目的 探究牛膝多糖（Achyranthis Bidentatae Radix polysaccharides，ABPS）對(duì)髓核細(xì)胞退變的保護(hù)作用及其機(jī)制。方法 分離大鼠椎間盤(pán)髓核細(xì)胞，采用白細(xì)胞介素-1β（interlenkin1β，1L-1β）處理構(gòu)建髓核細(xì)胞退變模型，將細(xì)胞分為對(duì)照組、IL-1β組、ABPS（100 mg/L）＋I(xiàn)L-1β組、ABPS（200 mg/L）＋I(xiàn)L-1β組，研究ABPS對(duì)1L-1β處理大鼠髓核細(xì)胞增殖、氧化應(yīng)激、NOD樣受體蛋白3（NOD-Like Receptor Protein 3，NLRP3）和線粒體自噬的影響。采用慢病毒技術(shù)轉(zhuǎn)染帕金森疾病蛋白2（Parkinson disease protein 2，Parkin）shRNA，隨后采用IL-1β和ABPS處理，研究線粒體自噬在ABPS抑制1L-1β處理髓核細(xì)胞氧化應(yīng)激和NLRP3炎性小體活化中的作用。將細(xì)胞分為對(duì)照組、IL-1β組、ABPS＋I(xiàn)L-1β組、ABPS＋I(xiàn)L-1β＋沉默信息調(diào)節(jié)因子3（silent information regulators 3，SIRT3）抑制劑（3-TYP）組，研究SIRT3在ABPS調(diào)控細(xì)胞線粒體自噬中的作用。免疫熒光法檢測(cè)細(xì)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）以及微管相關(guān)蛋白1輕鏈3（microtubule-associated protein 1 light 3，LC3）與線粒體的共定位情況；流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期分布；Western blotting檢測(cè)II型膠原（type II Collagen）、蛋白聚糖（aggrecan）、活化型Caspase-3（cleaved Casase-3）、NLRP3、Caspase-1、Parkin、LC3、選擇性自噬接頭蛋白-1（sequestosome-1，SQSTM1/p62）、研究SIRT3、B細(xì)胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）以及Bcl-2關(guān)聯(lián)X蛋白（Bcl-2 associated X protein，Bax）蛋白表達(dá)；原位末端標(biāo)記法（TdT-mediated dUTP nick end labeling，TUNEL）檢測(cè)細(xì)胞凋亡；熒光探針檢測(cè)活性氧（reactive oxygen species，ROS）、線粒體膜電位（mitochondrial membrane potential，MMP）和線粒體活性氧（mitochondrial ROS，mtROS）；比色法檢測(cè)超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽（glutathione，GSH）和丙二醛（malondialdehyde，MDA）水平。結(jié)果 與對(duì)照組比較，IL-1β組髓核細(xì)胞存活率、type II Collagen和aggrecan水平以及Ki67熒光強(qiáng)度均顯著降低（P＜0.05），而G₀/G₁期細(xì)胞分布比例則明顯升高（P＜0.05）。與IL-1β組比較，ABPS處理組細(xì)胞存活率、type II collagen和aggrecan蛋白水平以及Ki67熒光強(qiáng)度升高（P＜0.05），G₀/G₁期細(xì)胞分布比例明顯降低（P＜0.05）。此外，與對(duì)照組比較，IL-1β組髓核細(xì)胞中SOD和GSH水平降低（P＜0.05），MDA、ROS、TUNEL陽(yáng)性細(xì)胞、NLRP3、Caspase-3、cleaved Caspase-3和Caspase-1蛋白水平明顯升高（P＜0.05）。ABPS處理逆轉(zhuǎn)了IL-1β處理對(duì)髓核細(xì)胞氧化應(yīng)激、凋亡和NLRP3炎性小體活化的影響。此外，ABPS處理減弱了IL-1β處理誘導(dǎo)的髓核細(xì)胞MMP降低和mtROS升高（P＜0.05）。ABPS增強(qiáng)了IL-1β處理髓核細(xì)胞中Parkin和LC3的表達(dá)（P＜0.05），SQSTM1/p62蛋白表達(dá)進(jìn)一步降低（P＜0.05）。Parkin shRNA阻斷了ABPS對(duì)IL-1β處理髓核細(xì)胞線粒體自噬、氧化應(yīng)激、凋亡和NLRP3炎性小體的影響（P＜0.05）。此外，IL-1β處理組髓核細(xì)胞中的SIRT3蛋白水平降低（P＜0.05），ABPS處理后細(xì)胞中的SIRT3蛋白水平升高（P＜0.05）。3-TYP阻斷了ABPS對(duì)IL-1β處理髓核細(xì)胞中Parkin介導(dǎo)線粒體自噬的調(diào)節(jié)作用（P＜0.05）。結(jié)論 ABPS可通過(guò)調(diào)控SIRT3/Parkin信號(hào)通路介導(dǎo)的線粒體自噬，減輕IL-1β誘導(dǎo)的髓核細(xì)胞氧化應(yīng)激和NLRP3炎性小體活化。

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年體育局課題研究項(xiàng)目（202320）;全國(guó)中醫(yī)臨床特色技術(shù)傳承人才項(xiàng)目[國(guó)中醫(yī)藥人教教育便函(2019)96號(hào)];2024年河南省中醫(yī)藥課題研究專項(xiàng)課題（2024ZY2066）