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

作　　者：万宏劲 胡逸斌 王昕[1] 张凯[1] 秦安[1] 马培翔 马辉 赵杰[1] 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)

机构地区：[1]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

出　　处：《上海交通大学学报(医学版)》2025年第3期261-270,共10页Journal of Shanghai Jiao tong University:Medical Science

基　　金：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)。

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1.5μmol/L Nef 5μL,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1.5μmol/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。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)pathwayrelated proteins under TNF-αstimulation 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 5µL of 1.5μmol/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 1.5μmol/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 extracellul

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 

分 类 号：R681.5[医药卫生—骨科学]