通路，促进STAT3磷酸化，进而诱导BMDMs向M2型极化。本研究阐明了GLP-1受体调控骨代谢、改善骨重建平衡的分子机制，为防治OP提供了新靶标。