转化生长因子β3对脂多糖诱导的成骨细胞中IL-6表达的影响

目的:探讨转化生长因子β3 (transforming growth factor β3,TGF-β3)对成骨细胞内炎性细胞因子IL-6表达的影响,及其发挥抗炎作用的机制.方法:20 μg/mL牙髓卟啉单胞菌脂多糖(lipopolysaccharide of Porphyromonas endodontalis,Pe-LPS)刺激人成骨肉瘤细胞系MG63,构建成骨细胞炎症模型.取不同浓度(5～20 ng/mL)的人重组蛋白生长因子TGF-β3和TGF-β1,分别与20 μg/mL P.e-LPS共同作用于MG63细胞24 h后,利用实时荧光定量PCR检测细胞内IL-6 mRNA的表达,ELISA法检测培养液上清中IL-6的表达水平.以10 ng/mL TGF-β3预处理细胞30 ain后,再与20 μg/mL P.e-LPS共同作用20 min,Western印迹法检测细胞内ERK1/2蛋白磷酸化的表达.采用SPSS13.0软件包对数据进行统计学分析.结果:实时荧光定量PCR结果显示,单独20 μg/mL P.e-LPS作用下,MG63细胞内IL-6的表达显著增高(P＜0.01);TGF-β1在低浓度条件下(5～10 ng/mL)对IL-6的表达无显著作用,仅在20 ng/mL时可显著抑制IL-6的表达(P＜0.05).不同浓度的TGF-β3与Re-LPS共同作用均可显著抑制IL-6的表达(P＜0.01).ELISA结果显示,10～20 ng/mL TGF-β3可在蛋白水平上对IL-6有显著抑制作用(P＜0.05).单独P.e-LPS作用时,可使MG63细胞内ERK1/2蛋白的磷酸化水平升高(P＜0.01);而当TGF-β3与P.e-LPS共同作用时,ERK1/2的磷酸化被抑制(P＜0.05).结论:相同浓度条件下,TGF-β3比TGF-β1对成骨细胞炎症的抑制作用更为显著,ERK1/2信号机制参与了TGF-β3的抗炎过程.

Inhibiting effect of transforming growth factor β3 on IL-6 expression in MG63 induced by lipopolysaccharide

PURPOSE:To explore the effect of transforming growth factor β3 (TGF-β3) on IL-6 expression in inflammatory MG63,and the mechanism by which TGF-β3 exert its anti-inflammatory effect.METHODS:Cell line MG63 was stimulated by 20 μg/mL lipopolysaccharide of Porphyromonas endodontalis (P.e-LPS) to establish the inflammatory model of osteoblast.TGF-β3 or TGFβ1 varying fmm 5 to 20 ng/mL was added together with P.e-LPS for 24 h,then the mRNA expression of IL-6 was detected by real-time PCR,the role of TGF-β3 on IL-6 protein was further verified by ELISA.MG63 was pretreated with 10 ng/mL TGF-β3 for 30 min in RPMI 1640 medium without fetal bovine serum (FBS),then the cells were cultured for another 20 min with 20 μg/mL P.e-LPS,the phosphorylation level of ERK1/2 was measured by Western blot.Statistical analysis was performed using one-way ANOVA with SPSS13.0 software package.RESULTS:The results of real-time PCR revealed that,when MG63 was treated with 20 μg/mL P.e-LPS alone,the mRNA expression of IL-6 increased significantly (P＜0.01).When TGF-β1 was added with P.e-LPS,it could barely decrease IL-6 prominently at the highest concentration (P＜0.05).Whereas,the inhibition effect of TGF-β3 on IL-6 was dramatic (P＜0.01),ELISA results showed that 10-20 ng/mL TGF-β3 blocked the IL-6 expression at protein level (P＜0.05).20 μg/mL P.e-LPS promoted the phosphorylation level of ERK1/2 in MG63 (P＜0.01),while with 10 ng/mL TGF-β3,the effect of P.e-LPS on ERK1/2 was blocked(P＜0.05).CONCLUSIONS:TGF-β3 is more potent than TGF-β1 in inhibiting MG63,and ERK1/2 is involved in its anti-inflammatory effect.