牛杀菌/通透性增加蛋白对脂多糖介导的炎性细胞因子表达的影响

Effects on the expression of lipopolysaccharide-induced inflammatory cytokines mediated by bovine bactericidal/ permeability-increasing protein

摘要:

杀菌/通透性增加蛋白 (Bactericidal/permeability-increasing protein, BPI) 能结合并特异地中和来自革兰氏阴性菌外膜的脂多糖 (Lipopolysaccharide, LPS)。为了研究牛源BPI蛋白及其 N端结构域在LPS介导的免疫应答中的作用，本文将BPI全长1 449 bp编码区序列 (BPI) 和其N端714 bp的编码区序列 (BPI714) 分别导入mHEK293细胞，分析了稳定表达的BPI或BPI714对LPS介导的炎性细胞因子表达的影响。首先将构建的pLEX-BPI/pLEX-BPI714载体分别转染mHEK293细胞，获得稳定表达牛源BPI或BPI714的mHEK293细胞；然后用LPS刺激上述细胞，分别收集刺激前、刺激后1 h、3 h、6 h、12 h、24 h、36 h和48 h的细胞，并同时收集未表达BPI或BPI714的 mHEK293细胞在各时间点的样品作为对照；采用定量RT-PCR检测上述细胞中炎性细胞因子IL-8、IL-1β、TNF-α、NF-κB-1、NF-κB-2的相对表达水平，比较LPS刺激前后表达BPI/BPI714和对照细胞中上述基因转录水平的变化规律。研究表明，LPS刺激后，对照细胞中IL-8、IL-1β、TNF-α、NF-κB-2表达水平在不同时间点均显著提高 (P<0.05)，并呈现规律性变化；而稳定表达BPI/BPI714的细胞在同样刺激条件下，IL-8、IL-1β、TNF-α、NF-κB-2基因的转录水平均未发生显著变化 (P>0.05)。根据我们的实验结果，在mHKE293细胞模型中BPI或BPI714均能显著降低LPS介导的炎性细胞因子表达，抑制LPS介导的免疫应答。这不仅为进一步研究BPI抑菌机制和利用其抑菌功能提供了可靠的实验依据，也为分析抗菌蛋白的抗菌效果提供了一种可靠的实验方法。

Abstract:

Bactericidal/permeability-increasing protein (BPI) can bind to and specifically neutralize lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria. In order to evaluate potent LPS-neutralizing activity of bovine BPI, the full-length coding sequence (1 449bp) or 714 bp N-terminal coding sequence (BPI714) of bovine BPI was transfected into mHEK293 cells and the expression of LPS-induced inflammatory cytokines was studied. First, we constructed the lentiviral expression vectors and generated mHEK293 cells stably expressing recombinant bovine BPI or BPI714. Then, we detected the expression of IL-8, IL-1β, TNF-α, NF-κB-1 and NF-κB-2 genes by real-time PCR at 0, 1, 3, 6, 12, 24, 36 and 48 h post of LPS induction in cells with or without recombinant bovine BPI or BPI714 ectopic expression, respectively. In response to LPS, the robust abundance of inflammatory cytokines including IL-8, IL-1β, TNF-α and NF-κB-2 was observed in wild type mHEK293 cells at eachtime point. On the contrary, mRNA abundance of IL-8, TNF-α and NF-κB-2 in transfected mHEK293 cells showed no significant changes at each indicated time point. Our results demonstrated that recombinant bovine full length BPI or BPI714 down-regulated the expression of inflammatory cytokines and revealed that either of bovine BPI or BPI714 was able to inhibit the immune respond stimulated by LPS. This study provides evidence for further investigating the mechanisms and application of BPI/LPS-neutralizing activity and also documents a reliable approach for analysis of the efficacy of antibacterial proteins.