抑制NF-κB活性对TNF-α联合IFN-γ诱导肾小管上皮细胞表达趋化因子的影响

Effect of inhibiting NF-κB on expression of chemokines in renal tubular epithelial cells induced by TNF-α combined with IFN-γ

目的 :探讨抑制核转录因子-κB(nuclear transcription factor-kappa B,NF-κB)活性对肿瘤坏死因子(tumor necrosis factor,TNF)-α联合干扰素(interferon,IFN)-γ诱导肾小管上皮细胞表达趋化因子的影响,及过氧化小体增殖剂激活型受体-γ(peroxisome proliferator activated receptor-gamma,PPAR-γ)激动剂15-脱氧-Δ(12,14)-前列腺素J2(15-deoxy-Δ12,14-prostaglandin J2,15d-PGJ2)抑制趋化因子表达的机制。方法 :将肾小管上皮细胞HK-2分为空白组、TNF-α联合IFN-γ刺激组(刺激组)、NF-κB的特异性抑制剂吡咯烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate,PDTC)干预组及15d-PGJ2干预组。实时荧光定量PCR检测各组趋化因子CXCL9、CXCL10、CXCL11 m RNA的相对表达量;ELISA检测各组上清液中趋化因子CXCL9、CXCL10、CXCL11的蛋白分泌量;Western blot检测空白组、刺激组及15d-PGJ2干预组中NF-κB信号通路相关蛋白p65、IκBα及其磷酸化蛋白p-p65、p-IκBα的表达量。结果 :TNF-α联合IFN-γ刺激HK-2细胞后,CXCL9、CXCL10、CXCL11 m RNA表达量及蛋白分泌量显著增高(P<0.05);而经PDTC预处理后,趋化因子CXCL9、CXCL10、CXCL11的m RNA相对表达量分别下降了85.44%、45.94%、45.03%(P<0.05),蛋白分泌量分别下降了60.87%、47.59%及53.42%(P<0.05);15d-PGJ2预处理后,CXCL9、CXCL10、CXCL11的m RNA相对表达量分别下降了93.87%、92.40%、86.81%(P<0.01),蛋白分泌量分别下降了49.74%、67.13%、62.39%(P<0.01),且Western blot结果显示HK-2细胞中p65及IκBα蛋白的磷酸化水平与刺激组相比明显降低(P<0.01)。结论 :抑制NF-κB活性使HK-2细胞分泌趋化因子CXCL9、CXCL10、CXCL11减少;15d-PGJ2通过抑制NF-κB信号通路的活化,下调趋化因子CXCL9、CXCL10、CXCL11的表达。

Objective：To study the effect of inhibiting nuclear transcription factor- kappa B （NF-κB） on expression of chemokines in renal tubular epithelial cells induced by tumor necrosis factor （TNF）-α combined with interferon （IFN）-γ and the mechanism of 15-deoxy-Δ12,14-prostaglandin J2 （15d-PGJ2） of peroxisome proliferator activated receptor-gamma （PPAR-γ） inhibits the expression of chemokines. Methods：HK-2 cells were divided into four groups：the control group,the TNF-α plus with IFN-γ treatment group,the pyrrolidine dithiocarbamate （PDTC） treatment group and the 15d-PGJ2 treatment group. The expressions of CXCL9,CXCL10 and CXCL11 mRNA were measured by real-time quantitative PCR and the levels of CXCL9,CXCL10 and CXCL11 in culture supernatant were examined by ELISA. The expressions of p65,IκB-α and p-p65,and p-IκBα were measured by Western blotting assay. Results：The mRNA expression and protein secretion of CXCL9,CXCL10 and CXCL11 in HK-2 cells were up-regulated after TNF-α and IFN-γ stimulation （P 〈 0.05）. CXCL9,CXCL10 and CXCL11 were down-regulated by 85.44%,45.94% and 45.03% at mRNA level （P 〈 0.05） and 60.87%,47.59% and 53.42% at protein level （P 〈 0.05） when pretreated with PDTC;when pretreated with 15d-PGJ2,CXCL9,CXCL10 and CXCL11 were down-regulated by 93.87%,92.4% and 86.81% at mRNA level （P 〈 0.01） and 60.87%,47.59% and 53.42% at protein level （P 〈 0.01）,and Western blot results showed that the phosphorylation levels of p65 and IκBα were down-regulated （P 〈 0.01）. Conclusion：Inhibition of NF-κB decreases the expression of CXCL9,CXCL10 and CXCL11. The expressions of CXCL9,CXCL10 and CXCL11 could be inhibited by 15d-PGJ2 through the NF-κB signal pathway.