淫羊藿苷调控Nrf2/HO-1信号通路对软骨细胞铁死亡的影响*

作者：张 阔1，张镭潇1，郑泽炜1，徐金凡1，张庆文2

单位：1.广州中医药大学第三临床医学院，广东 广州 510006； 2.广州中医药大学第三附属医院，广东 广州 510405

引用：引用：张阔，张镭潇，郑泽炜，徐金凡，张庆文.淫羊藿苷调控Nrf2/HO-1信号通路对软骨细胞铁死亡的影响[J].中医药导报,2025,31(9):17-23.

DOI：10.13862/j.cn43-1446/r.2025.09.003

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摘要：

目的：探讨淫羊藿苷对Atdc5软骨细胞铁死亡状态的影响及作用机制。方法：体外培养软骨细胞，运用CCK-8法检测0.0、1.0、2.0、4.0、8.0、16.0、32.0、64.0 μmol/L淫羊藿苷对Atdc5软骨细胞增殖的影响，以确定最佳干预浓度。将Atdc5软骨细胞分为空白对照组、模型组、淫羊藿苷低浓度组和淫羊藿苷高浓度组，空白对照组软骨细胞未进行药物干预，模型组软骨细胞采用白细胞介素-1β（IL-1β）（10 ng/mL）干预，淫羊藿苷低浓度组软骨细胞采用IL-1β（10 ng/mL）及低浓度淫羊藿苷（8.0 μmol/L）干预，淫羊藿苷高浓度组软骨细胞采用IL-1β（10 ng/mL）及高浓度淫羊藿苷（16.0 μmol/L）干预，各组均干预24 h。采用荧光显微镜检测细胞荧光强度，分析细胞活性氧（ROS）含量，采用试剂盒检测细胞丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）含量。采用定量逆转录聚合酶链反应（qRT-PCR）检查细胞Ⅱ型胶原α1链（COL2A1） mRNA、聚集蛋白聚糖（ACAN） mRNA、谷胱甘肽过氧化物酶4（GPX4） mRNA、溶质载体家族7成员11抗体（SLC7A11） mRNA、核因子E2相关因子2（Nrf2） mRNA、血红素氧合酶1（HO-1） mRNA表达水平，采用蛋白质印迹法（Western blotting）检测细胞Nrf2、HO-1、GPX4、SLC7A11蛋白表达水平。结果：8.0 μmol/L和16.0 μmol/L的淫羊藿苷对Atdc5软骨细胞无细胞毒性，且能显著促进其增殖（P＜0.01）。8.0 μmol/L和16.0 μmol/L的淫羊藿苷在促进细胞增殖方面效果相近（P＞0.05）。模型组细胞ROS、MDA含量高于空白对照组（P＜0.01），GSH-Px含量低于空白对照组P＜0.01）；淫羊藿苷低、高浓度组细胞ROS、MDA含量低于模型组（P＜0.01），GSH-Px含量高于模型组（P＜0.01）。模型组细胞COL2A1 mRNA、ACAN mRNA、GPX4 mRNA、SLC7A11 mRNA、Nrf2 mRNA、HO-1 mRNA相对表达量低于空白对照组（P＜0.01）。淫羊藿苷低、高浓度组细胞COL2A1 mRNA、ACAN mRNA、GPX4 mRNA、SLC7A11 mRNA、Nrf2 mRNA、HO-1 mRNA相对表达量高于模型组（P＜0.01）；淫羊藿苷高浓度组细胞COL2A1 mRNA、ACAN mRNA相对表达量高于淫羊藿苷低浓度组（P＜0.05）。模型组细胞Nrf2、HO-1、GPX4、SLC7A11蛋白相对表达量低于空白对照组（P＜0.01）；淫羊藿苷低、高浓度组细胞Nrf2、HO-1、GPX4、SLC7A11蛋白相对表达量高于模型组（P＜0.01）；淫羊藿苷高浓度组细胞Nrf2、HO-1蛋白相对表达量高于淫羊藿苷低浓度组（P＜0.01）。结论：淫羊藿苷可能通过激活Nrf2/HO-1信号通路，抑制软骨细胞铁死亡，改善软骨细胞功能状态。

关键词：膝骨关节炎；淫羊藿苷；软骨细胞；铁死亡；Nrf2/HO-1信号通路

Abstract：

Objective: To explore the effect of icariin on the ferroptosis status of Atdc5 chondrocytes and its underlying mechanisms. Methods: In vitro culture of chondrocytes was performed to assess the effect of icariin at concentrations of 0.0, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0 μmol/L on the proliferation of Atdc5 chondrocytes using the CCK-8 method, in order to determine the optimal intervention concentration. The Atdc5 chondrocytes were divided into blank control group, model group, low concentration icariin group, and high concentration icariin group. The blank control group did not receive any drug intervention, while the model group was treated with IL-1β (10 ng/mL). The low concentration icariin group was treated with IL-1β (10 ng/mL) and icariin (8.0 μmol/L), and the high concentration icariin group received IL-1β (10 ng/mL) and icariin (16.0 μmol/L). All groups were treated for 24 hours. Fluorescence microscopy was used to detect the fluorescence intensity of the cells, and analyze the content of reactive oxygen species (ROS). A kit was utilized to measure the levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to investigate the expression levels of type Ⅱ collagen α1 chain (COL2A1) mRNA, aggrecan (ACAN) mRNA, glutathione peroxidase 4 (GPX4) mRNA, solute carrier family 7 member 11 (SLC7A11) mRNA, nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA, and heme oxygenase 1 (HO-1) mRNA. Additionally, protein expression levels of Nrf2, HO-1, GPX4, and SLC7A11 in the cells were assessed using Western blotting. Results: The concentrations of 8.0 μmol/L and 16.0 μmol/L of icariin demonstrated no cytotoxicity to Atdc5 chondrocytes and significantly promoted their proliferation (P<0.01), with similar effects on cell proliferation between the 8.0 μmol/L and 16.0 μmol/L groups (P>0.05). The model group showed higher levels of ROS and MDA than blank control group (P<0.01), while lower levels of GSH-Px  than blank control group (P<0.01). The low and high concentration icariin groups showed lower levels of ROS and MDA than model group (P<0.01), while higher levels of GSH-Px than model group (P<0.01). The model group showed lower relative expression levels of COL2A1 mRNA, ACAN mRNA, GPX4 mRNA, SLC7A11 mRNA, Nrf2 mRNA, and HO-1 mRNA than blank control group (P<0.01). In contrast, the low and high concentration icariin groups showed higher relative expression levels of COL2A1 mRNA, ACAN mRNA, GPX4 mRNA, SLC7A11 mRNA, Nrf2 mRNA, and HO-1 mRNA than model group (P<0.01). Additionally, the high concentration icariin group showed higher relative expression levels of COL2A1 mRNA and ACAN mRNA  than low concentration icariin group (P<0.05). The model group showed lower relative expression levels of Nrf2, HO-1, GPX4, and SLC7A11 proteins than blank control group (P<0.01). The low and high concentration icariin groups showed higher relative expression levels of Nrf2, HO-1, GPX4, and SLC7A11 proteins than model group (P<0.01). The high concentration icariin group showed higher relative expression levels of Nrf2 and HO-1 protein than low concentration icariin group (P<0.01). Conclusion: Icariin may inhibit ferroptosis in chondrocytes and improve their functional status by activating the Nrf2/HO-1 antioxidant signaling pathway.

Key words：knee osteoarthritis; Icariin; chondrocyte; ferroptosis; Nrf2/HO-1 signaling pathway

发布时间：2026-01-08

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