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。
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-1βand 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 result were reversed after overexpression of circ_0032131.In addition,the mean fluorescence intensity of p-IKBαand 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.
作者
周巧
刘健
万磊
汪元
王桂珍
齐亚军
胡月迪
朱艳
ZHOU Qiao;LIU Jian;WAN Lei;WAN Yuan;WANG Guizheng;QI Yajun;HU Yuedi;ZHU Yan(The Second Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230061,China;The First Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230031,China;Anhui University of Chinese Medicine,Hefei 230012,China)
出处
《中国骨质疏松杂志》
北大核心
2025年第3期388-396,共9页
Chinese Journal of Osteoporosis
基金
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)。
