乳源酪蛋白糖巨肽对NF-κB信号通路中关键蛋白的调控作用

摘要

以结肠癌细胞HT-29为细胞系,在确定乳源性酪蛋白糖巨肽(casein glycomacropeptide,CGMP)对脂多糖(lipopolysaccharides,LPS)诱导的HT-29细胞核因子-κB(nuclear factor-κB,NF-κB)亚单位p65蛋白影响的基础上,在最适作用时间条件下,利用Western blotting技术进一步检测乳源CGMP对NF-κB信号通路上关键蛋白IκBα、p-IκBα、E3RSIκB、UBC5表达水平的影响,以阐述乳源CGMP调控NF-κB信号通路中关键蛋白的作用机制.结果表明:乳源CGMP组的3种质量浓度(0.001、0.010、0.100μg/mL)均可在一定程度上抑制LPS诱导的HT-29细胞NF-κB信号通路上IκBα蛋白的降解,0.100μg/mL作用较为明显,与空白对照组比较有显著性差异(P<0.01).研究明确地证实了乳源CGMP可通过抑制p-IκBα、E3RSIκB、UBC5蛋白的表达来抑制IκBα蛋白的降解,进而抑制NF-κB信号通路的激活.结论:乳源CGMP可显著降低NF-κB信号通路关键蛋白IκBα的降解,其机制是抑制了IκBα的磷酸化和泛素化,使p-IκBα和泛素化关键蛋白E3RSIκB和UBC5的表达均有所下降,进而减少了IκBα的降解,增加了IκBα-p65-p50蛋白三聚体的数量,使p65蛋白核移位效应降低,进而减少下游基因的表达.因此,研究结果科学地阐释了乳源CGMP是通过调控NF-κB信号通路发挥抗炎的作用.%This study was undertaken to determine the effect of casein glycomacropeptide (CGMP) on the p65 subunit of nuclear factor-κB (NF-κB) of HT-29 human colon cancer cells challenged with lipopolysaccharide (LPS). Furthermore, Western blotting technology was used to detect and compared the expression levels of the key proteins involved in the NF-κB signaling pathway such as IκBα, p-IκBα, E3RSIκB and UBC5 in the control group, milk-derived CGMP group and LPS group to uncover the molecular mechanism of CGMP in regulating the NF-κB signaling pathway. The results showed that all three doses of CGMP (0.001, 0.010 and 0.100 μg/mL) could suppress the degradation of IκBα in some degree, which is involved in the NF-κB signaling pathway of HT-29 cells challenged with LPS and this effect was more significant at the dose of 0.100 μg/mL, which was significantly different when compared with the control group (P < 0.01). Therefore, it was confirmed that CGMP could suppress the degradation of IκBα by repressing the expression of p-IκBα, E3RSIκB and UBC5, thereby inhibiting the activation of the NF-κB signaling pathway. As a result, CGMP can significantly reduce the degradation of IκBα in the NF-κB signal pathway, increase the amount of protein trimer-IκBα-p65-p50 and decrease nuclear translocation of p65 and downstream gene expression by inhibiting the phosphorylation and ubiquitination of IκBα and reducing the expression of p-IκBα and key ubiquitin proteins (E3RSIκB and UBC5). This study illustrates that milk-derived CGMP plays an anti-inflammatory role by regulating the NF-κB signal pathway.