安石榴苷在白细胞介素-1β诱导软骨细胞中的作用及其机制

Role of punicalagin in interleukin-1β-induced osteoarthritis chondrocytes and mechanism

目的探讨安石榴苷在白细胞介素(IL)-1β诱导软骨细胞中的作用及其机制。方法不同浓度(10、20、40、80、160μmol/L)安石榴苷处理软骨细胞,噻唑蓝(MTT)法检测细胞活性。采用酶联免疫吸附试验(ELISA)法检测上清液中环氧化酶(COX)-2、一氧化氮(NO)、基质金属蛋白酶(MMP)-1和MMP-13的表达;实时定量反转录聚合酶链反应(RT-qPCR)检测MMP-1和基质金属蛋白酶组织抑制剂-1(TIMP-1)信使RNA(mRNA)表达的变化;蛋白质印迹法(Western blot)检测核因子-κB(NF-κB)通路相关蛋白表达。多组间比较用单因素方差分析,两组间比较采用t检验。结果PUN+IL-1β组COX-2、NO、MMP-1、MMP-13表达水平低于IL-1β组(71.80±4.15、53.80±4.09、33.80±3.03比85.00±4.36,t=4.906、11.676、21.559,P<0.05;207.00±15.89、160.00±10.37、105.80±7.46比278.80±11.82,t=8.107、16.896、27.674,P<0.05;614.20±16.41、445.40±19.01、214.00±13.44比692.80±20.77,t=6.641、19.651、43.288,P<0.05;63.40±4.28、42.40±2.70、31.00±2.24比81.20±5.17,t=5.933、14.879、19.937,P<0.05)。PUN+IL-1β组MMP-1 mRNA相对表达量低于IL-1β组(6.76±0.26、2.63±0.26、1.79±0.22比10.06±0.48,t=13.547、30.472、34.965,P<0.05)。PUN+IL-1β组TIMP-1 mRNA相对表达量高于IL-1β组(0.24±0.03、0.46±0.03、0.80±0.04比0.12±0.03,t=6.399、19.575、32.224,P<0.05)。PUN+IL-1β组p-p65、p-IκB含量低于IL-1β组(1.14±0.03、1.06±0.03、0.97±0.03比1.29±0.03,t=6.272、9.714、13.571,P<0.05;1.30±0.04、1.16±0.03、1.00±0.04比1.41±0.02,t=4.384、11.005、15.367,P<0.05)。结论安石榴苷通过抑制炎性介质COX-2、NO、MMP-1和MMP-13的表达来抑制NF-κB通路的活化,进而减缓软骨细胞的炎性反应。

Objective To explore the role and mechanism of punicalagin in interleukin(IL)-1β-induced chondrocytes.Methods Chondrocytes were treated with different concentrations of punicalagin(10,20,40,80,160μmol/L),and methyl thiazolyl tetrazolium(MTT)method was used to detect cell viability.Enzyme linked immunosorbent assay(ELISA)was conducted to detect the expression of cyclooxygenase-2(COX-2),nitric oxide(NO),matrix metalloproteinase(MMP)-1 and MMP-13 in the supernatant liquid.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of MMP-1 and tissue inhibitor of metalloproteinase-1(TIMP-1).Western blotting was used to detect the expression of nuclear factor-κB(NF-κB)pathway related protein.One-way analysis of variance was used for comparison between multiple groups,and T-test was used for comparison between two groups.One-way analysis of variance was used for comparison between multiple groups,and T-test was used for comparison between two groups.Results The expression level of COX-2,NO,MMP-1 and MMP-13 in the PUN+IL-1βgroup were lower than that in the IL-1βgroup(71.80±4.15,53.80±4.09,33.80±3.03 to 85.00±4.36,t=4.906,11.676,21.559,P<0.05;207.00±15.89,160.00±10.37,105.80±7.46 to 278.80±11.82,t=8.107,16.896,27.674,P<0.05;614.20±16.41,445.40±19.01,214.00±13.44 to 692.80±20.77,t=6.641,19.651,43.288,P<0.05;63.40±4.28,42.40±2.70,31.00±2.24 to 81.20±5.17,t=5.933,14.879,19.937,P<0.05).The relative expression level of MMP-1 mRNA in PUN+IL-1βgroup was lower than that in the IL-1βgroup(6.76±0.26,2.63±0.26,1.79±0.22 to 10.06±0.48,t=13.547,30.472,34.965,P<0.05).The relative expression level of TIMP-1 mRNA in PUN+IL-1βgroup was higher than that in the IL-1βgroup(0.24±0.03,0.46±0.03,0.80±0.04 to 0.12±0.03,t=6.399,19.575,32.224,P<0.05).The protein content of p-p65 and p-IκB in the PUN+IL-1βgroup were lower than that in the IL-1βgroup(1.14±0.03,1.06±0.03,0.97±0.03 to 1.29±0.03,t=6.272,9.714,13.571,P<0.05;1.30±0.04,1.16±0.03,1.00±0.04 to 1.41±0.02,t=4.384,11.005,15.367,P<0.05).Conclusion Punicarin could inhibit the activation of the NF-κB pathway by inhibiting the expression of inflammatory mediators COX-2,NO,MMP-1 and MMP-13,and then slowing down the inflammatory response of chondrocytes.