目的 探究牛膝多糖（Achyranthis Bidentatae Radix polysaccharides，ABPS）对髓核细胞退变的保护作用及其机制。方法 分离大鼠椎间盘髓核细胞，采用白细胞介素-1β（interlenkin1β，1L-1β）处理构建髓核细胞退变模型，将细胞分为对照组、IL-1β组、ABPS（100 mg/L）＋IL-1β组、ABPS（200 mg/L）＋IL-1β组，研究ABPS对1L-1β处理大鼠髓核细胞增殖、氧化应激、NOD样受体蛋白3（NOD-Like Receptor Protein 3，NLRP3）和线粒体自噬的影响。采用慢病毒技术转染帕金森疾病蛋白2（Parkinson disease protein 2，Parkin）shRNA，随后采用IL-1β和ABPS处理，研究线粒体自噬在ABPS抑制1L-1β处理髓核细胞氧化应激和NLRP3炎性小体活化中的作用。将细胞分为对照组、IL-1β组、ABPS＋IL-1β组、ABPS＋IL-1β＋沉默信息调节因子3（silent information regulators 3，SIRT3）抑制剂（3-TYP）组，研究SIRT3在ABPS调控细胞线粒体自噬中的作用。免疫荧光法检测细胞增殖核抗原（proliferation related Ki 67 antigen，Ki67）和半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）以及微管相关蛋白1轻链3（microtubule-associated protein 1 light 3，LC3）与线粒体的共定位情况；流式细胞术检测细胞周期分布；Western blotting检测II型胶原（type II Collagen）、蛋白聚糖（aggrecan）、活化型Caspase-3（cleaved Casase-3）、NLRP3、Caspase-1、Parkin、LC3、选择性自噬接头蛋白-1（sequestosome-1，SQSTM1/p62）、研究SIRT3、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）以及Bcl-2关联X蛋白（Bcl-2 associated X protein，Bax）蛋白表达；原位末端标记法（TdT-mediated dUTP nick end labeling，TUNEL）检测细胞凋亡；荧光探针检测活性氧（reactive oxygen species，ROS）、线粒体膜电位（mitochondrial membrane potential，MMP）和线粒体活性氧（mitochondrial ROS，mtROS）；比色法检测超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽（glutathione，GSH）和丙二醛（malondialdehyde，MDA）水平。结果 与对照组比较，IL-1β组髓核细胞存活率、type II Collagen和aggrecan水平以及Ki67荧光强度均显著降低（P＜0.05），而G₀/G₁期细胞分布比例则明显升高（P＜0.05）。与IL-1β组比较，ABPS处理组细胞存活率、type II collagen和aggrecan蛋白水平以及Ki67荧光强度升高（P＜0.05），G₀/G₁期细胞分布比例明显降低（P＜0.05）。此外，与对照组比较，IL-1β组髓核细胞中SOD和GSH水平降低（P＜0.05），MDA、ROS、TUNEL阳性细胞、NLRP3、Caspase-3、cleaved Caspase-3和Caspase-1蛋白水平明显升高（P＜0.05）。ABPS处理逆转了IL-1β处理对髓核细胞氧化应激、凋亡和NLRP3炎性小体活化的影响。此外，ABPS处理减弱了IL-1β处理诱导的髓核细胞MMP降低和mtROS升高（P＜0.05）。ABPS增强了IL-1β处理髓核细胞中Parkin和LC3的表达（P＜0.05），SQSTM1/p62蛋白表达进一步降低（P＜0.05）。Parkin shRNA阻断了ABPS对IL-1β处理髓核细胞线粒体自噬、氧化应激、凋亡和NLRP3炎性小体的影响（P＜0.05）。此外，IL-1β处理组髓核细胞中的SIRT3蛋白水平降低（P＜0.05），ABPS处理后细胞中的SIRT3蛋白水平升高（P＜0.05）。3-TYP阻断了ABPS对IL-1β处理髓核细胞中Parkin介导线粒体自噬的调节作用（P＜0.05）。结论 ABPS可通过调控SIRT3/Parkin信号通路介导的线粒体自噬，减轻IL-1β诱导的髓核细胞氧化应激和NLRP3炎性小体活化。

Objective The aim of this study is to investigate the protective effect of Achyranthes Bidentata polysaccharides (ABPS) against degeneration of nucleus pulposus cells (NCPs) and its possible mechanisms. Methods Rat intervertebral disc NCPs were isolated and treated with interleukin-1β (1L-1β) to construct a model of NCP degeneration. The cells were divided into control group, IL-1β group, ABPS (100 mg/L) + IL-1β group, and ABPS (200 mg/L) + IL-1β group to investigate the effects of ABPS on proliferation, oxidative stress, NOD-like receptor protein 3 (NLRP3) and mitophagy in 1L-1β-treated rat NCPs,. To investigate the role of mitophagy in ABPS-mediated inhibition of IL-1β-induced oxidative stress and NLRP3 inflammasome activation in NCPs, lentiviral vector-mediated transfection of Parkinson disease protein 2 (Parkin) short hairpin RNA (shRNA) was performed, followed by treatment with IL-1β and ABPS. To examine the role of silent information regulators 3 (SIRT3) in ABPS regulation of mitophagy, cells were divided into four groups: control, IL-1β, ABPS + IL-1β, and ABPS + IL-1β + 3-TYP. Immunofluorescence was employed to examine the co-localization of cell proliferation-related Ki 67 antigen (Ki67), cysteine-aspartate protease-3 (Caspase-3), and microtubule-associated protein 1 light 3 (LC3) with mitochondria. Flow cytometry was performed for cell cycle analysis. Western blotting was used to detect protein expression levels of type II Collagen, aggrecan, cleaved Caspase-3, NLRP3, Caspase-1, Parkin, LC3, sequestosome-1 (SQSTM1/p62), SIRT3, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein (Bax). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was employed to detect cell apoptosis. Fluorescent probes were used to assess reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial ROS (mtROS). Colorimetric assays were performed to measure the levels of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA). Results Compared with the control group, the viability of NCPs, type II Collagen and aggrecan levels, and proliferation related Ki67 fluorescence intensity were significantly decreased in the IL-1β group (P < 0.05), while the distribution ratio of cells in G₀/G₁ phase was significantly increased (P < 0.05). Compared with the 1L-1β group, cell viability, type II Collagen and aggrecan protein levels, and Ki67 fluorescence intensity were increased in the ABPS-treated group (P < 0.05), while the distribution ratio of cells in G₀/G₁ phase was significantly decreased (P < 0.05). Compared with the control group, the levels of SOD and GSH in NCPs were decreased in the IL-1β group (P < 0.05), and the levels of MDA, ROS, TUNEL-positive cells, NLRP3, Caspase3, cleaved Caspase-3, and Caspase1 were significantly increased (P < 0.05). ABPS treatment reversed the effects of IL-1β treatment on oxidative stress, apoptosis and NLRP3 inflammasome activation in NCPs. In addition, ABPS treatment attenuated the IL-1β-induced reduction of MMP and increase of mtROS in NCPs (P < 0.05). ABPS enhanced the expression of Parkin and LC3 in IL-1β-treated NCPs (P < 0.05), and SQSTM1/p62 protein expression was further reduced (P < 0.05). Parkin shRNA blocked the effects of ABPS on mitophagy, oxidative stress, apoptosis, and NCPs3 inflammasome in IL-1β-treated NCPs (P < 0.05). The level of SIRT3 protein in NCPs from IL-1β-treated group was decreased (P < 0.05) and increased (P < 0.05) in ABPS-treated cells. 3-TYP blocked the regulation of ABPS on Parkin-mediated mitophagy in IL-1β-treated NCPs (P < 0.05). Conclusion ABPS attenuates IL-1β-induced NCPs oxidative stress and NLRP3 inflammasome activation by modulating SIRT3/Parkin signaling pathway-mediated mitophagy.

R285.5

2023年体育局课题研究项目（202320）;全国中医临床特色技术传承人才项目[国中医药人教教育便函(2019)96号];2024年河南省中医药课题研究专项课题（2024ZY2066）