目的 探讨菝葜皂苷元（sarsasapogenin，SAR）对糖皮质激素性骨质疏松症（glucocorticoid-induced osteoporosis，GIOP）成骨细胞铁死亡的影响及作用机制。方法 地塞米松处理小鼠MC3T3-E1成骨细胞建立GIOP细胞模型，设置对照组、模型组及SAR（1、2、4 μmol/L）组和SAR（4μmol/L）＋铁死亡激活剂erastin（10 μmol/L）组、SAR（4μmol/L）＋磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）抑制剂LY294002（10 μmol/L）组。CCK-8法检测细胞活力；采用C11 BODIPY 581/591、二氢乙锭（dihydroethidium，DHE）和Ferro Orange染色检测脂质过氧化、活性氧（reactive oxygen species，ROS）和亚铁离子水平；试剂盒检测丙二醛（malondialdehyde，MDA）和谷胱甘肽（glutathione，GSH）含量；碱性磷酸酯酶（alkaline phosphatase，ALP）和茜素红S染色检测成骨分化；Western blotting检测乙酰辅酶A合成酶长链家族成员4（acetyl coenzyme A synthetase long-chain family member 4，ACSL4）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、PI3K/蛋白激酶B（protein kinase B，Akt）信号通路及骨形成相关蛋白[ALP、I型胶原A1（collagen type I α1，COL1A1）、骨形态发生蛋白2（bone morphogenetic protein 2，BMP2）、Runt相关转录因子2（Runt-related transcription factor 2，RUNX2）]表达。ip地塞米松建立小鼠GIOP模型，随机分为对照组、模型组、SAR（10 mg/kg）组和SAR（10 mg/kg）＋LY294002（10 mg/kg）组。采用Micro-CT对小鼠股骨远端进行骨形态分析；苏木素-伊红（hematoxylin eosin，HE）和Masson染色评价股骨组织形态学改变；钙黄绿素-茜素红双标记实验测定矿化沉积率（mineral apposition rate，MAR）；Western blotting检测ACSL4、GPX4、PI3K/Akt信号通路相关蛋白表达。结果 与对照组比较，模型组细胞活力显著降低（P＜0.001），脂质过氧化、ROS、亚铁离子、MDA水平及ACSL4蛋白表达显著升高（P＜0.001），GSH水平和GPX4蛋白表达显著降低（P＜0.001），ALP含量及活性、钙结节形成、骨形成相关蛋白表达及PI3K、Akt的磷酸化水平显著降低（P＜0.001）；与模型组比较，SAR处理可呈剂量相关性地增加细胞活力，抑制铁死亡，促进骨形成，并上调PI3K和Akt的磷酸化水平（P＜0.01）；与SAR组比较，SAR＋erastin组骨形成能力显著降低（P＜0.05），SAR＋LY294002组铁死亡明显增多（P＜0.05）。动物实验结果表明，与对照组比较，模型组小鼠股骨远端骨小梁出现严重的骨丢失，骨形态严重损坏，p-PI3K、p-Akt和GPX4蛋白表达显著降低（P＜0.001），ACSL4蛋白表达显著升高（P＜0.001）；与模型组比较，SAR组骨形态明显改善，p-PI3K、p-Akt和GPX4蛋白表达显著升高（P＜0.01），ACSL4蛋白表达显著降低（P＜0.001）；与SAR组比较，SAR＋LY294002组骨形态损坏，p-PI3K、p-Akt和GPX4蛋白表达显著降低（P＜0.05），ACSL4蛋白表达显著升高（P＜0.05）。结论 SAR可有效减轻GIOP，其机制可能是通过激活PI3K/Akt信号通路抑制成骨细胞铁死亡，进而促进骨形成。

Objective To investigate the effect and mechanism of sarsasapogenin (SAR) on ferroptosis of osteoblasts in glucocorticoid-induced osteoporosis (GIOP). Methods GIOP cell model was established by treating mouse osteoblasts (MC3T3-E1) with dexamethasone (Dex), control group, model group, SAR (1, 2, 4 μmol/L) group, SAR (4 μmol/L) + ferroptosis activator erastin (10 μmol/L) group, SAR (4 μmol/L) + phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 μmol/L) group were set up. Cell viability was measured by CCK-8 assay; Levels of lipid peroxidation, reactive oxygen species (ROS) and ferrous ions were detected by C11 BODIPY 581/591, DHE and Ferro Orange staining; The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by kits; Osteogenic differentiation was detected by alkaline phosphatase (ALP) and alizarin red S staining; The expressions of acetyl coenzyme A synthetase long-chain family member (ACSL4), glutathione peroxidase 4 (GPX4), PI3K/protein kinase B (Akt) signaling pathway, and bone formation related proteins (ALP, collagen type I alpha 1 (COL1A1), bone morphogenetic protein 2 (BMP2), Runt-related transcription factor 2 (RUNX2) were detected by Western blotting. Mouse GIOP model was established by intraperitoneal injection of Dex. The mice were randomly divided into control group, model group, SAR (10 mg/kg) group and SAR (10 mg/kg) + LY294002 (10 mg/kg) group. The bone morphology of distal femurs of mice was analyzed by Micro-CT; The morphologic changes of femur were evaluated by hematoxylin eosin (HE) and Masson staining; The mineral apposition rate (MAR) was determined by calcein-alizarin red double labeling experiment; The expressions of ACSL4, GPX4 and PI3K/Akt signaling pathway related proteins were detected by Western blotting. Results Compared with control group, cell viability in model group was significantly decreased (P < 0.001), the levels of lipid peroxidation, ROS, ferrous ion, MDA and ACSL4 protein expression were significantly increased (P < 0.001), while GSH content and GPX4 protein expression were significantly decreased (P < 0.001), the content and activity of ALP, calcium nodules, bone formation related protein expressions and phosphorylation levels of PI3K and Akt were significantly decreased (P < 0.001). Compared with model group, SAR dose-dependently increased cell viability, reduced ferroptosis, promoted bone formation, and increased the phosphorylation levels of PI3K and Akt (P < 0.01). Compared with SAR group, bone formation ability was significantly decreased in SAR + erastin group (P < 0.05), while ferroptosis in SAR + LY294002 group was significantly enhanced (P < 0.05). The results of animal experiments showed that compared with control group, there was serious bone loss in distal bone trabecula of femur, bone morphology was severely damaged, p-PI3K, p-Akt and GPX4 protein expressions were significantly decreased, while ACSL4 protein expression was significantly increased in model group (P < 0.001). Compared with model group, bone morphology was significantly improved, p-PI3K, p-Akt and GPX4 protein expressions were significantly increased (P < 0.01), while ACSL4 protein expression was significantly decreased in SAR group (P < 0.01). Compared with SAR group, bone morphology was damaged, p-PI3K, p-Akt and GPX4 protein expressions were significantly decreased (P < 0.05), while ACSL4 protein expression was significantly increased in SAR + LY294002 group (P < 0.05).Conclusion SAR could effectively alleviate GIOP, and its mechanism might be involved in the activation of PI3K/Akt signaling pathway to inhibit ferroptosis of osteoblasts and thus promote bone formation.

R285.5

河南省中医药科学研究专项课题（2021ZY2120，2024ZY2050）；河南省卫生健康委国家中医药传承创新中心科研专项（2023ZXZX1172）