鼠尾草酸对白细胞介素-1β诱导的软骨细胞基质降解的作用及机制

Effect and mechanism of carnosic acid on interleukin-1βinduced degradation of chondrocyte extracellular matrix

目的:探讨鼠尾草酸(CAD)对白细胞介素-1β(IL-1β)诱导的大鼠软骨细胞基质降解的作用及机制。方法:体外培养原代大鼠软骨细胞并随机分成4组:对照组(Control)、IL-1β组(10 ng/mL)、CAD(4μmol/L)+IL-1β组和CAD(8μmol/L)+IL-1β组。处理24 h后,CCK-8检测不同浓度CAD(0、1、2、4、8、16、32和64μmol/L)对软骨细胞增殖的效应;实时荧光定量PCR(RT-qPCR)检测软骨细胞肿瘤坏死因子-α(TNF-α)、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)、性别决定区域Y框蛋白9(Sox9)、Ⅱ型胶原蛋白α1链(Col2a1)和聚集蛋白聚糖(ACAN)mRNA的表达;免疫细胞荧光检测软骨细胞MMP13、Col2a1和P65蛋白的表达;Western Blot检测软骨细胞P65的蛋白表达及定量分析。采用分子对接验证CAD潜在的作用靶点。结果:与对照组相比,高浓度CAD(16、32和64μmol/L)以浓度依赖性的方式抑制软骨细胞增殖(P<0.01);进一步与对照组相比,IL-1β显著促进软骨细胞TNF-α、MMP13和ADAMTS5的表达并抑制Sox9、Col2a1和ACAN的表达,而CAD(4和8μmol/L)处理能够显著改善IL-1β诱导的软骨细胞基质降解(P<0.05,P<0.01);机制层面,CAD能够显著抑制IL-1β诱导的P65核转位;分子对接显示CAD与P65具有较好的结合。结论:CAD能够显著改善IL-1β诱导的软骨细胞基质降解并通过P65抑制核因子-κB(NF-κB)信号通路。

Objective:To investigate the effect and mechanism of carnosic acid(CAD)on interleukin-1β(IL-1β)induced degradation of chondrocyte extracellular matrix.Methods:Primary rat chondrocytes were cultured in vitro and randomly divided into four groups:control group,IL-1βgroup(10 ng/mL),CAD(4μmol/L)+IL-1βgroup,and CAD(8μmol/L)+IL-1βgroup.After 24 hours of treatment,a CCK-8 kit was used to detect the effect of different concentrations of CAD(0,1,2,4,8,16,32,and 64μmol/L)on chondrocyte proliferation.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of tumor necrosis factor-α(TNF-α),matrix metalloproteinase 13(MMP13),a disintegrin and metalloproteinase with thrombospondin 5(ADAMTS5),SRY-box transcription factor 9(Sox9),collagen typeⅡalpha 1 chain(Col2a1)and aggrecan(ACAN)mRNA in chondrocytes.MMP13,Col2a1,and P65 protein expression levels in chondrocytes were detected by immunofluorescence.Western Blot was used to detect the protein expression and quantitative analysis of P65 in chondrocytes.Molecular docking was used to verify the potential targets of CAD.Results:Compared with the control group,high concentrations of CAD(16,32,and 64μmol/L)inhibited the proliferation of chondrocytes in a concentration-dependent manner.Moreover,compared with the control group,IL-1βsignificantly promoted the expression of TNF-α,MMP13 and ADAMTS5,and inhibited the expression of Sox9,Col2a1 and ACAN in chondrocytes.However,CAD(4 and 8μmol/L)could significantly ameliorate the matrix degradation of chondrocytes induced by IL-1β(P<0.05).At the mechanism level,CAD could significantly inhibit the nuclear translocation of P65 induced by IL-1β,and molecular docking showed that CAD had a good binding to P65.Conclusion:CAD could significantly reverse the matrix degradation of chondrocytes induced by IL-1βand inhibit the nuclear factor-kappa B(NF-κB)signal pathway through P65.