乳铁蛋白对脂多糖激活的U937细胞TLR4信号通路相关膜分子表达的影响

Effects of lactoferrin on the expression of TLR4 signaling pathway-associated membrane molecule in LPS-activated U937 macrophages

摘要:

目的：研究重组人乳铁蛋白（recombinant human lactoferrin，rhLF）对脂多糖（lipopolysaccharide，LPS）激活的 U937细胞Toll样受体4（toll-like receptors 4，TLR4）信号通路相关膜分子 TLR4、CD14、MD2表达的影响。方法：经佛波酯（phorbol 12-myristate 13-acetate，PMA）诱导成熟的 U937细胞随机分为6组：对照组、LPS组（给予100 ng/ml LPS）、LPS+50 μg/ml rhLF组、LPS+100 μg/ml rhLF组、LPS+200 μg/ml rhLF组、LPS+500 μg/ml rhLF组，采用 RT-PCR和 Ｗestern blot检测各组细胞 TLR4、CD14、MD2 mRNA和蛋白的表达，ELISA检测各组细胞上清液中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的含量。结果：LPS组U937细胞 TLR4、CD14、MD2 mRNA和蛋白的表达均明显高于对照组（P＜0.05），LPS+50 μg/ml rhLF组三者mRNA和蛋白的表达与 LPS组差异不明显（P ＞0.05），LPS+100 μg/ml rhLF组、LPS+200 μg/ml rhLF组、LPS+500 μg/ml rhLF组均明显降低了 LPS诱导的 TLR4、CD14、MD2 mRNA和蛋白的表达（P＜0.05）；LPS组 TNF-α的含量明显高于对照组（P＜0.05），LPS+50 μg/ml rhLF组、LPS+100 μg/ml rhLF组、LPS+200 μg/ml rhLF组、LPS+500 μg/ml rhLF组均明显降低了 LPS诱导的 TNF-α的产生（P＜0.05）。结论：rhLF通过抑制 TLR4介导的 LPS跨膜信号转导和炎症因子 TNF-α的分泌，对 LPS所致的脓毒血症具有潜在的治疗作用。

Abstract:

Objective：To investigate the effects of recombinant human lactoferrin（rhLF） on the expression of toll-like receptors 4 （TLR4） signaling pathway-associated membrane molecule（TLR4，CD14 and MD2） in lipopolysaccharide（LPS）-activated U937 macrophages. Methods：U937 was induced to maturation by phorbol 12-myristate 13-acetate（PMA） and then divided into six groups：control group，LPS group（100 ng/ml LPS），LPS+50 μg/ml rhLF group，LPS+100 μg/ml rhLFgroup，LPS+200 μg/ml rhLF group and LPS+500 μg/ml rhLF group. The mRNA and protein expressions of TLR4，CD14 and MD2 were determined by RT-PCR and Western blot. The concentration of tumor necrosis factor-α（TNF-α） was detected by ELISA. Results：The mRNA and protein expres－sion of TLR4，CD14 and MD2 increased significantly in LPS group than in control group（P<0.05）. The mRNA and protein expression of TLR4，CD14 and MD2 had no significant change in LPS+50 μg/ml rhLF group（P >0.05），but reduced significantly in LPS+100 μg/ml rhLF group，LPS+200 μg/ml rhLF group and LPS+500 μg/ml rhLF group than in LPS group（P<0.05）. The concentration of TNF-α increased significantly in the LPS group than in the control group（P<0.05）. The levels of TNF-α reduced significantly in LPS+50 μg/ml rhLF group，LPS+100 μg/ml rhLF group，LPS+200 μg/ml rhLF group and LPS+500 μg/ml rhLF group than in LPS group（P<0.05）. Conclusion：rhLF might have a potential protective effect on LPS induced sepsis through suppressing TLR4 transmembrane signal transduction pathway and reducing the secretion of TNF-α.