Liao Taiyang, Ma Zhenyuan, Liu Deren, Shi Lei, Mao Jun, Wang Peimin, Ding Liang
Department of Orthopedics and Traumatology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China.
Key Laboratory for Metabolic Diseases in Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2024 Dec 25;53(6):669-679. doi: 10.3724/zdxbyxb-2024-0091.
To investigate the effect of nuclear protein (Nupr) 1 on the pathological progression of osteoarthritis and its relationship with ferroptosis of chondrocytes.
Chondrocytes from mouse knees were divided into small interfering RNA (siRNA) control group, small interfering RNA targeting Nupr1 (siNupr1) group, siRNA control+IL-1β group (siRNA control interference for 24 h followed by 10 ng/mL IL-1β) and siNupr1+IL-1β group (siNupr1 interference for 24 h followed by 10 ng/mL IL-1β). The protein and mRNA expressions of Nupr1 were detected by Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation viabilities were measured using the cell counting kit-8 method. The levels of ferrous ions were detected by FerroOrange staining. Lipid peroxidation levels were detected by C11-BODIPY-591 fluorescence imaging. The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by enzyme-linked immunosorbent assay. The protein expressions of acyl-CoA synthetase long-chain family (ACSL) 4, P53, glutathione peroxidase (GPX) 4 and solute carrier family 7 member 11 gene (SLC7A11) were detected by Western blotting. The osteoarthritis model was constructed by destabilization of the medial meniscus (DMM) surgery in 7-week-old male C57BL/6J mice. The mice were randomly divided into four groups with 10 animals in each group: sham surgery (Sham)+adeno-associated virus serotype 5 (AAV5)-short hairpin RNA (shRNA) control group, Sham+AAV5-shRNA control targeting (shNupr1) group, DMM+AAV5-shRNA control group, and DMM+AAV5-shNupr1 group. Hematoxylin and eosin staining and Safranin O-Fast Green staining were used to observe the morphological changes in cartilage tissue. The Osteoarthritis Research Society International (OARSI) osteoarthritis cartilage histopathology assessment system was used to evaluate the degree of cartilage degeneration in mice. The mRNA expressions of matrix metallopeptidase (MMP) 13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5, cyclooxy-genase (COX) 2, and GPX4 were detected by qRT-PCR.
experiments showed that knocking down alleviated the decrease of chondrocyte proliferation activity induced by IL-1β, reduced iron accumulation in mouse chondrocytes, lowered lipid peroxidation, downregulated ACSL4 and P53 protein expression and upregulated GPX4 and SLC7A11 protein expression (all <0.01), thereby inhibiting ferroptosis in mouse chondrocytes. Meanwhile, animal experiments demonstrated that knocking down delayed the degeneration of articular cartilage in osteoarthritis mice, improved the OARSI score, slowed down the degradation of the extracellular matrix in osteoarthritis cartilage, and reduced the expression of the key ferroptosis regulator GPX4 (all <0.01).
Knockdown of can delay the pathological progression of osteoarthritis through inhibiting ferroptosis in mouse chondrocytes.
探讨核蛋白（Nupr）1对骨关节炎病理进程的影响及其与软骨细胞铁死亡的关系。
将小鼠膝关节软骨细胞分为小干扰RNA（siRNA）对照组、靶向Nupr1的小干扰RNA（siNupr1）组、siRNA对照组+白细胞介素-1β（IL-1β）组（siRNA对照干扰24小时后加入10 ng/mL IL-1β）和siNupr1+IL-1β组（siNupr1干扰24小时后加入10 ng/mL IL-1β）。采用蛋白质免疫印迹法和定量逆转录聚合酶链反应（qRT-PCR）检测Nupr1的蛋白和mRNA表达。使用细胞计数试剂盒-8法检测细胞增殖活力。通过FerroOrange染色检测亚铁离子水平。采用C11-BODIPY-591荧光成像检测脂质过氧化水平。采用酶联免疫吸附测定法检测丙二醛（MDA）和谷胱甘肽（GSH）含量。采用蛋白质免疫印迹法检测酰基辅酶A合成酶长链家族（ACSL）4、P53、谷胱甘肽过氧化物酶（GPX）4和溶质载体家族7成员11基因（SLC7A11）的蛋白表达。通过内侧半月板不稳定（DMM）手术构建7周龄雄性C57BL/6J小鼠骨关节炎模型。将小鼠随机分为四组，每组10只：假手术（Sham）+腺相关病毒5型（AAV5）-短发夹RNA（shRNA）对照组、Sham+靶向Nupr1的AAV5-shRNA对照组（shNupr1）组、DMM+AAV5-shRNA对照组和DMM+AAV5-shNupr1组。采用苏木精-伊红染色和番红O-固绿染色观察软骨组织形态学变化。采用国际骨关节炎研究学会（OARSI）骨关节炎软骨组织病理学评估系统评估小鼠软骨退变程度。采用qRT-PCR检测基质金属蛋白酶（MMP）13、含血小板反应蛋白基序的解聚素和金属蛋白酶（ADAMTS）5、环氧化酶（COX）2和GPX4的mRNA表达。
实验表明，敲低Nupr1可减轻IL-−1β诱导的软骨细胞增殖活性降低，减少小鼠软骨细胞内铁蓄积，降低脂质过氧化，下调ACSL4和P53蛋白表达，上调GPX4和SLC7A11蛋白表达（均P<0.01），从而抑制小鼠软骨细胞铁死亡。同时，动物实验表明，敲低Nupr1可延缓骨关节炎小鼠关节软骨退变，改善OARSI评分，减缓骨关节炎软骨细胞外基质降解，并降低关键铁死亡调节因子GPX4的表达（均P<0.01）。
敲低Nupr-1可通过抑制小鼠软骨细胞铁死亡延缓骨关节炎的病理进程。
