School of Graduate, Guangxi University of Chinese Medicine, Nanning 530001, China; Department of Endocrinology, The First Affiliated Hospital of Guangxi University of Chinese Medicine, 89-9 Dongge Road, Nanning 530023, China.
School of Graduate, Guangxi University of Chinese Medicine, Nanning 530001, China.
Phytomedicine. 2024 Jul 25;130:155718. doi: 10.1016/j.phymed.2024.155718. Epub 2024 May 20.
Senile osteoporosis (SOP) is an age-related systemic metabolic bone disorder. Previous studies have proved that Zhuang-Gu-Fang (ZGF) modulates myokines, stimulates osteogenic differentiation, and mitigates osteoporosis.
To elucidate the mechanism by which ZGF promotes osteogenic differentiation via myoblast and myoblast exosomal microRNAs (miRNAs) and investigate its potential implications in senile osteoporosis.
Characterization of ZGF and ZGF serum using UHPLC-MS/MS. An alkaline phosphatase (ALP) activity assay and staining techniques were employed to corroborate the impacts of ZGF on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) via myoblasts. Subsequently, exosomes derived from myoblasts were isolated through ultracentrifugation. The effects of ZGF on the BMSCs' osteogenic differentiation were substantiated through ALP activity, alizarin red staining, and a quantitative real-time polymerase reaction system (qRT-PCR). Selected miRNAs were identified via high-throughput sequencing and subjected to differential expression analysis, and subsequently validated through qRT-PCR. The senescence-accelerated (SAMP6) mice were selected as the SOP models. qRT-PCR analyses were further conducted to confirm the expression levels of these selected miRNAs in the muscle and bone tissues of the SAMP6 mice, and the protein expression of osteogenesis-related transcription factors OCN and Osterix in its bone tissue was evaluated by immunofluorescence staining analysis (IF).
ZGF may enhance the osteogenic differentiation of BMSCs through myoblasts and myoblast-derived exosomes. High-throughput sequencing, differential expression analysis, and subsequent qRT-PCR validation identified four miRNAs that stood out due to their significant differential expression: miR-5100, miR-142a-3p, miR-126a-3p, miR-450b-5p and miR-669a-5p. Moreover, the mice experiment corroborated these findings, which revealed that ZGF not only up-regulated the expression of miR-5100, miR-450b-5p and miR-126a-3p in muscle and bone tissues but also concurrently down-regulated the expression of miR-669a-5p in these tissues. IF staining analysis indicated that ZGF can significantly increase the protein expression of the osteogenic transcription factors OCN and Osterix in the bone tissue of mice with SOP.
ZGF can promote osteogenic differentiation of osteoblasts, regulate bone metabolism, and thereby delay the process of SOP. Perhaps, its mechanism is to upregulate myoblast-derived exosomes miR-5100, miR-126a-3p, and miR-450b-5p or downregulate miR-669a-5p. This study reports for the first time that myoblast exosomes miR-669a-5p and miR-450b-5p are novel targets for the regulation of osteoblastic differentiation and the treatment of SOP.
老年性骨质疏松症（SOP）是一种与年龄相关的系统性代谢性骨疾病。先前的研究已经证明壮骨方（ZGF）可以调节肌因子，刺激成骨分化，减轻骨质疏松症。
阐明 ZGF 通过成肌细胞及其衍生的外泌体 microRNAs（miRNAs）促进成骨分化的机制，并研究其在老年性骨质疏松症中的潜在意义。
使用 UHPLC-MS/MS 对 ZGF 和 ZGF 血清进行表征。通过碱性磷酸酶（ALP）活性测定和染色技术，证实 ZGF 通过成肌细胞对骨髓间充质干细胞（BMSCs）的成骨分化的影响。随后，通过超速离心分离出成肌细胞衍生的外泌体。通过 ALP 活性、茜素红染色和实时定量聚合酶链反应系统（qRT-PCR）证实 ZGF 对 BMSCs 成骨分化的影响。通过高通量测序鉴定选定的 miRNAs，并进行差异表达分析，随后通过 qRT-PCR 进行验证。选择快速老化（SAMP6）小鼠作为 SOP 模型。进一步进行 qRT-PCR 分析，以确认这些选定的 miRNAs 在 SAMP6 小鼠肌肉和骨骼组织中的表达水平，并通过免疫荧光染色分析（IF）评估其骨组织中成骨相关转录因子 OCN 和 Osterix 的蛋白表达。
ZGF 可能通过成肌细胞和成肌细胞衍生的外泌体增强 BMSCs 的成骨分化。高通量测序、差异表达分析和随后的 qRT-PCR 验证确定了四个因显著差异表达而脱颖而出的 miRNAs：miR-5100、miR-142a-3p、miR-126a-3p、miR-450b-5p 和 miR-669a-5p。此外，小鼠实验证实了这些发现，表明 ZGF 不仅上调了肌肉和骨骼组织中 miR-5100、miR-450b-5p 和 miR-126a-3p 的表达，还同时下调了这些组织中 miR-669a-5p 的表达。IF 染色分析表明，ZGF 可显著增加 SOP 小鼠骨骼组织中成骨转录因子 OCN 和 Osterix 的蛋白表达。
ZGF 可促进成骨细胞的成骨分化，调节骨代谢，从而延缓 SOP 的进程。其机制可能是上调成肌细胞衍生的外泌体 miR-5100、miR-126a-3p 和 miR-450b-5p，或下调 miR-669a-5p。本研究首次报道，成肌细胞外泌体 miR-669a-5p 和 miR-450b-5p 是调节成骨细胞分化和治疗 SOP 的新靶点。
