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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 investigate the effect of inhibiting the activity of nuclear transcription factor-kappa B (NF-κB) on renal tubular epithelial cells induced by tumor necrosis factor (TNF) -α and interferon (IFN) -γ And the expression of chemokines, and the effect of 15-deoxy-Δ (12,14) -prostaglandin J2 (PPAR-γ) agonist -deoxy-Δ12, 14-prostaglandin J2, 15d-PGJ2) inhibit the expression of chemokines. Methods: The renal tubular epithelial cells HK-2 were divided into blank group, TNF-α combined with IFN-γ stimulation group (stimulation group), the specific inhibitor of NF-κB pyrrolidine dithiocarbamate PDTC intervention group and 15d-PGJ2 intervention group. Real-time fluorescence quantitative PCR was used to detect the relative expression of chemokines CXCL9, CXCL10 and CXCL11 m RNA in each group; ELISA was used to detect the protein secretion of chemokines CXCL9, CXCL10 and CXCL11 in each group; Western blot was used to detect the expression of CXCL9, The expression of p65, IκBα and phosphorylated protein p-p65, p-IκBα in NF-κB signaling pathway group and 15d-PGJ2 intervention group were observed. Results: The expressions of CXCL9, CXCL10 and CXCL11 mRNA and protein were significantly increased in HK-2 cells stimulated with TNF-α and IFN-γ (P <0.05). After pretreatment with PDTC, the chemotactic factors CXCL9 and CXCL10 , And the expression of m RNA in CXCL11 decreased by 85.44%, 45.94% and 45.03%, respectively (P <0.05), and protein secretion decreased by 60.87%, 47.59% and 53.42% The relative expression levels of m RNA in CXCL9, CXCL10 and CXCL11 decreased by 93.87%, 92.40% and 86.81% (P <0.01), and the protein secretion decreased by 49.74%, 67.13% and 62.39%, respectively The results of Western blot showed that phosphorylation levels of p65 and IκBα in HK-2 cells were significantly lower than those in stimulation group (P <0.01). Conclusion: Inhibition of NF-κB activity induces the secretion of chemokines CXCL9, CXCL10 and CXCL11 in HK-2 cells. 15d-PGJ2 down-regulates the expression of chemokines CXCL9, CXCL10 and CXCL11 by inhibiting the activation of NF-κB signaling pathway.