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中华损伤与修复杂志(电子版) ›› 2025, Vol. 20 ›› Issue (05) : 412 -420. doi: 10.3877/cma.j.issn.1673-9450.2025.05.008

论著

对硒代甲硫氨酸逆转糖皮质激素介导的成骨细胞凋亡和成骨阻抑治疗大鼠激素性股骨头坏死的机制探索
马小杰1, 张贵慧2, 李润泽3, 王秋入4, 陈带领5, 马清伟6, 张磊6,(), 陈长军6,()   
  1. 1 250012 济南,山东中医药大学第二临床医学院 山东省千佛山医院骨外科
    2 250012 济南,山东第一医科大学第一附属医院(山东省千佛山医院)病理科
    3 271000 泰安八十八医院骨外科
    4 610041 成都,四川大学华西医院骨外科
    5 250012 济南,山东第二医科大学临床医学院 山东省千佛山医院骨外科
    6 250012 济南,山东第一医科大学第一附属医院(山东省千佛山医院)骨外科
  • 收稿日期:2025-03-29 出版日期:2025-10-01
  • 通信作者: 张磊, 陈长军
  • 基金资助:
    山东省自然科学基金(ZR2023QH517)

Mechanistic exploration of Selenomethionine in reversing glucocorticoid-mediated osteoblast apoptosis and osteogenesis inhibition for treating glucocorticoid-induced osteonecrosis of the femoral head in rats

Xiaojie Ma1, Guihui Zhang2, Runze Li3, Qiuru Wang4, Dailing Chen5, Qingwei Ma6, Lei Zhang6,(), Changjun Chen6,()   

  1. 1 Second Clinical Medical College of Shandong University of Traditional Chinese Medicine,Department of Orthopaedics Surgery,Shandong Provincial Qianfoshan Hospital,Ji'nan 250012,China
    2 Department of Pathology,the First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital,Ji'nan 250012,China
    3 Department of Orthopaedics Surgery,Taian eighty-eight Hospital,Taian 271000,China
    4 Department of Orthopaedics Surgery,West China Hospital,Sichuan University,Chengdu 610041,China
    5 Clinical Medical College of Shandong Second Medical University,Department of Orthopaedics Surgery,Shandong Provincial Qianfoshan Hospital,Ji'nan 250012,China
    6 Department of Orthopaedic Surgery,the First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital,Ji'nan 250012,China
  • Received:2025-03-29 Published:2025-10-01
  • Corresponding author: Lei Zhang, Changjun Chen
引用本文:

马小杰, 张贵慧, 李润泽, 王秋入, 陈带领, 马清伟, 张磊, 陈长军. 对硒代甲硫氨酸逆转糖皮质激素介导的成骨细胞凋亡和成骨阻抑治疗大鼠激素性股骨头坏死的机制探索[J/OL]. 中华损伤与修复杂志(电子版), 2025, 20(05): 412-420.

Xiaojie Ma, Guihui Zhang, Runze Li, Qiuru Wang, Dailing Chen, Qingwei Ma, Lei Zhang, Changjun Chen. Mechanistic exploration of Selenomethionine in reversing glucocorticoid-mediated osteoblast apoptosis and osteogenesis inhibition for treating glucocorticoid-induced osteonecrosis of the femoral head in rats[J/OL]. Chinese Journal of Injury Repair and Wound Healing(Electronic Edition), 2025, 20(05): 412-420.

目的

研究硒代甲硫氨酸(Se)对地塞米松(DEX)诱导的成骨细胞(OB)凋亡及成骨抑制的保护作用,并分析其对激素性股骨头坏死(SONFH)大鼠的保护效果。

方法

体外培养MC3T3-E1细胞,将MC3T3-E1细胞分为对照组(不进行任何干预)、DEX组(DEX 10 μmol/L)以及DEX+Se组(DEX 10 μmol/L + Se 10 μmol/L),使用CCK-8法测定各组细胞增殖活力;同时采用Real-time PCR和Western blot法检测OB成骨分化的变化;通过2,7-二氯荧光素二乙酸酯(DCFH-DA)探针法观察细胞活性氧(ROS)的累积情况,并运用流式细胞术、Real-time PCR以及Western blot检测细胞凋亡的改变;采用Western blot法检测Se对DEX作用下PI3K/Akt/GSK-3β信号通路相关分子表达的影响。取12只8周龄雄性SD大鼠分为3组,每组4只,均进行脂多糖肌内注射后,分为生理盐水对照组、甲泼尼龙(MPS)模型组、MPS+Se干预组,通过HE染色观察空骨陷窝的发生和治疗情况。

结果

在DEX作用下,MC3T3-E1细胞中ROS水平显著升高,同时细胞凋亡也显著增加,成骨分化能力显著下降。然而,Se可以通过减少DEX诱导的OB中ROS积累,抑制细胞凋亡,并激活PI3K/Akt/GSK-3β信号通路,有效缓解DEX引起的成骨功能障碍。经Se灌胃的SONFH模型大鼠,骨小梁的排列和数量相比MPS模型组大鼠得到显著的改善,空骨陷窝数量减少,骨坏死表现介于生理盐水对照组与MPS模型组之间。

结论

Se能够逆转DEX诱导的氧化应激损伤和细胞凋亡,并通过激活PI3K/Akt/GSK-3β信号通路缓解OB的成骨抑制作用,为SONFH的治疗提供潜在策略。

Objective

To investigate the protective effects of Selenomethionine (Se) against Dexamethasone (DEX)-induced apoptosis and osteogenic inhibition in osteoblasts (OB),and evaluate its protective role in steroid-induced osteonecrosis of femoral head (SONFH) in rats.

Methods

MC3T3-E1 cells were cultured in vitro and divided into 3 groups: control group (no intervention),DEX group (10 μmol/L DEX),and DEX+Se group (DEX 10 μmol/L + Se 10 μmol/L). Cell proliferation was assessed using the CCK-8 assay. Osteogenic differentiation was evaluated via Real-time PCR and Western blot analysis. Intracellular reactive oxygen species (ROS) accumulation was measured using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. Apoptosis was analyzed by flow cytometry,Real-time PCR,and Western blot. The effects of Se on the PI3K/Akt/GSK-3β signaling pathway under DEX exposure were examined using Western blot. Tweleve eight-week-old male SD rats were divided into 3 groups with 4 rats in each group. They were divided into normal saline control group, methylprednisolone (MPS) model group and MPS+Se intervention group respectively for intervention after all received intramuscular injection of lipopolysaccharide. Histological evaluation via hematoxylin-eosin (HE) staining was performed to assess the occurrence and treatment of empty bone lacunae.

Results

Treatment with DEX increased ROS production,enhanced apoptosis levels and significantly suppressed osteogenic differentiation capacity in MC3T3-E1 cells. However, Se could inhibit cell apoptosis by reducing the accumulation of ROS in DEX-mediated OB potentially through activation of the PI3K/Akt/GSK-3β signaling pathway and also effectively alleviated DEX-mediated ostengenic dysfunction. In rats administered Selenium by gavage, the arrangement and quantity of trabecular bone were significantly improved compared with those in the MPS model group, the number of empty bone lacunae was reduced, and manifestation of osteonecrosis was between that of control group and MPS model group.

Conclusion

Selenomethionine may protect against SONFH by activating the PI3K/Akt/GSK-3β signaling pathway,thereby reversing DEX-induced osteogenic inhibition,oxidative stress damage,and apoptosis in osteoblasts. Se could serve as a potential therapeutic agent for the prevention and treatment of SONFH.

表1 实时定量PCR引物序列
图1 不同浓度Se作用后各组MC3T3-E1细胞存活率。Se10、15、20 µmol/L时细胞存活率与0 µmol/L相比差异有统计学意义(F10 µmol/L=6.580、F15 µmol/L=4.316、F20 µmol/L=4.079),*P<0.05,**P<0.01,***P<0.001
图2 Se对DEX作用下MC3T3-E1细胞氧化应激和细胞凋亡的影响。A、B示使用DCFH-DA荧光探针法检测各组MC3T3-E1细胞中ROS水平的变化并进行统计分析;C、D示采用Annexin V-FITC/PI双染流式细胞术评估不同处理对MC3T3-E1细胞凋亡的影响并进行统计分析;E示利用RT-PCR技术检测各组MC3T3-E1细胞中与凋亡相关的基因(Caspase-3、Bax和Bcl-2)表达变化并进行统计分析。与对照组相比,DEX组Caspase-3、Bax表达增加,Bcl-2表达下降(FCaspase-3=4.223、FBax=14.33、FBcl-2=5.393)。与DEX相比,DEX+Se组Caspase-3、Bax表达下降,Bcl-2表达增加(FCaspase-3=5.465、FBax=10.07、FBcl-2=5.096);F、G示运用Western blot方法分析不同处理对MC3T3-E1细胞中凋亡相关蛋白(cleaved Caspase-3、Bax和Bcl-2)表达水平的影响并进行统计分析。与对照组相比,DEX组cleaved Caspase-3、Bax表达增加,Bcl-2表达下降(Fcleaved Caspase-3=10.024、FBax=14.42、FBcl-2=14.58),与DEX组相比,DEX+Se组cleaved Caspase-3、Bax表达下降,Bcl-2表达增加(Fcleaved Caspase-3=4.841、FBax=4.476、FBcl-2=6.45)。*P<0.05,**P<0.01,***P<0.001
图3 Se对DEX作用下MC3T3-E1细胞成骨性能的影响。A示使用RT-PCR法检测各组MC3T3-E1细胞中成骨相关基因(Runx-2、ALP、Collagen Ⅰ和Osteocalcin)的表达变化,并进行统计分析。与对照组相比,DEX组Runx-2、ALP、Collagen Ⅰ、osteocalcin表达下降(FRunx-2=8.504、FALP=5.275、FCollagen Ⅰ=8.624、Fosteocalcin=13.37)。与DEX组相比,DEX+Se组Runx-2、ALP、Collagen Ⅰ、osteocalcin表达增加(FRunx-2=17.95、FALP=12.00、FCollagen Ⅰ=6.964、Fosteocalcin=7.346);B、C示采用Western blot法评估不同处理条件对MC3T3-E1细胞中成骨相关蛋白(ALP、Runx-2、Collagen Ⅰ)表达的影响,并进行统计分析。与对照组相比,DEX组Runx-2、ALP、Collagen Ⅰ表达下降(FRunx-2=9.766、FALP=11.45、FCollagen Ⅰ=9.905)。与DEX组相比,DEX+Se组Runx-2、ALP、Collagen Ⅰ表达增加(FRunx-2=10.286、FALP=7.37、FCollagen Ⅰ=6.046)。*P<0.05,**P<0.01,***P<0.001
图4 Se对糖皮质激素介导的MC3T3-E1细胞中PI3K/Akt/p-GSK3β信号通路的抑制作用。 A-D示利用 Western blot 方法检测不同处理条件对 MC3T3-E1 细胞中 PI3K/Akt/p-GSK3β信号通路的影响。与对照组相比,DEX组p-PI3K/PI3K、p-Akt/Akt、p-GSK-3β/GSK3β表达下降(t=7.812、8.672、8.302)。与DEX组相比,DEX-Se组p-PI3K/PI3K、p-Akt/Akt、p-GSK-3β/GSK-3β表达增加(t=4.780、5.565、4.940);E-F示Akt抑制剂MK2206和PI3K抑制剂LY294002对PI3K/Akt/GSK-3β信号通路的影响,及统计学分析。*P<0.05,**P< 0.01,***P<0.001
图5 Se对糖皮质激素诱导的大鼠股骨头坏死的影响。A示3组大鼠股骨头病理变化;B示3组大鼠股骨头空骨陷窝率统计分析与比较。***P<0.001
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