目的 探討菝葜皂苷元（sarsasapogenin，SAR）對糖皮質(zhì)激素性骨質(zhì)疏松癥（glucocorticoid-induced osteoporosis，GIOP）成骨細(xì)胞鐵死亡的影響及作用機制。方法 地塞米松處理小鼠MC3T3-E1成骨細(xì)胞建立GIOP細(xì)胞模型，設(shè)置對照組、模型組及SAR（1、2、4 μmol/L）組和SAR（4μmol/L）＋鐵死亡激活劑erastin（10 μmol/L）組、SAR（4μmol/L）＋磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）抑制劑LY294002（10 μmol/L）組。CCK-8法檢測細(xì)胞活力；采用C11 BODIPY 581/591、二氫乙錠（dihydroethidium，DHE）和Ferro Orange染色檢測脂質(zhì)過氧化、活性氧（reactive oxygen species，ROS）和亞鐵離子水平；試劑盒檢測丙二醛（malondialdehyde，MDA）和谷胱甘肽（glutathione，GSH）含量；堿性磷酸酯酶（alkaline phosphatase，ALP）和茜素紅S染色檢測成骨分化；Western blotting檢測乙酰輔酶A合成酶長鏈家族成員4（acetyl coenzyme A synthetase long-chain family member 4，ACSL4）、谷胱甘肽過氧化物酶4（glutathione peroxidase 4，GPX4）、PI3K/蛋白激酶B（protein kinase B，Akt）信號通路及骨形成相關(guān)蛋白[ALP、I型膠原A1（collagen type I α1，COL1A1）、骨形態(tài)發(fā)生蛋白2（bone morphogenetic protein 2，BMP2）、Runt相關(guān)轉(zhuǎn)錄因子2（Runt-related transcription factor 2，RUNX2）]表達(dá)。ip地塞米松建立小鼠GIOP模型，隨機分為對照組、模型組、SAR（10 mg/kg）組和SAR（10 mg/kg）＋LY294002（10 mg/kg）組。采用Micro-CT對小鼠股骨遠(yuǎn)端進(jìn)行骨形態(tài)分析；蘇木素-伊紅（hematoxylin eosin，HE）和Masson染色評價股骨組織形態(tài)學(xué)改變；鈣黃綠素-茜素紅雙標(biāo)記實驗測定礦化沉積率（mineral apposition rate，MAR）；Western blotting檢測ACSL4、GPX4、PI3K/Akt信號通路相關(guān)蛋白表達(dá)。結(jié)果 與對照組比較，模型組細(xì)胞活力顯著降低（P＜0.001），脂質(zhì)過氧化、ROS、亞鐵離子、MDA水平及ACSL4蛋白表達(dá)顯著升高（P＜0.001），GSH水平和GPX4蛋白表達(dá)顯著降低（P＜0.001），ALP含量及活性、鈣結(jié)節(jié)形成、骨形成相關(guān)蛋白表達(dá)及PI3K、Akt的磷酸化水平顯著降低（P＜0.001）；與模型組比較，SAR處理可呈劑量相關(guān)性地增加細(xì)胞活力，抑制鐵死亡，促進(jìn)骨形成，并上調(diào)PI3K和Akt的磷酸化水平（P＜0.01）；與SAR組比較，SAR＋erastin組骨形成能力顯著降低（P＜0.05），SAR＋LY294002組鐵死亡明顯增多（P＜0.05）。動物實驗結(jié)果表明，與對照組比較，模型組小鼠股骨遠(yuǎn)端骨小梁出現(xiàn)嚴(yán)重的骨丟失，骨形態(tài)嚴(yán)重?fù)p壞，p-PI3K、p-Akt和GPX4蛋白表達(dá)顯著降低（P＜0.001），ACSL4蛋白表達(dá)顯著升高（P＜0.001）；與模型組比較，SAR組骨形態(tài)明顯改善，p-PI3K、p-Akt和GPX4蛋白表達(dá)顯著升高（P＜0.01），ACSL4蛋白表達(dá)顯著降低（P＜0.001）；與SAR組比較，SAR＋LY294002組骨形態(tài)損壞，p-PI3K、p-Akt和GPX4蛋白表達(dá)顯著降低（P＜0.05），ACSL4蛋白表達(dá)顯著升高（P＜0.05）。結(jié)論 SAR可有效減輕GIOP，其機制可能是通過激活PI3K/Akt信號通路抑制成骨細(xì)胞鐵死亡，進(jìn)而促進(jìn)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

河南省中醫(yī)藥科學(xué)研究專項課題（2021ZY2120，2024ZY2050）；河南省衛(wèi)生健康委國家中醫(yī)藥傳承創(chuàng)新中心科研專項（2023ZXZX1172）