AGEs调节NF-κB通路对软骨细胞炎症因子IL-1β的影响

Effects of AGEs on IL-1βExpression in Chondrocytes via IL-κB Pathway

目的观察AGEs调节NF-κB通路对软骨细胞炎症因子IL-1β表达的影响。方法将软骨细胞传代培养,取对数生长的软骨细胞接种于96孔板,分为对照组、低剂量AGEs(20μg/L)组、高剂量AGEs(40μg/L)组、NF-κB通路抑制剂PDTC(5μg/L)组和AGEs(40μg/L)+PDTC(5μg/L)组,共培养24 h后ELISA检测各组上清液中IL-1β的含量,Real-time PCR检测各组IL-1β和P65基因表达,Western-blot检测IL-1β和P65蛋白表达。结果对照组软骨细胞上清液中IL-1β的浓度明显高于低剂量AGEs组和高剂量AGEs组,差异有统计学意义(P<0.05);PDTC组和AGEs+PDTC组软骨细胞上清液中IL-1β含量与对照组比较明显下降(P<0.05)。对照组软骨细胞中IL-1β和P65 mRNA相对表达量高于低剂量组和高剂量组(P<0.05),低于PDTC组(P<0.05)。PDTC组IL-1β和P65 mRNA相对表达量低于AGEs+PDTC组(P<0.05)。对照组软骨细胞中IL-1β和P65蛋白相对表达量低于低剂量组和高剂量组(P<0.05),但高于PDTC组(P<0.05)。PDTC组软骨细胞中IL-1β和P65蛋白相对表达量较AGEs+PDTC组减少(P<0.05)。结论AGEs上调软骨细胞IL-1β的表达,NF-κB通路可能是参与骨关节炎发生发展的重要通路之一。

Objective To study effects of AGEs modulating NF-κB pathway on inflammatory factor IL-1βin chondrocytes.Methods Chondrocytes were subcultured.Chondrocytes in logarithmic growth phase were seeded into 96-well plates and were divided into control group,low-dose AGEs(20μg/L)group,high-dose AGEs(40μg/L)group,NF-κB pathway inhibitor PDTC(5μg/L)group and high-dose AGEs(40μg/L)+inhibitor PDTC(5μg/L)group.After 24 hours of co-cultivation,IL-1βin supernatant was tested by ELISA.Real-time PCR was carried out to detect IL-1βand P65 gene.Moreover,western blot was conducted to exam IL-1βand P65 protein.Results IL-1βin chondrocyte supernatant in control group was prominently higher than that in low-dose AGEs group and high-dose AGEs group with statistically significant difference(P<0.05).IL-1βin chondrocyte supernatant of PDTC group and high-dose AGEs+PDTC group was markedly lower than that of control group(P<0.05).Relative expressions of IL-1βand P65 mRNA in chondrocytes in control group were higher than that in low-dose AGEs group and high-dose AGEs group(P<0.05),and lower than PDTC group as well(P<0.05).Relative expressions of IL-1βand P65 mRNA in PDTC group were lower than that in AGEs+PDTC group(P<0.05).Relative expressions of IL-1βand P65 protein in chondrocytes in control group were lower than those in low-dose and high-dose AGEs group(P<0.05),but higher than those in PDTC group(P<0.05).Besides,relative expressions of IL-1βand P65 protein in chondrocytes in PDTC group were reduced in AGEs+PDTC group(P<0.05).Conclusion AGEs boost IL-1βecpression through NF-κB pathway that may be one of the important pathways involved in the occurrence and progression of osteoarthritis.