摘要
目的探讨长链非编码RNA NORAD(long non-coding RNA NORAD,lncRNA NORAD)对白介素-1β(interleukin-1β,IL-1β)诱导的骨关节炎软骨细胞炎症损伤的作用和机制。方法采用IL-1β刺激软骨细胞建立骨关节炎的细胞模型。软骨细胞分为对照组、IL-1β组(10 ng/mU的IL-1β刺激软骨细胞24 h)、IL-1β+si-NC组(转染NC siRNA 48 h后,进行IL-1β刺激)和IL-1β+si-NORAD组(转染NORAD siRNA 48 h后,进行IL-1β刺激)。实时荧光定量逆转录聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction,RT-qPCR)检测NORAD的表达水平。Calcein AM细胞活性检测试剂盒检测软骨细胞的存活率。一步法TUNEL细胞凋亡检测试剂盒检测软骨细胞凋亡。Western blot检测Bax、Bcl-2、MMP-13、COL2A1和NF-κB p65的蛋白表达水平。ELISA试剂盒检测细胞炎性因子IL-6和TNF-α的浓度。结果与对照组比较,IL-1β组的NORAD表达水平显著升高(P<0.01)。与IL-1β+si-NC组比较,IL-1β+si-NORAD组的NORAD表达水平显著降低(P<0.01)。与对照组比较,IL-1β组和IL-1β+si-NC组软骨细胞的存活率显著降低(P<0.01),细胞凋亡水平显著升高(P<0.01),细胞外基质降解显著增加(P<0.01),IL-6和TNF-α浓度显著升高(P<0.01),NF-κB p65蛋白的磷酸化水平显著升高(P<0.01)。与IL-1β组和IL-1β+si-NC组比较,IL-1β+si-NORAD组软骨细胞的存活率显著提高(P<0.01),细胞凋亡水平显著下降(P<0.01),细胞外基质降解显著降低(P<0.01),IL-6和TNF-α浓度显著下降(P<0.01),NF-κB p65蛋白的磷酸化水平显著降低(P<0.01)。结论基因沉默lncRNA NORAD可以缓解IL-1β诱导的软骨细胞炎症损伤,其保护作用可能是通过下调NF-κB信号通路实现的。
Objective To explore the role of long non-coding RNA NORAD(lncRNA NORAD)in IL-1β-induced inflammatory injury of osteoarthritis chondrocytes and the mechanism.Methods An in vitro osteoarthritis model was established by IL-1βinduction in chondrocytes.The chondrocytes were divided into four groups,including control group,IL-1βgroup(stimulation of chondrocytes with 10ng/mL of IL-1βfor 24h),IL-1β+si-NC group(transfection with siRNA NC for 48h followed by IL-1βstimulation),and IL-1β+si-NORAD group(transfection with NORAD siRNA for 48 h followed by IL-1βstimulation).The expression level of NORAD was detected by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).The survival rate of chondrocytes was measured using the Calcein AM Cell Viability Assay Kit.The apoptotic level of chondrocytes was assessed by the One Step TUNEL Apoptosis Assay Kit.The protein levels of Bax,B-cell lymphoma 2(Blc-2),metalloproteinase 13(MMP-13),collagen typeⅡalpha 1 chain(COL2A1),and nuclear factor kappa B(NF-κB)p65 were examined by Western blot.The concentrations of interleukin 6(IL-6)and tumor necrosis factor-alpha(TNF-α)were determined by enzyme-linked immunosorbent assay(ELISA).Results Compared with that of the control group,NORAD was significantly upregulated in IL-1βgroup(P<0.01).Compared with that of the IL-1β+si-NC group,NORAD was significantly downregulated in IL-1β+si-NORAD group(P<0.01).Compared with those of the control group,significantly lower survival,and significantly higher apoptotic rate,degradation of extracellular matrix,concentrations of IL-6 and TNF-α,and phosphorylated NF-κB p65 level were detected in IL-1βgroup and IL-1β+si-NC group(all P<0.01).Compared with those of the IL-1βgroup and IL-1β+si-NC group,significantly higher survival,and significantly lower apoptotic rate,degradation of extracellular matrix,concentrations of IL-6 and TNF-α,and phosphorylated NF-κB p65 level were detected in the chondrocytes of IL-1β+si-NORAD group(all P<0.01).Conclusion Knockdown of lnc
作者
陈永锋
宋和强
王鹏
郭建伟
陈晓超
CHEN Yongfeng;SONG Heqiang;WANG Peng(Department of Orthopedics,the First Affiliated Hospital of AiRForce Medical University,Shaanxi,Xi’an 710032,China)
出处
《河北医药》
CAS
2024年第2期186-191,共6页
Hebei Medical Journal
基金
陕西省重点研发计划(编号:2023-ZDLSF-14)。
