Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Bologna, Italy; Dipartimento di Scienze Mediche e Chirurgiche, Università di Bologna, Bologna, Italy.
Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Bologna, Italy.
Free Radic Biol Med. 2020 Jun;153:159-172. doi: 10.1016/j.freeradbiomed.2020.03.029. Epub 2020 Apr 17.
Oxidative stress (OS) contributes to Osteoarthritis (OA) pathogenesis and its effects are worsened by the impairment of homeostatic mechanisms such as autophagy in OA chondrocytes. Rescue of an efficient autophagic flux could therefore reduce the bulk of damaged molecules, and at the same time improve cell function and viability. As a promising dietary or intra-articular supplement to rescue autophagy in OA chondrocytes, we tested spermidine (SPD), known to induce autophagy and to reduce OS in several other cellular models. Chondrocytes were obtained from OA cartilage and seeded at high-density to keep their differentiated phenotype. The damaging effects of OS and the chondroprotective activity of SPD were assessed by evaluating the extent of cell death, oxidative DNA damage and caspase 3 activation. The autophagy promoting activity of SPD was evaluated by assessing pivotal autophagic effectors, i.e. Beclin-1 (BECN-1), microtubule-associated protein 1 light chain 3 II (LC3-II) and p62. BECN-1 protein expression was significantly increased by SPD and reduced by HO treatment. SPD also rescued the impaired autophagic flux consequent to HO exposure by increasing mRNA and protein expression of LC3-II and p62. SPD induction of mitophagy was revealed by immunofluorescent co-localization of LC3-II and TOM20. The key protective role of autophagy was confirmed by the loss of SPD chondroprotection upon autophagy-related gene 5 (ATG5) silencing. Significant SPD tuning of the HO-dependent induction of degradative (MMP-13), inflammatory (iNOS, COX-2) and hypertrophy markers (RUNX2 and VEGF) was revealed by Real Time PCR and pointed at the SPD ability of reducing NF-κB activation through autophagy induction. Conversely, blockage of autophagy led to parallel increases of oxidative markers and p65 nuclear translocation. SPD also increased the proliferation of slow-proliferating primary cultures. Taken together, our findings highlight the chondroprotective, anti-oxidant and anti-inflammatory activity of SPD and suggest that the protection afforded by SPD against OS is exerted through the rescue of the autophagic flux.
氧化应激（OS）有助于骨关节炎（OA）的发病机制，并且 OA 软骨细胞中自噬等内稳态机制的损伤会使其恶化。因此，有效自噬流的恢复可以减少受损分子的总量，同时改善细胞功能和活力。作为一种有前途的膳食或关节内补充剂，以恢复 OA 软骨细胞中的自噬，我们测试了 spermidine（SPD），已知其在几种其他细胞模型中诱导自噬并减少 OS。软骨细胞从 OA 软骨中获得，并以高密度播种以保持其分化表型。通过评估细胞死亡程度、氧化 DNA 损伤和 caspase 3 激活来评估 OS 的破坏作用和 SPD 的软骨保护活性。通过评估关键的自噬效应物，即 Beclin-1（BECN-1）、微管相关蛋白 1 轻链 3 II（LC3-II）和 p62，评估 SPD 促进自噬的活性。SPD 蛋白表达显著增加，而 HO 处理则减少。SPD 还通过增加 LC3-II 和 p62 的 mRNA 和蛋白表达来挽救由于 HO 暴露而受损的自噬流。LC3-II 和 TOM20 的免疫荧光共定位揭示了 SPD 诱导的线粒体自噬。自噬相关基因 5（ATG5）沉默后 SPD 软骨保护作用的丧失证实了自噬的关键保护作用。实时 PCR 显示，SPD 对 HO 依赖性诱导的降解（MMP-13）、炎症（iNOS、COX-2）和肥大标志物（RUNX2 和 VEGF）的显著调节作用，并指出了 SPD 通过诱导自噬减少 NF-κB 激活的能力。相反，自噬的阻断导致氧化标志物和 p65 核转位的平行增加。SPD 还增加了缓慢增殖的原代培养物的增殖。总之，我们的研究结果强调了 SPD 的软骨保护、抗氧化和抗炎活性，并表明 SPD 对 OS 的保护作用是通过恢复自噬流来发挥的。
