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杨军, 彭力田, 毛滔, 彭文.基于JNK通路探讨补肾活血方对大鼠骨关节炎软骨细胞凋亡的影响[J].湖南中医药大学学报,2024,44(9):1575-1582[点击复制] |
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| 基于JNK通路探讨补肾活血方对大鼠骨关节炎软骨细胞凋亡的影响 |
| 杨军,彭力田,毛滔,彭文 |
| (湖南中医药大学第二附属医院, 湖南 长沙 410005) |
| 摘要: |
| 目的 探讨补肾活血方（Bushen Huoxue Formula, BSHXF）通过c-Jun氨基末端激酶（c-Jun N-terminal kinase, JNK）信号通路对大鼠骨关节炎（Osteoarthritis, OA）软骨细胞凋亡的影响。方法 采用白细胞介素-1β (interleukin-1β, IL-1β)诱导体外分离培养的大鼠软骨细胞构建体外OA模型，并使用不同浓度的BSHXF干预处理。使用MTT法测定细胞活力；CCK-8法检测细胞增殖能力；流式细胞术评估细胞凋亡情况；Western bolt检测凋亡相关蛋白裂解的胱天蛋白酶-3（Cleaved Caspase-3）、B淋巴细胞瘤-2（B-cell lymphoma 2, Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein, Bax）水平和JNK、磷酸化c-Jun 氨基末端激酶（phosphorylated c-Jun N-terminal kinase, p-JNK）蛋白水平。试剂盒检测细胞中活性氧（reactive oxygen species, ROS）、丙二醛（malondialdehyde, MDA）、谷胱甘肽（Glutathione, GSH）水平；ELISA法检测细胞上清液中促炎因子肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）、白细胞介素-6（interleukin-6, IL-6）含量。结果 （1）与对照组相比，模型组软骨细胞活力显著降低，细胞凋亡率显著升高（P＜0.01）；模型组Cleaved Caspase-3、Bax、p-JNK蛋白表达水平显著升高（P＜0.01），Bcl-2蛋白表达水平显著降低（P＜0.01）。与模型组相比，BSHXF治疗组软骨细胞活力升高（P＜0.05），凋亡率显著降低（P＜0.01）；BSHXF治疗组Cleaved Caspase-3、Bax、p-JNK蛋白表达水平显著降低（P＜0.01），Bcl-2蛋白表达水平升高（P＜0.01）。（2）与对照组相比，模型组细胞中ROS和MDA的相对含量显著升高（P＜0.01），GSH的相对含量显著降低（P＜0.01）；模型组细胞中TNF-α和IL-6的含量均显著升高（P＜0.01）。与模型组相比，BSHXF治疗组细胞中ROS和MDA的相对含量显著降低（P＜0.01），GSH的相对含量显著升高（P＜0.01）；BSHXF治疗组细胞中TNF-α和IL-6的含量均显著降低（P＜0.01）。（3）与溶剂对照组相比，激活剂对照组的p-JNK、Cleaved Caspase-3、Bax蛋白水平显著上调（P＜0.05），Bcl-2蛋白表达水平显著降低（P＜0.01），细胞增殖能力下降（P＜0.05），细胞凋亡率显著增加（P＜0.01）；BSHXF治疗组和溶剂对照组两组间细胞增殖、凋亡率和凋亡相关蛋白表达（Cleaved Caspase-3、Bax、Bcl-2）情况差异均无统计学意义（P＞0.05）。（4）与溶剂对照组相比，激活剂对照组的ROS和MDA水平显著升高（P＜0.01），而GSH水平显著降低（P＜0.01），TNF-α、IL-6含量升高（P＜0.05）；BSHXF治疗组和溶剂对照组两组间ROS相对含量、MDA、GSH水平差异均无统计学意义（P＞0.05）。结论 BSHXF通过抑制JNK信号通路激活抑制IL-1β诱导的OA软骨细胞凋亡，改善氧化应激和炎症反应。 |
| 关键词: 骨关节炎 补肾活血方 JNK信号通路 软骨细胞凋亡 氧化应激 炎症反应 |
| DOI：10.3969/j.issn.1674-070X.2024.09.004 |
| 投稿时间：2024-01-14 |
| 基金项目:湖南省中医药科研计划项目（E2022114）。 |
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| Effects of Bushen Huoxue Formula on chondrocyte apoptosis in rats with osteoarthritis based on JNK pathway |
| YANG Jun, PENG Litian, MAO Tao, PENG Wen |
| (The Second Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410005, China) |
| Abstract: |
| Objective To investigate the effects of Bushen Huoxue Formula (BSHXF) on chondrocyte apoptosis in rats with osteoarthritis (OA) through the c-Jun N-terminal kinase (JNK) signaling pathway. Methods An in vitro OA model was constructed using interleukin-1β (IL-1β) to induce apoptosis in rat chondrocytes cultured ex vivo. Different concentrations of BSHXF were used for intervention. Cell viability was determined using MTT assay; CCK-8 assay was used to examine cell proliferation ability; flow cytometry was employed to assess cell apoptosis; Western blot was used to check the levels of apoptosis-related proteins, including cleaved cysteine protease-3 (Cleaved Caspase-3), B-cell lymphoma 2 (Bcl-2), and Bcl-2 associated X protein (Bax), as well as the levels of JNK and phosphorylated c-Jun N-terminal kinase (p-JNK) proteins. The reagent kit was used to measure the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) in cells; ELISA was used to check the content of pro-inflammatory factor of tumor necrosis factor-α (TNF-α) and IL-6 in the cell supernatant. Results (1) Compared with the control group, the model group showed a significant decrease in chondrocyte viability and a significant increase in cell apoptosis rate (P＜0.01); the protein expression levels of Cleaved Caspase-3, Bax, and p-JNK in the model group significantly increased (P＜0.01), while the expression of Bcl-2 significantly decreased (P＜0.01). Compared with the model group, the BSHXF treatment group showed an increase in chondrocyte viability (P＜0.05) and a significant decrease in apoptosis rate (P＜0.01); the protein expression levels of Cleaved Caspase-3, Bax, and p-JNK were significantly reduced (P＜0.01) and the protein expression level of Bcl-2 increased (P＜0.01) in the BSHXF treatment group. (2) Compared with the control group, the relative content of ROS and MDA in the model group cells significantly increased (P＜0.01), while the relative content of GSH significantly decreased (P＜0.01); the content of TNF-α and IL-6 in the model group cells significantly increased (P＜0.01). Compared with the model group, the BSHXF treatment group showed a significant decrease in the relative content of ROS and MDA (P＜0.01) and a significant increase in the relative content of GSH (P＜0.01); the content of TNF-α and IL-6 in cells treated with BSHXF was significantly reduced (P＜0.01). (3) Compared with the solvent control group, the activator control group showed a significant upregulation of p-JNK, Cleaved Caspase-3, and Bax protein levels (P＜0.05), a significant reduction in Bcl-2 protein expression (P＜0.01), a decrease in cell proliferation ability (P＜0.05), and a significant increase in cell apoptosis rate (P＜0.01); there were no significant differences in cell proliferation, apoptosis rate, and expressions of apoptosis-related proteins (Cleaved Caspase-3, Bax, and Bcl-2) between the BSHXF treatment group and the solvent control group (P＞0.05). (4) Compared with the solvent control group, the activator control group exhibited a significant increase in ROS and MDA levels (P＜0.01) and a significant decrease in GSH level (P＜0.01), with increased content of TNF-α and IL-6 (P＜0.05); there were no significant differences in the relative content of ROS and the levels of MDA and GSH between the BSHXF treatment group and the solvent control group (P＞0.05). Conclusion BSHXF inhibits IL-1β-induced apoptosis in OA chondrocytes by suppressing the activation of JNK signaling pathway, thereby relieving oxidative stress and inflammatory responses. |
| Key words: osteoarthritis Bushen Huoxue Formula JNK signaling pathway chondrocyte apoptosis oxidative stress inflammatory response |
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