牛膝多糖通过SIRT3/Parkin调控的线粒体自噬减轻IL-1β诱导的大鼠髓核细胞氧化应激和NLRP3炎性小体活化  

Achyranthis Bidentatae Radix polysaccharides attenuate IL-1β-induced oxidative stress and NLRP3 inflammasome activation in rat myeloid cells via SIRT3/Parkin-regulated mitophagy

作　　者：黄家俊 吴迪友 陶广义 赵宇 黄俊卿[2] 杨彬[2] HUANG Jiajun;WU Diyou;TAO Guangyi;ZHAO Yu;HUANG Junqing;YANG Bin(College of Orthopedics and Traumatology,Henan University of Chinese Medicine,Zhengzhou 450000,China;Henan Provincial Hospital of Traditional Chinese Medicine,Zhengzhou 450000,China)

机构地区：[1]河南中医药大学骨伤学院,河南郑州450000 [2]河南省中医院,河南郑州450000

出　　处：《中草药》2025年第5期1652-1666,共15页Chinese Traditional and Herbal Drugs

基　　金：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)。

摘　　要：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(typeⅡCollagen)、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(Td T-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,mt ROS);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、typeⅡCollagen和agObjective 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-1βgroup,ABPS(100 mg/L)+IL-1βgroup,and ABPS(200 mg/L)+IL-1βgroup 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-1βand 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 prolifer

关 键 词：牛膝多糖 髓核细胞 沉默信息调节因子3 帕金森蛋白 线粒体自噬 

分 类 号：R285.5[医药卫生—中药学]