甲基莲心碱通过KEAP1/NRF2/GPX4和NF-κB信号通路减轻椎间盘退行性变

doi:10.3969/j.issn.1674-8115.2025.03.002

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (3): 261-270.doi: 10.3969/j.issn.1674-8115.2025.03.002

甲基莲心碱通过KEAP1/NRF2/GPX4和NF-κB信号通路减轻椎间盘退行性变

万宏劲(), 胡逸斌, 王昕, 张凯, 秦安, 马培翔, 马辉(), 赵杰()

上海交通大学医学院附属第九人民医院骨科，上海市骨科内植物重点实验室，上海 200011

收稿日期:2024-09-09 接受日期:2025-01-10 出版日期:2025-03-28 发布日期:2025-03-28
通讯作者: 赵　杰，主任医师，博士；电子信箱：profzhaojie@126.com
马　辉，主任医师，博士；电子信箱：sh9_spine@163.com。
作者简介:万宏劲（1999—），男，博士生；电子信箱：whjswts5882@sjtu.edu.cn。
基金资助:
国家自然科学基金(82130073);上海市自然科学基金(23ZR1447400);上海交通大学医学院附属第九人民医院种子基金(JYZZ215)

Neferine alleviates intervertebral disc degeneration through KEAP1/NRF2/GPX4 and NF-κB signaling pathways

WAN Hongjin(), HU Yibin, WANG Xin, ZHANG Kai, QIN An, MA Peixiang, MA Hui(), ZHAO Jie()

Department of Orthopedics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Orthopedic Implants, Shanghai 200011, China

Received:2024-09-09 Accepted:2025-01-10 Online:2025-03-28 Published:2025-03-28
Contact: ZHAO Jie, E-mail: profzhaojie@126.com
MA Hui, E-mail: sh9_spine@163.com.
Supported by:
National Natural Science Foundation of China(82130073);Natural Science Foundation of Science and Technology Committee of Shanghai(23ZR1447400);Fundamental Research Program Funding of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYZZ215)

摘要/Abstract

摘要：

目的·研究甲基莲心碱（neferine，Nef）在椎间盘退行性变（intervertebral disc degeneration，IDD）中的治疗作用以及潜在的调控通路。方法·通过CCK-8（cell counting kit-8）实验检测Nef对髓核细胞活力及细胞增殖的影响。通过分子对接软件分析Nef与Kelch样ECH相关蛋白1（kelch-like ECH-associated protein 1，KEAP1）Kelch结构域的潜在结合位点。用肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）诱导髓核细胞铁死亡和炎症反应。应用Western blotting检测在TNF-α刺激下，加入或不加Nef时核因子红系2相关因子2/谷胱甘肽过氧化物酶4（nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4，NRF2/GPX4）信号通路和核因子κB（nuclear factor-κB，NF-κB）通路相关蛋白的表达水平。通过髓核细胞高密度培养检测Nef对髓核细胞的细胞外基质代谢的影响。构建大鼠尾椎针刺退变模型，尾椎Co3/4椎间盘（IDD+Nef组）内注射2次1.5 μmol/L Nef 5 μL，Co2/3椎间盘（IDD组）注射等体积PBS；4周后通过X线检测椎间隙高度，通过磁共振成像检测椎间盘退变情况，通过组织学染色方法检测椎间盘结构。结果·CCK-8实验结果显示Nef在1.5 μmol/L浓度及以下不会抑制髓核细胞活力和增殖。分子对接结果表明，Nef可能通过直接结合KEAP1蛋白Kelch结构域，降低KEAP1与NRF2之间相互作用从而激活NRF2。Western blotting结果显示，Nef提高了铁死亡关键抑制蛋白NRF2和GPX4的表达量，同时抑制了NF-κB通路磷酸化P65蛋白的表达量（均P<0.05）。髓核细胞高密度培养结果显示，Nef减轻了TNF-α诱导的髓核细胞细胞外基质降解（P<0.05）。动物实验结果显示，与IDD组相比，IDD+Nef组椎间隙高度较高，Pfirrmann分级较低（均P<0.05），组织学退变程度较低。结论·Nef可能通过激活KEAP1/NRF2/GPX4通路抑制TNF-α诱导的髓核细胞铁死亡，通过抑制NF-κB信号通路减轻TNF-α诱导的髓核细胞炎症和细胞外基质降解，从而减轻大鼠IDD。

关键词: 甲基莲心碱, 椎间盘退行性变, KEAP1/NRF2信号通路, 铁死亡, NF-κB信号通路, 炎症

Abstract:

Objective ·To investigate the therapeutic effects of neferine (Nef) on intervertebral disc degeneration (IDD) and the underlying regulatory pathways. Methods ·The effects of Nef on the viability and proliferation of nucleus pulposus cells were assessed using the cell counting kit-8 (CCK-8) assay. Molecular docking software was employed to analyze the potential binding sites of Nef within the Kelch domain of kelch-like ECH-associated protein 1 (KEAP1). Tumor necrosis factor-α (TNF-α) was used to induce ferroptosis and inflammation in nucleus pulposus cells. Western blotting was performed to detect the expression levels of nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4 (NRF2/GPX4) pathway- and nuclear factor-κB (NF-κB) pathway-related proteins under TNF-α stimulation with or without Nef. The effect of Nef on the metabolism of extracellular matrix in nucleus pulposus cells was evaluated using high-density cell culture. A needle puncture-induced IDD rat model was established, and 5 µL of 1.5 μmol/L Nef was injected twice into the intervertebral disc at the Co3/4 level (IDD+Nef group), while an equivalent volume of PBS was injected into the Co2/3 disc (IDD group). After 4 weeks, the intervertebral space height was detected by X-ray, disc degeneration was detected by magnetic resonance imaging, and disc structure was evaluated by histological staining. Results ·The CCK-8 assay revealed that Nef at concentrations of 1.5 μmol/L and below did not inhibit the viability and proliferation of nucleus pulposus cells. Molecular docking results suggested that Nef might activate NRF2 by directly binding to the KEAP1 Kelch domain, thereby reducing the interaction between KEAP1 and NRF2. Western blotting indicated that Nef significantly increased the expression of the key ferroptosis-inhibiting proteins NRF2 and GPX4, while decreasing the expression of the phospho-P65 protein in the NF-κB pathway (all P<0.05). The high-density culture of nucleus pulposus cells demonstrated that Nef mitigated the TNF-α- induced degradation of the extracellular matrix (P<0.05). Animal study results showed that compared to the IDD group, the IDD+Nef group exhibited a greater intervertebral disc space height, a lower Pfirrmann grade (both P<0.05), and a reduced degree of histological degeneration. Conclusion ·Nef may inhibit TNF-α-induced ferroptosis in nucleus pulposus cells by activating the KEAP1/NRF2/GPX4 pathway and reduce TNF-α-induced inflammation and extracellular matrix degradation by suppressing the NF-κB pathway, thereby alleviating IDD in rats.

Key words: neferine, intervertebral disc degeneration, KEAP1/NRF2 signaling pathway, ferroptosis, NF-κB signaling pathway, inflammation

中图分类号:

R681.5

万宏劲, 胡逸斌, 王昕, 张凯, 秦安, 马培翔, 马辉, 赵杰. 甲基莲心碱通过KEAP1/NRF2/GPX4和NF-κB信号通路减轻椎间盘退行性变[J]. 上海交通大学学报（医学版）, 2025, 45(3): 261-270.

WAN Hongjin, HU Yibin, WANG Xin, ZHANG Kai, QIN An, MA Peixiang, MA Hui, ZHAO Jie. Neferine alleviates intervertebral disc degeneration through KEAP1/NRF2/GPX4 and NF-κB signaling pathways[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(3): 261-270.

图/表 5

图1 Nef对髓核细胞活力和增殖的影响Note： A. The effect of Nef on cell viability measured by CCK-8 test. B. The effect of Nef on cell proliferation measured by CCK-8 test. ①P=0.025, ②P=0.006, ③P<0.001, compared with the corresponding 0 μmol·L－1 group.

Fig 1 Effects of Nef on the viability and proliferation of nucleus pulposus cells

图2 Nef与KEAP1分子对接分析以及对TNF-α诱导的髓核细胞铁死亡的抑制作用Note： A. Molecular docking analysis showed the binding of Nef to the Kelch domain of KEAP1. B. Western blotting bands of KEAP1 and NRF2 proteins. C. Western blotting bands of GPX4, xCT, and ACSL4 proteins. D. Statistical analysis of relative expression of the above proteins. ①P<0.001, ②P=0.001, compared with the nucleus pulposus cells treated only with TNF-α.

Fig 2 Molecular docking analysis of Nef with KEAP1 and its inhibitory effect on TNF-α-induced ferroptosis in nucleus pulposus cells

图3 Nef对NF-κB信号通路及TNF-α诱导的髓核细胞细胞外基质降解的抑制作用Note： A. Western blotting bands of IκBα, P65 and p-P65 proteins. B. Western blotting bands of MMP3, MMP9, MMP13, and SOX9 proteins. C. Statistical analysis of relative expression of the above proteins. D. The representative Alcian blue staining images and quantification of extracellular matrix in the high-density culture of nucleus pulposus cells. ①P<0.001, ②P=0.001, ③P=0.032, ④P=0.048, compared with the nucleus pulposus cells treated only with TNF-α. IOD—integrated optical density.

Fig 3 Inhibitory effect of Nef on the NF-κB signaling pathway and TNF-α-induced extracellular matrix degradation in nucleus pulposus cells

图4 Nef对大鼠IDD模型的体内治疗作用Note： A. The representative MRI image of a rat IDD model. B. The Pfirrmann grade (n=5). C. The representative X-ray image of a rat IDD model. D. The height of intervertebral space (n=5). E. The representative images of H-E and safranin O-fast green (SO-FG) staining of the intervertebral discs. Scale bar=500 μm. ①P<0.001, ②P=0.002, compared with the IDD group.

Fig 4 In vivo therapeutic effect of Nef on IDD in rat models

图5 Nef治疗IDD的潜在分子机制示意图Note： ROS—reactive oxygen species; GSSG—glutathione disulfide; ECM—extracellular matrix; GSH—glutathione; system Xc－—antiporter of cystine and glutamate.

Fig 5 Potential molecular mechanism involved in the therapeutic effects of Nef in IDD

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甲基莲心碱通过KEAP1/NRF2/GPX4和NF-κB信号通路减轻椎间盘退行性变

Neferine alleviates intervertebral disc degeneration through KEAP1/NRF2/GPX4 and NF-κB signaling pathways

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