连翘苷对IL-1β诱导的人关节软骨细胞凋亡及细胞外基质降解的影响

The effects of phillyrin on IL-β-induced human articular chondrocyte apoptosis and extracellular matrix degradation

目的探究连翘苷(phillyrin,PHN)对IL-1β(interleukin-1β,IL-1β)诱导的人关节软骨细胞凋亡及细胞外基质降解的作用及机制。方法将细胞分为Control组、IL-1β组、PHN(2 mg/ml)组、PHN(5 mg/ml)组和PHN(10 mg/ml)组,用IL-1β处理软骨细胞30 min后加入相应浓度的PHN处理细胞,MTT检测各组细胞活性,流式检测细胞凋亡,RT-PCR检测炎症介导因子以及collagenⅡ、基质金属蛋白酶(MMP-13)、聚集蛋白聚糖(aggrecan)和AMAMTS-5的基因表达水平,Western blot检测细胞增殖、凋亡、细胞外基质和NF-κB相关蛋白的表达,免疫荧光检测NF-κB的入核情况。结果与Control组比较,IL-1β组细胞活性及PCNA蛋白表达水平均明显降低,细胞凋亡率及促进细胞凋亡蛋白的表达水平显著升高;与IL-1β组比较,PHN(2、5、10 mg/ml)组细胞活性和PCNA、Bcl-2、PARP蛋白表达水平显著升高,细胞凋亡率及p53、Bad、Bax、cl-caspase-3的表达水平明显降低。IL-1β促进炎症介导因子的表达,而PHN抑制炎症接到因子的表达。同时,与Control组比较,IL-1β组细胞collagenⅡ和aggrecan的蛋白和基因表达水平明显降低,SOX-9、TIMP-1、MMP-9、MMP-13、ADAMTS-5基因及蛋白表达水平明显升高;PHN(2、5、10 mg/ml)组collagenⅡ和aggrecan基因和蛋白表达水平显著高于IL-1β组,PHN(5、10 mg/ml)组SOX-9、TIMP-1、MMP-9、MMP-13、ADAMTS-5基因和蛋白表达水平明显降低。此外,IL-1β能显著抑制IκBα表达,促进p-IκBα和NF-κB p65表达,并促进NF-κB转移入核;PHN(5、10mg/ml)能显著减弱IL-1β对IκBα、p-IκBα、NF-κBp65表达及NF-κB转移入核的调控作用。结论连翘苷能抑制IL-1β诱导的人软骨细胞凋亡及细胞外基质降解,作用机制可能与抑制NF-κB信号通路激活有关。

This study was designed to investigate the effects of phillyrin(PHN) on IL-β-induced human articular chondrocyte cell apoptosis and extracellular matrix degration, and its mechanism. Articular chondrocytes were divided into control group, IL-1β group, PHN(2 mg/ml) group, PHN(5 mg/ml) group and PHN(10 mg/ml)group. The cells in PHN groups were pretreated with IL-1β for 30 min, and then treated with PHN at corresponding concentration. The viability and apoptosis of articular chondrocytes were measured by MTT and flow cytometry,respectively. RT-PCR was performed for measuring the mRNA levels of inflammatory mediators and collagenⅡ, matrix metalloproteinase-13(MMP-13), aggrecan and AMAMTS-5;Western blot was used to detect cell proliferation, cell apoptosis, extracellular matrix degradation and NF-κB related proteins expression.The nuclear localization signal of NF-κB was determined by immunofluorescence. Data showed that as compared with control group, the cell viability and protein levels of PCNA were decreased significantly, the apoptosis rate and the expression levels of apoptotic proteins were significantly increased in IL-1β group. Compared with IL-1β group, the cell viability and protein levels of PCNA, Bcl-2, PARP in PHN(2, 5, 10 mg/ml) groups were increased significantly, while the apoptosis rate, the levels of p53, Bad, Bax and cl-caspase-3 were decreased significantly. IL-1β promotes the expression of inflammatory mediators, while PHN inhibits the expression of inflammatory receptors. Meanwhile, the protein and mRNA levels of collagen Ⅱ and aggrecan in IL-1β group were decreased compared with control group, but the gene and protein levels of SOX-9, TIMP-1, MMP-9, MMP-13 and ADAMTS-5 were increased notably;the protein and mRNA levels of collagen Ⅱ and aggrecan in PHN(2, 5, 10 mg/ml) group were higher than those in IL-1β group, but the gene and protein levels of SOX-9, TIMP-1, MMP-9,MMP-13 and ADAMTS-5 were decreased markedly. In addition, IL-1β inhibited the expression of IκBα, but induced the expression levels of p-IκBα and NF-κB p65, and induced nuclear import of NF-κB, while PHN(5, 10 mg/ml) attenuated the effects of IL-1β on the expression levels of IκBα, p-IκBα, NF-κB p65 and the nuclear import of NF-κB. In conclusion, PHN can inhibit IL-1β-induced cell apoptosis and degradation of extracellular matrix of human articular chondrocytes, and the mechanism may relate to NF-κB signaling pathway inhibition.