阿拉曼丁通过 AMPK/eNOS 轴促进成骨细胞分化来减轻卵巢切除诱导的骨质疏松症。
Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.
机构信息
Department of Orthopaedics, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong City, 226001, Jiangsu Province, PR China.
Laboratory Animal Center of Nantong University, Medical School of Nantong University, Nantong City, 226001, Jiangsu Province, PR China.
出版信息
BMC Musculoskelet Disord. 2024 Jan 10;25(1):45. doi: 10.1186/s12891-023-07159-2.
BACKGROUND
Alamandine is a newly characterized peptide of renin angiotensin system. Our study aims to investigate the osteo-preservative effects of alamandine, explore underlying mechanism and bring a potential preventive strategy for postmenopausal osteoporosis in the future.
METHODS
An ovariectomy (OVX)-induced rat osteoporosis model was established for in vivo experiments. Micro-computed tomography and three-point bending test were used to evaluate bone strength. Histological femur slices were processed for immunohistochemistry (IHC). Bone turnover markers and nitric oxide (NO) concentrations in serum were determined with enzyme-linked immunosorbent assay (ELISA). The mouse embryo osteoblast precursor (MC3T3-E1) cells were used for in vitro experiments. The cell viability was analysed with a Cell Counting Kit‑8. We performed Alizarin Red S staining and alkaline phosphatase (ALP) activity assay to observe the differentiation status of osteoblasts. Western blotting was adopted to detect the expression of osteogenesis related proteins and AMP-activated protein kinase/endothelial nitric oxide synthase (AMPK/eNOS) in osteoblasts. DAF-FM diacetate was used for semi-quantitation of intracellular NO.
RESULTS
In OVX rats, alamandine alleviated osteoporosis and maintained bone strength. The IHC showed alamandine increased osteocalcin and collagen type I α1 (COL1A1) expression. The ELISA revealed alamandine decreased bone turnover markers and restored NO level in serum. In MC3T3-E1 cells, alamandine promoted osteogenic differentiation. Western blotting demonstrated that alamandine upregulated the expression of osteopontin, Runt-related transcription factor 2 and COL1A1. The intracellular NO was also raised by alamandine. Additionally, the activation of AMPK/eNOS axis mediated the effects of alamandine on MC3T3-E1 cells and bone tissue. PD123319 and dorsomorphin could repress the regulating effect of alamandine on bone metabolism.
CONCLUSION
Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.
背景
阿马林是肾素-血管紧张素系统中一种新鉴定的肽。我们的研究旨在探讨阿马林的骨保护作用，探索其潜在的作用机制，并为绝经后骨质疏松症带来一种潜在的预防策略。
方法
建立了去卵巢（OVX）诱导的大鼠骨质疏松模型进行体内实验。使用微计算机断层扫描和三点弯曲试验评估骨强度。对股骨组织切片进行免疫组织化学（IHC）处理。使用酶联免疫吸附试验（ELISA）测定血清中骨转换标志物和一氧化氮（NO）浓度。采用小鼠胚胎成骨前体细胞（MC3T3-E1）细胞进行体外实验。使用细胞计数试剂盒-8 分析细胞活力。我们进行茜素红 S 染色和碱性磷酸酶（ALP）活性测定，观察成骨细胞的分化状态。采用 Western blot 检测成骨细胞中骨生成相关蛋白和 AMP 激活的蛋白激酶/内皮型一氧化氮合酶（AMPK/eNOS）的表达。使用 DAF-FM 二乙酸酯对半定量细胞内 NO。
结果
在 OVX 大鼠中，阿马林减轻了骨质疏松症并维持了骨强度。IHC 显示阿马林增加了骨钙素和Ⅰ型胶原α1（COL1A1）的表达。ELISA 显示阿马林降低了骨转换标志物并恢复了血清中的 NO 水平。在 MC3T3-E1 细胞中，阿马林促进成骨分化。Western blot 表明阿马林上调了骨桥蛋白、Runt 相关转录因子 2 和 COL1A1 的表达。细胞内 NO 也被阿马林上调。此外，AMPK/eNOS 轴的激活介导了阿马林对 MC3T3-E1 细胞和骨组织的作用。PD123319 和 Dorsomorphin 可抑制阿马林对骨代谢的调节作用。
结论
阿马林通过激活 AMPK/eNOS 轴促进成骨分化，从而减轻去卵巢诱导的骨质疏松症。
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