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中华老年骨科与康复电子杂志

中华老年骨科与康复电子杂志 ›› 2026, Vol. 12 ›› Issue (02) : 85 -92. doi: 10.3877/cma.j.issn.2096-0263.2026.02.004

基础研究

槲皮素对H2O2诱导骨关节炎软骨细胞氧化损伤的保护作用与机制研究
李军1,2, 沈姝霖1,2, 张荣炜1,2, 黄章睿1,2, 梁锐明1,2,3, 罗世兴1,4,†()   
  1. 1530021　南宁，广西医科大学再生医学与医用生物资源开发应用省部共建协同创新中心
    2530021　南宁，广西组织器官修复医用生物材料工程技术研究中心
    3530021　南宁，广西医科大学生命科学研究院
    4536000　北海，广西医科大学第九附院医院骨科
  • 收稿日期:2026-03-23 出版日期:2026-04-05
  • 通信作者: 罗世兴
  • 基金资助:
    国家自然科学基金(82360425); 广西科技创新平台计划(桂科LT2600640002)

Protective Effect of Quercetin against H2O2-Induced Oxidative Damage in Osteoarthritic Chondrocytes

Jun Li1,2, Shulin Shen1,2, Rongwei Zhang1,2, Zhangrui Huang1,2, Ruiming Liang1,2,3, Shixing Luo1,4,†()   

  1. 1Collaborative Innovation Centre of Regenerative Medicine and Medical Bio-Resource Development and Application Co-constructed by the Province and Ministry, Guangxi Medical University, Nanning 530021, China
    2Guangxi Engineering Research Centre for Medical Biomaterials of Tissue and Organ Repair, Nanning 530021, China
    3Life Sciences Institute, Guangxi Medical University, Nanning, 530021, China
    4Department of Orthopedics, The Ninth Affiliated Hospital of Guangxi Medical University, Beihai 536000, China
  • Received:2026-03-23 Published:2026-04-05
  • Corresponding author: Shixing Luo
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引用本文：

李军, 沈姝霖, 张荣炜, 黄章睿, 梁锐明, 罗世兴. 槲皮素对H2O2诱导骨关节炎软骨细胞氧化损伤的保护作用与机制研究[J/OL]. 中华老年骨科与康复电子杂志, 2026, 12(02): 85-92.

Jun Li, Shulin Shen, Rongwei Zhang, Zhangrui Huang, Ruiming Liang, Shixing Luo. Protective Effect of Quercetin against H2O2-Induced Oxidative Damage in Osteoarthritic Chondrocytes[J/OL]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2026, 12(02): 85-92.

目的

研究槲皮素（quercetin, Que）对过氧化氢（H2O2）诱导的骨关节炎（osteoarthritis, OA）软骨细胞氧化损伤的保护作用及相关分子机制。

方法

体外分离培养乳鼠关节软骨细胞，随机分为正常对照组、模型组（H2O2诱导）及处理组（Que干预组）。采用CCK-8法筛选Que的安全浓度；利用Calcein-AM染色检测细胞活力；以DCFH-DA、DHE、DAF-FM DA及JC-1荧光探针分别检测细胞内ROS、超氧阴离子、NO水平及线粒体膜电位变化；采用免疫荧光和RT-qPCR检测炎症因子及基质降解酶的表达。

结果

10~40 μg/mL Que对软骨细胞无明显毒性。与模型组相比，40 μg/mL Que显著提高了细胞活力（P<0.05），降低胞内ROS、超氧阴离子及NO水平，并改善H2O2导致的线粒体膜电位下降（P<0.05）。同时，Que可显著下调MMP-13、IL-6蛋白表达，以及MMP-3、MMP-13、IL-1β、IL-6 mRNA的表达（P<0.05）。

结论

槲皮素可通过清除活性氧、抑制NO生成、维持线粒体功能，减轻H2O2诱导的软骨细胞氧化应激与炎症反应，对OA软骨细胞具有保护作用，有望成为骨关节炎治疗的天然候选药物。

关键词: 槲皮素, 骨关节炎, 氧化应激, 线粒体, 软骨细胞
Objective

To investigate the protective effects and related molecular mechanisms of quercetin (Que) against oxidative stress, inflammatory response, and mitochondrial dysfunction induced by hydrogen peroxide H2O2 osteoarthritis (OA) chondrocytes.

Methods

Articular chondrocytes were isolated from neonatal rats and randomly divided into a normal control group, a model group (H2O2 induced), and a treatment group (Que intervention). The safe concentration of Que was determined by CCK-8 assay. Cell viability was evaluated using Calcein-AM staining. The levels of intracellular ROS, superoxide anion, NO, and mitochondrial membrane potential were detected by DCFH-DA, DHE, DAF-FM DA, and JC-1 fluorescent probes, respectively. The expression of inflammatory factors and matrix-degrading enzymes was measured by immunofluorescence and RT-qPCR.

Results

Que at 10-40 μg/mL showed no obvious cytotoxicity to chondrocytes. Compared with the model group, 40 μg/mL Que significantly increased cell viability (P<0.05), reduced intracellular levels of ROS, superoxide anion, and NO, and ameliorated the H2O2 induced decrease in mitochondrial membrane potential (P<0.05). Meanwhile, Que significantly downregulated the protein expression of MMP-13 and IL-6 as well as the mRNA expression of MMP-3, MMP-13, IL-1β, and IL-6 (P<0.05).

Conclusion

Quercetin attenuates H2O2 induced oxidative stress and inflammation in chondrocytes by scavenging reactive oxygen species, inhibiting NO production, and maintaining mitochondrial function. It exerts a protective effect on OA chondrocytes and is expected to be a natural candidate for the treatment of osteoarthritis.

Key words: Quercetin, Osteoarthritis, Oxidative Stress, Mitochondria, Chondrocytes
表1 PCR引物序列
基因 上游（5’~3’） 下游（5’~3’）
GAPDH CACGACATACTCAGCACCAG TCCAGTATGACTCTACCCACG
IL-6 TCTGCGCAGCTTTAAGGAGT CCCAGTGGACAGGTTTCTGA
IL-1β GCACAGTTCCCCAACTGGTA GGAGACTGCCCATTCTCGAC
MMP-3 GGCTGTGTGCTCATCCTACC TGGAAAGGTACTGAAGCCACC
MMP-13 ACCATCCTGTGACTCTTGCG TTCACCCACATCAGGCACTC
图1 CCK8检测Que对软骨细胞的毒性作用注：软骨细胞经0-100 μg/mL Que处理24 h后，采用CCK-8试剂盒测定细胞相对增殖率（以0 μg/mL组为100%）。数据表示为均值±标准差（n=3）均值±SD（n=3）。*P<0.05，**P<0.01，***P<0.001，****P<0.0001
图2 Calcein-AM/PI染色结果注：（A）活/死染色荧光图（绿=活细胞）。比例尺=50 μm。（B）基于（A）图ImageJ计数的相对增殖率（%），均值±SD（n=3）。*P<0.05，**P<0.01，***P<0.001，****P<0.0001
图3 氧化应激及线粒体功能染色结果注：A，C，E：分别采用DCFH-DA、DHE和DAF-FM DA荧光探针染色观察各组细胞内总活性氧（ROS）、超氧阴离子（O2-）和一氧化氮（NO）水平（标尺= 50 μm）；B，D，F：分别为A、C、E对应指标的平均荧光强度定量分析结果。G：JC-1染色观察线粒体膜电位变化，红色荧光代表高膜电位状态（J-聚集体），绿色荧光代表低膜电位状态（单体）（标尺= 50 μm）；H，I：分别为JC-1聚集体（红色）与单体（绿色）的平均荧光强度定量分析。*P<0.05，**P<0.01，***P<0.001，****P<0.0001
图4 IL-6及MMP13免疫荧光染色结果注：A，C：代表性免疫荧光图像（DAPI染核呈蓝色，IL-6呈红色，MMP-13呈绿色；标尺=50 μm）；B，D：对应蛋白的平均荧光强度定量分析。数据以均值±SD表示（n=3），*P<0.05，**P<0.01，***P<0.001，****P<0.0001
图5 炎症与基质降解相关基因mRNA表达水平检测注：A-D）qRT-PCR检测IL-6、IL-1β、MMP-3和MMP-13的相对mRNA表达量（以正常组为基准归一化）。数据以均值±SD表示（n=3）。*P<0.05，**P<0.01，***P<0.001，****P<0.0001；ns=无显著差异
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