Supplementary Material for: Glucagon-Like Peptide-2 Ameliorates Age-Associated Bone Loss and Gut Barrier Dysfunction in Senescence-Accelerated Mouse Prone 6 Mice

Introduction: Senile osteoporosis is one of the most common age-related diseases worldwide. Glucagon like peptide-2 (GLP-2), a naturally occurring gastrointestinal peptide, possesses therapeutic effects on bone loss in postmenopausal women and ovariectomized rats. However, the role of GLP-2 in senile osteoporosis and underlying mechanisms has not been explored. Methods: GLP-2 was subcutaneously injected into the 6-month-old male senile osteoporosis model of senescence-accelerated mouse prone 6 (SAMP6) mice for 6 weeks. SAMP6 subjected to normal saline and senescence-accelerated mouse resistant 1 served as control groups. Micro-computed tomography was performed to evaluate the bone mass and microarchitecture of the mice. Osteoblastic and osteoclastic activities were determined by biochemical, quantitative real-time PCR, histological, and histomorphometric analyses combined with hematoxylin-eosin, toluidine blue, and tartrate-resistant acid phosphatase staining. We also examined the proteins and structure of intestinal tight junction using immunohistochemical assay as well as a transmission electron microscope. Serum inflammation marker levels were measured using ELISA. Additionally, anti-oxidative enzymes GPX-4 and SOD-2 and receptors of GLP-2 and vitamin D expression in the ileum and colon were detected under immunofluorescence staining. Results: Six-week GLP-2 treatment attenuated bone loss in SAMP6 mice, as evidenced by increased bone mineral density, improved microarchitecture in femora, and enhanced osteogenic activities. In contrast, the activity of osteoclastic activity was not obviously inhibited. Moreover, GLP-2 ameliorated tight junction structure and protein expression in the intestinal barrier, which was accompanied by the reduction of TNF-α level. The expression of receptors of intestinal GLP-2 and vitamin D in the ileum was elevated. Furthermore, the oxidative stress in the intestines was improved by increasing the GPX-4 and SOD-2 signaling. Conclusion: Our findings suggest that GLP-2 could ameliorate age-associated bone loss, tight junction structure, and improved antioxidant enzyme activity in the gut in SAMP6 mice. Amelioration of gut barrier dysfunction may potentially contribute to improving bone formation and provide evidence for targeting the entero-bone axis in the treatment of senile osteoporosis.引言：老年性骨质疏松症是全球范围内最常见的年龄相关性疾病之一。胰高血糖素样肽-2（Glucagon like peptide-2, GLP-2）是一种天然存在的胃肠肽，对绝经后女性及去卵巢大鼠的骨丢失具有治疗作用。然而，GLP-2在老年性骨质疏松症中的作用及其潜在机制尚未得到探索。 方法：将GLP-2皮下注射给6月龄雄性快速老化小鼠易感品系6（senescence-accelerated mouse prone 6, SAMP6）老年性骨质疏松模型小鼠，持续6周。以给予生理盐水的SAMP6小鼠及快速老化小鼠抵抗品系1（senescence-accelerated mouse resistant 1, SAMR1）小鼠作为对照组。采用显微计算机断层扫描（Micro-computed tomography, micro-CT）评估小鼠的骨量及骨微结构。通过生化检测、定量实时PCR、组织学及组织形态计量学分析，结合苏木精-伊红、甲苯胺蓝及抗酒石酸酸性磷酸酶染色，评估成骨细胞与破骨细胞活性。同时采用免疫组织化学检测及透射电子显微镜，观察肠紧密连接的蛋白表达与结构。采用酶联免疫吸附测定（ELISA）检测血清炎症标志物水平。此外，通过免疫荧光染色检测回肠与结肠中谷胱甘肽过氧化物酶4（GPX-4）、超氧化物歧化酶2（SOD-2）的表达，以及GLP-2受体与维生素D受体的表达。 结果：为期6周的GLP-2治疗可减轻SAMP6小鼠的骨丢失，具体表现为骨密度升高、股骨微结构改善以及成骨活性增强。与之相反，破骨细胞活性未受到明显抑制。此外，GLP-2可改善肠屏障的紧密连接结构与蛋白表达，同时伴随肿瘤坏死因子α（TNF-α）水平降低。回肠内肠道GLP-2受体与维生素D受体的表达水平升高。此外，通过增强GPX-4与SOD-2信号通路，可改善肠道氧化应激状态。 结论：本研究结果表明，GLP-2可减轻SAMP6小鼠的年龄相关性骨丢失，改善肠道紧密连接结构，并提升肠道抗氧化酶活性。肠屏障功能障碍的改善或可促进骨形成，同时为靶向肠-骨轴治疗老年性骨质疏松症提供了实验依据。