| 摘要: |
| 目的 观察低氧对IL-1β诱导软骨细胞自噬及焦亡相关蛋白表达的影响，并探讨其保护软骨的作用机制。方法 通过生物信息学方法分析骨关节炎患者软骨与正常软骨中Bcl2/腺病毒E1B相互作用蛋白3样蛋白（BNIP3L/NIX）的表达差异情况。提取C57BL/6J乳鼠膝关节原代软骨细胞，将细胞分为对照组、IL-1β组、低氧组、IL-1β＋低氧组，以10 ng/mL IL-1β处理细胞24 h模拟骨关节炎样软骨细胞损伤，低氧处理采用1% O2培养24 h。采用蛋白质印迹法检测各组细胞中Ⅱ型胶原蛋白α1（COL2α1）、基质金属蛋白酶13（MMP13）、血小板反应蛋白解整合素金属肽酶5（ADAMTS5）、核苷酸结合寡聚化结构域样受体3（NLRP3）、消皮素D氨基末端结构域（GSDMD-N）、含CARD结构的凋亡相关斑点样蛋白（ASC）、IL-18、低氧诱导因子1α（HIF1α）、NIX、Beclin1、微管相关蛋白轻链3（LC3）、p62蛋白的表达。结果 生物信息学分析结果显示，骨关节炎患者软骨细胞中NIX的表达比正常软骨降低。蛋白质印迹法验证结果显示，与对照组比较，IL-1β组软骨细胞中COL2α1、NIX蛋白表达水平降低（均P＜0.05），MMP13、ADAMTS5、NLRP3、GSDMD-N、ASC、IL-18、HIF1α蛋白表达水平增高（均P＜0.01）；与IL-1β组相比，IL-1β＋低氧组软骨细胞中COL2α1、HIF1α、NIX、Beclin1表达水平及LC3Ⅱ/LC3Ⅰ升高（均P＜0.01），MMP13、ADAMTS5、NLRP3、GSDMD-N、ASC、IL-18、p62表达水平降低（均P＜0.01）；与对照组相比，低氧组软骨细胞中HIF1α、NIX、Beclin1蛋白表达水平及LC3Ⅱ/LC3Ⅰ增高（均P＜0.01），p62、NLRP3、GSDMD-N、IL-18蛋白表达水平降低（均P＜0.05）。结论 低氧可能通过HIF1α/NIX介导的线粒体自噬清除NLRP3炎症小体、抑制软骨细胞焦亡，从而减轻IL-1β诱导的软骨细胞损伤。 |
| 关键词: 低氧 线粒体自噬 骨关节炎 软骨细胞 焦亡 |
| DOI：10.16781/j.CN31-2187/R.20240552 |
| 投稿时间：2024-08-07修订日期：2024-12-16 |
| 基金项目:国家自然科学基金青年科学基金（81802234），重庆市中青年医学高端人才项目，重庆市科卫联合医学科研项目（2024GDRC002），重庆医科大学附属第二医院“宽仁英才”项目（kryc-gg-2116）. |
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| Hypoxia inhibits chondrocyte pyroptosis via HIF1α/NIX-mediated mitochondrial autophagy |
| LUO Shiqi,WEI Xia,JIA Lang* |
(Department of Rehabilitation, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
*Corresponding author) |
| Abstract: |
| Objective To observe the effect of hypoxia (HX) on autophagy and pyroptosis-related protein expression of interleukin (IL)-1β-induced chondrocytes, and explore its mechanism of cartilage protection. Methods The expression of Bcl2/adenovirus E1B interacting protein 3-like (BNIP3L/NIX) in normal and osteoarthritis chondrocytes was analyzed by bioinformatics method. The primary chondrocytes from the knee joints of C57BL/6J neonatal mice were extracted and assigned to control group, IL-1β group, HX group, or IL-1β＋HX group. The cells were treated with 10 ng/mL IL-1β for 24 h to simulate osteoarthritis-like chondrocyte injury, and HX treatment was by incubation with 1% O2 for 24 h. The expression levels of collagen typeⅡ α1 (COL2α1), matrix metalloproteinase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5), nucleotide-binding oligomerization domain-like receptor 3 (NLRP3), gasdermin D N-terminal domain (GSDMD-N), apoptosis-associated speck-like protein containing a CARD (ASC), IL-18, hypoxia induced factor 1α (HIF1α), NIX, Beclin1, microtubule-associated protein-light chain 3 (LC3), and p62 proteins were detected by Western blotting in each group. Results Bioinformatics analysis showed that the expression of NIX was lower in osteoarthritis chondrocytes than in normal chondrocytes. Western blotting showed that compared to the control group, the IL-1β group showed significant decreases in COL2α1 and NIX protein expression (both P＜0.05) and significant increases in MMP13, ADAMTS5, NLRP3, GSDMD-N, ASC, IL-18 and HIF1α protein expression (all P＜0.01). Compared to the IL-1β group, the IL-1β＋HX group showed significant increases in COL2α1, HIF1α, NIX, Beclin1, and LC3Ⅱ/LC3Ⅰ (all P＜0.01) and significant decreases in MMP13, ADAMTS5, NLRP3, GSDMD-N, ASC, IL-18 and p62 (all P＜0.01). Compared to the control group, the HX group exhibited significant increases in HIF1α, NIX, Beclin1, and LC3Ⅱ/LC3Ⅰ (all P＜0.01) and decreases in p62, NLRP3, GSDMD-N and IL-18 (all P＜0.05). Conclusion Hypoxia may eliminate NLRP3 inflammasome and inhibit chondrocyte pyroptosis through HIF1α/NIX-mediated mitochondrial autophagy, thereby reducing IL-1β-induced chondrocyte injury. |
| Key words: hypoxia mitochphagy osteoarthritis chondrocytes pyroptosis |
