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| 环状RNA 0032131调控NF-κB通路对骨关节炎软骨变性的影响 |
| Effect of modulation of the NF-kB pathway by cyclic RNA 0032131 on cartilage degeneration in osteoarthritis |
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| DOI：10.3969/j.issn.1006-7108.2025.03.011 |
| 中文关键词: 环状RNA 0032131 NF-κB 骨关节炎 软骨变性 诊断标志物 |
| 英文关键词:circRNA_0032131, NF-kB, osteoarthritis cartilage degeneration, diagnostic marker |
| 基金项目:国家自然基金面上项目(82274310)；安徽省名中医刘健工作室建设项目(中医药发展秘〔2018〕11号)；安徽省第12批“115”创新团队[（皖人才办）〔2019〕1号]；高水平中医药重点学科建设项目-中医痹病学[zdxk20220129]；安徽省高校自然科学重大项目（2023AH040112）；安徽省高等学校科学研究项目（自然科学类）重点项目（2022AH050449）；2024年中央财政医疗服务与保障能力提升项目国家优势科室（优势专科）-老年病科（皖财社〔2024〕453号，2024lnbkzk08） |
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| 中文摘要: |
| 目的 旨在探讨hsa_circRNA_0032131对白细胞介素（IL）-1β诱导的骨关节炎（OA）软骨变性的作用。方法 IL-1β处理的软骨细胞（CHON-001）用作 OA 的体外模型。细胞转染建立小干扰（si-RNA）和过表达质粒（OE）模型，分为IL-1β、IL-1β+OE-NC、IL-1β+OE-circ_0032131、IL-1β+si-NC和IL-1β+si-circ_0032131共5组。定量聚合酶链反应（RT-qPCR）检测circ_0032131、ADAMTS5、MMP13、SOX9、COL2A1和NF-κB p65 mRNA的表达。CCK-8和流式细胞术评估细胞活力。蛋白免疫印迹法（WB）测定ADAMTS5、MMP13、SOX9、COL2A1蛋白水平。免疫荧光检测p-NF-κB p65和p-IKBα的平均荧光强度。酶联免疫吸附试验（ELISA）检测IL-1β和IL-4的表达。相关性分析circ_0032131与炎症因子、ECM产物和NF-κB的关系。ROC曲线计算circ_0032131曲线下面积。结果 与未处理细胞相比，IL-1β诱导的CHON-001细胞中的circ_0032131上调，IL-4水平下降，ADAMTS5、MMP13和NF-κB p65 mRNA水平升高，SOX9和COL2A1 mRNA水平下降。ROC曲线结果，circ_0032131曲线下的面积为0.9156（0.8136~0.9271），与SOX9、COL2A1和 IL-4呈负相关，与MMP13、IL-1β和NF-κB p65呈正相关。circ_0032131敲低减轻 IL-1β 诱导软骨细胞损伤，与IL-1β+si-NC相比，IL-1β+si-circ_0032131组细胞活力升高，细胞凋亡下降，SOX9和COL2A1蛋白水平升高，ADAMTS5和MMP13 蛋白水平下降。过表达circ_0032131后结果相反。此外，与IL-1β+OE-NC组相比，OE-circ_0032131组p-IKBα和p-NF-κB p65平均荧光强度升高，而circ_0032131敲低削弱了NF-κB信号的活性。结论 circ_0032131在IL-1β 诱导的人软骨细胞中高表达，敲低circ_0032131通过靶向调控NF-кB通路减轻IL-1β 诱导的软骨细胞凋亡、ECM 降解和炎症。 |
| 英文摘要: |
| Objective To investigate the role of hsa_circRNA_0032131 (circ_0032131) in interleukin (IL)-1β-induced cartilage degeneration in osteoarthritis (OA). Methods IL-1β-treated chondrocytes (CHON-001) were used as an in vitro cellular model of OA. Cells were transfected to establish small interference gene (si-RNA) and overexpression plasmid (OE) models. Cells were divided into five groups, IL-1β, IL-1β+OE-NC, IL-1β+OE-circ_0032131, IL-1β+si-NC, and IL-1β+si-circ_0032131 group. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of circ_0032131, ADAMTS5, MMP13, SOX9, COL2A1 and NF-κB p65 mRNA. Cell viability and apoptosis were assessed with CCK-8 and flow cytometry. Western blotting was performed to determine ADAMTS5, MMP13, SOX9, and COL2A1 protein levels. Immunofluorescence assay detected the average fluorescence intensity of p-NF-κB p65 and p-IKBα. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of IL-1β and IL-4. Correlation analysis was performed to investigate the relationship between circ_0032131 and inflammatory factors, ECM metabolites, and NF-κB pathway. ROC curves were calculated to calculate the area under the circ_0032131 curve. Results Compared to that in untreated cells, circ_0032131 was up-regulated in IL-1β-induced CHON-001 cells, IL-4 expression levels decreased, ADAMTS5, MMP13 and NF-κB p65 mRNA levels increased, and chondrogenic synthesis products SOX9 and COL2A1 mRNA levels decreased. As a result of the ROC curve, the area under the circ_0032131 curve had an AUC of 0.9156 (0.8136-0.9271), which was negatively correlated with SOX9, COL2A1, and IL-4, and positively correlated with MMP13, IL-1β, and NF-κB p65. Circ_0032131 knockdown attenuated the IL-1β-induced chondrocyte injury. Compared to IL-1β+si-NC group, the IL-1β+si-circ_0032131 group showed increased cell viability, decreased apoptosis, increased SOX9 and COL2A1 protein levels, and decreased ADAMTS5 and MMP13 protein levels. The results were reversed after overexpression of circ_0032131. In addition, the mean fluorescence intensity of p-IKBα and p-NF-κB p65 was significantly higher in the OE-circ_0032131 group compared to that in the IL-1β+OE-NC group, whereas circ_0032131 knockdown attenuated NF-κB signalling activity. Conclusion Circ_0032131 is highly expressed in IL-1β-induced osteoarthritic chondrocytes Knockdown of circ_0032131 may attenuate IL-1β-induced chondrocyte apoptosis, ECM degradation, and inflammation by targeting and regulating the NF-кB signalling pathway. |
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