Zhang Xinglong, Yang Zhenhuan, Xu Qian, Xu Chunlei, Shi Wei, Pang Ran, Zhang Kai, Liang Xinyu, Li Hui, Li Zhijun, Zhang Huafeng
Department of Orthopaedics, General Hospital of Tianjin Medical University, Tianjin, China.
Department of Orthopaedics, Tianjin Nankai Hospital, Tianjin, China.
Orthop Surg. 2024 Mar;16(3):733-744. doi: 10.1111/os.14010. Epub 2024 Feb 21.
Glucocorticoid (GC) overuse is strongly associated with steroid-induced osteonecrosis of the femoral head (SINFH). However, the underlying mechanism of SINFH remains unclear. This study aims to investigate the effect of dexamethasone (Dex)-induced oxidative stress on osteocyte apoptosis and the underlying mechanisms.
Ten patients with SINFH and 10 patients with developmental dysplasia of the hips (DDH) were enrolled in our study. Sixty rats were randomly assigned to the Control, Dex, Dex + N-Acetyl-L-cysteine (NAC), Dex + Dibenziodolium chloride (DPI), NAC, and DPI groups. Magnetic resonance imaging (MRI) was used to examine edema in the femoral head of rats. Histopathological staining was performed to assess osteonecrosis. Immunofluorescence staining with TUNEL and 8-OHdG was conducted to evaluate osteocyte apoptosis and oxidative damage. Immunohistochemical staining was carried out to detect the expression of NOX1, NOX2, and NOX4. Viability and apoptosis of MLO-Y4 cells were measured using the CCK-8 assay and TUNEL staining. 8-OHdG staining was conducted to detect oxidative stress. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure reactive oxygen species (ROS). The expression of NOX1, NOX2, and NOX4 in MLO-Y4 cells was analyzed by Western blotting. Multiple comparisons were performed using one-way analysis of variance (ANOVA).
In patients and the rat model, hematoxylin-eosin (HE) staining revealed a significantly higher rate of empty lacunae in the SINFH group than in the DDH group. Immunofluorescence staining indicated a significant increase in TUNEL-positive cells and 8-OHdG-positive cells in the SINFH group compared to the DDH group. Immunohistochemical staining demonstrated a significant increase in the expression of NOX1, NOX2, and NOX4 proteins in SINFH patients compared to DDH patients. Moreover, immunohistochemical staining showed a significant increase in the proportion of NOX2-positive cells compared to the Control group in the femoral head of rats. In vitro, Dex significantly inhibited the viability of osteocyte cells and induced apoptosis. After Dex treatment, the intracellular ROS level increased. However, Dex treatment did not alter the expression of NOX proteins in vitro. Additionally, NAC and DPI inhibited the generation of intracellular ROS and partially alleviated osteocyte apoptosis in vivo and in vitro.
This study demonstrates that GC promotes apoptosis of osteocyte cells through ROS-induced oxidative stress. Furthermore, we found that the increased expression of NOXs induced by GC serves as an important source of ROS generation.
糖皮质激素（GC）过度使用与类固醇诱导的股骨头坏死（SINFH）密切相关。然而，SINFH的潜在机制仍不清楚。本研究旨在探讨地塞米松（Dex）诱导的氧化应激对骨细胞凋亡的影响及其潜在机制。
本研究纳入10例SINFH患者和10例发育性髋关节发育不良（DDH）患者。60只大鼠随机分为对照组、Dex组、Dex+N-乙酰半胱氨酸（NAC）组、Dex+二苯碘鎓氯化物（DPI）组、NAC组和DPI组。采用磁共振成像（MRI）检查大鼠股骨头水肿情况。进行组织病理学染色以评估骨坏死。采用TUNEL和8-羟基脱氧鸟苷（8-OHdG）免疫荧光染色评估骨细胞凋亡和氧化损伤。进行免疫组织化学染色检测NOX1、NOX2和NOX4的表达。使用CCK-8法和TUNEL染色检测MLO-Y4细胞的活力和凋亡情况。进行8-OHdG染色检测氧化应激。采用2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）染色检测活性氧（ROS）。通过蛋白质印迹法分析MLO-Y4细胞中NOX1、NOX2和NOX4的表达。采用单因素方差分析（ANOVA）进行多重比较。
在患者和大鼠模型中，苏木精-伊红（HE）染色显示，SINFH组的空骨陷窝率显著高于DDH组。免疫荧光染色表明，与DDH组相比，SINFH组TUNEL阳性细胞和8-OHdG阳性细胞显著增加。免疫组织化学染色显示，与DDH患者相比，SINFH患者中NOX1、NOX2和NOX4蛋白的表达显著增加。此外，免疫组织化学染色显示，与对照组相比，大鼠股骨头中NOX2阳性细胞比例显著增加。在体外，Dex显著抑制骨细胞活力并诱导凋亡。Dex处理后，细胞内ROS水平升高。然而，Dex处理在体外未改变NOX蛋白的表达。此外，NAC和DPI在体内和体外均抑制细胞内ROS的产生并部分减轻骨细胞凋亡。
本研究表明，GC通过ROS诱导的氧化应激促进骨细胞凋亡。此外，我们发现GC诱导的NOXs表达增加是ROS产生的重要来源。
