Huang Guijiang, Yin Wenjie, Zhao Xin, Xu Muli, Wang Peijin, Li Rong, Zhou Li, Tang Wei, Jiao Jianlin
Department of Science and Education, The First Affiliated Hospital of Kunming Medical University, Kunming, 650600, China; Department of Pharmacy, Yan'an Hospital Affiliated to Kunming Medical University, Kunming, 650600, China.
Department of Pharmacy, Yan'an Hospital Affiliated to Kunming Medical University, Kunming, 650600, China.
J Ethnopharmacol. 2025 Jan 31;340:118961. doi: 10.1016/j.jep.2024.118961. Epub 2024 Dec 7.
In China, Osteoking is a commonly used treatment and preventive measure for osteoporosis. The pathophysiology of osteoporosis is closely associated with apoptosis; however, it remains unclear whether the role of Osteoking in promoting bone formation is linked to apoptosis.
This study aims to investigate whether Osteoking inhibits apoptosis of BMSCs in osteoporotic rats via the PI3K/AKT signaling pathway and to conduct a detailed exploration of this mechanism. The goal is to provide a theoretical basis for the clinical application of Osteoking in osteoporosis treatment.
A rat model of osteoporosis was established through bilateral ovariectomy (OVX), followed by treatment with Osteoking. After ten weeks of therapy, BMD was evaluated. The biomechanics of the left tibia were measured, the left femur was sequenced, and the right tibia was stained using histomorphometric and Masson's staining methods. Peripheral serum was collected to measure bone-related markers, including E2, PINP, and CTX. RNA-Seq results were verified using the remaining bone samples. Comparative analysis demonstrated the efficacy of Osteoking in treating osteoporosis and provided preliminary insights into the underlying mechanisms. Primary BMSCs were cultured using bone marrow apposition. CCK8 assays were conducted to screen the intervention conditions of Osteoking and LY294002. Various concentrations of Osteoking-containing serum and LY294002 were tested separately to determine the optimal intervention concentration for drug delivery. The impact of Osteoking on lipid formation was also evaluated. Following treatment of BMSCs from OVX rats with Sham serum, OVX serum, OVX + LY294002 serum, and Osteoking + LY294002 serum, the expression of PI3K/AKT/mTOR, osteogenesis-related regulatory factors, and apoptosis-related regulatory factors was assessed. Flow cytometry was employed to evaluate apoptosis in BMSCs.
Osteoking significantly improved whole-body BMD and bone biomechanical indices in OVX rats. It also significantly elevated the serum levels of E2 and PINP while reducing the level of CTX, which significantly improved bone microstructure and promoted new bone formation. RNA-seq analysis indicated that the therapeutic mechanism involved the PI3K/AKT signaling pathway. Osteoking increased the expression of RUNX2 and decreased the expression of PPAR-γ, a marker of lipogenesis, in OVX rats. Extraction of BMSCs for subsequent studies revealed a significant reduction in proliferation and osteogenic differentiation, along with an increase in lipogenic differentiation, in the OVX group. Osteoking treatment inhibited the expression of PPAR-γ and increased the expression of RUNX2 in BMSCs. Additionally, Osteoking reversed the LY294002-mediated inhibition of PI3K/AKT/mTOR signaling pathway activation, increased the expression of the apoptosis-protecting protein Bcl2, and decreased the expression of apoptosis-associated proteins Caspase3 and Bax.
Osteoking markedly improved bone microstructure, biomechanics, and bone density in OVX rats. Osteoking-containing serum reversed the imbalance in lineage differentiation in OVX rats, characterized by reduced osteogenic differentiation and increased lipid differentiation of BMSCs. Furthermore, Osteoking-containing serum significantly increased BMSC proliferation and prevented apoptosis in OVX rats through the PI3K/AKT signaling pathway.
在中国，骨刻灵是骨质疏松症常用的治疗和预防措施。骨质疏松症的病理生理学与细胞凋亡密切相关；然而，骨刻灵促进骨形成的作用是否与细胞凋亡有关仍不清楚。
本研究旨在探讨骨刻灵是否通过PI3K/AKT信号通路抑制骨质疏松大鼠骨髓间充质干细胞（BMSCs）的凋亡，并对该机制进行详细探究。目的是为骨刻灵在骨质疏松症治疗中的临床应用提供理论依据。
通过双侧卵巢切除（OVX）建立大鼠骨质疏松模型，随后用骨刻灵进行治疗。治疗10周后，评估骨密度（BMD）。测量左胫骨的生物力学性能，对左股骨进行测序，并用组织形态计量学和Masson染色方法对右胫骨进行染色。收集外周血清以测量骨相关标志物，包括雌二醇（E2）、I型前胶原氨基端前肽（PINP）和I型胶原交联C端肽（CTX）。使用剩余的骨样本验证RNA测序结果。比较分析证明了骨刻灵治疗骨质疏松症的疗效，并对潜在机制提供了初步见解。采用骨髓贴壁法培养原代BMSCs。进行CCK8试验以筛选骨刻灵和LY294002的干预条件。分别测试不同浓度的含骨刻灵血清和LY294002，以确定药物递送的最佳干预浓度。还评估了骨刻灵对脂质形成的影响。用假手术血清、OVX血清、OVX + LY294002血清和骨刻灵 + LY294002血清处理OVX大鼠的BMSCs后，评估PI3K/AKT/mTOR、成骨相关调节因子和凋亡相关调节因子的表达。采用流式细胞术评估BMSCs中的细胞凋亡。
骨刻灵显著改善了OVX大鼠的全身骨密度和骨生物力学指标。它还显著提高了血清E2和PINP水平，同时降低了CTX水平，显著改善了骨微结构并促进了新骨形成。RNA测序分析表明治疗机制涉及PI3K/AKT信号通路。骨刻灵增加了OVX大鼠中RUNX2的表达，并降低了脂质生成标志物PPAR-γ的表达。提取BMSCs用于后续研究发现，OVX组的增殖和成骨分化显著降低，同时脂质生成分化增加。骨刻灵治疗抑制了BMSCs中PPAR-γ的表达并增加了RUNX2的表达。此外，骨刻灵逆转了LY294002介导的对PI3K/AKT/mTOR信号通路激活的抑制，增加了凋亡保护蛋白Bcl2的表达，并降低了凋亡相关蛋白Caspase3和Bax的表达。
骨刻灵显著改善了OVX大鼠的骨微结构、生物力学和骨密度。含骨刻灵血清逆转了OVX大鼠谱系分化的不平衡，其特征是BMSCs的成骨分化减少和脂质分化增加。此外，含骨刻灵血清通过PI3K/AKT信号通路显著增加了OVX大鼠BMSCs的增殖并防止了细胞凋亡。
