Hu Xianfeng, Yang Liu, Du Yanhua, Meng Xiangping, Shi Yuanyuan, Zeng Juan
Department of General Practice, Wuhan Fourth Hospital, Wuhan, China.
Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, China.
Bone Joint Res. 2023 Aug 3;12(8):476-485. doi: 10.1302/2046-3758.128.BJR-2022-0248.R2.
Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation.
Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay.
The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs.
APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression.
黄芪多糖（APS）参与多种生理过程，如增强免疫力和抑制肿瘤。APS可通过调节人骨髓间充质干细胞（hBMSCs）的成骨分化来影响骨质疏松症（OP）。本研究旨在阐明APS在hBMSC增殖和成骨细胞分化中的作用机制。
采用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测OP组织和hBMSCs中微小RNA（miR）-760及含锚蛋白重复序列和FYVE结构域蛋白1（ANKFY1）的表达。使用细胞计数试剂盒-8检测细胞活力。采用定量逆转录聚合酶链反应（qRT-PCR）评估细胞周期蛋白D1和成骨标志物基因（骨钙素（OCN）、碱性磷酸酶（ALP）和 runt相关转录因子2（RUNX2））的表达。通过茜素红S染色检测矿化沉积。此外，在调节miR-760后，采用蛋白质免疫印迹法检测ANKFY1蛋白水平。使用荧光素酶报告基因检测法确定miR-760与ANKFY1之间的关系。
miR-760在OP组织中的表达上调，而ANKFY1表达下调。APS通过以下方式促进hBMSCs的分化和增殖：提高细胞活力；上调细胞周期蛋白D1、ALP、OCN和RUNX2的表达水平；诱导成骨细胞矿化。此外，APS下调miR-760的表达。发现miR-760过表达可抑制APS对hBMSC分化和增殖的促进作用，而敲低miR-760则产生相反的效果。发现ANKFY1是miR-760的直接靶点。此外，ANKFY1参与了APS介导的hBMSCs中miR-760功能的调节。
APS促进hBMSCs的成骨分化和增殖。此外，APS通过下调miR-760和上调ANKFY1表达来减轻OP的影响。
