变形链球菌细胞壁对EAhy926细胞TLR4、IL-6和IL-8表达的影响

Effects of the cell wall of Streptococcus mutans on the expression of TLR4、IL-6 and IL-8 by EAhy926 cells

摘要:

摘要：【目的】观察变形链球菌细胞壁对EAhy926细胞的增殖活性、TLR4的表达和炎性细胞因子IL-6和IL-8分泌的影响，初步探讨变形链球菌致血管内皮细胞TLR4表达、炎症反应及其两者之间的关系。【方法】变形链球菌细胞壁作用于EAhy926细胞，MTT法检测EAhy926细胞的增殖活性；RT-PCR法检测EAhy926细胞TLR4、IL-6和IL-8 mRNA的表达；流式细胞术检测EAhy926细胞表面TLR4的表达；细胞生物活性方法和ELISA分别检测EAhy926细胞IL-6和IL-8的分泌；抗体阻断实验观察IL-6和IL-8的表达与TLR4的关系。【结果】不同浓度的变形链球菌细胞壁作用6 h可促进EAhy926细胞的增殖(P<0.05)，但12 h后可明显抑制该细胞的生长(P<0.05)，呈现显著的时间和剂量依赖性。变形链球菌细胞壁作用于EAhy926细胞后，TLR4 mRNA和蛋白水平的表达量随着作用时间延长而逐渐增高，在16 h达到高峰，24 h后又逐渐下降(P<0.01)；IL-6和IL-8的表达也呈现明显的时间依赖性增高(P<0.05)。经TLR4抗体阻断后，变形链球菌细胞壁刺激EAhy926细胞IL-6和IL-8的产生明显减少(P<0.01)。【结论】变形链球菌细胞壁可明显抑制EAhy926细胞的生长，上调该细胞TLR4的表达，促进炎性细胞因子IL-6和IL-8的分泌；IL-6和IL-8的产生与TLR4的表达上调密切相关。

Abstract:

Abstract: [Objective] To investigate the effects of the cell wall of Streptococcus mutans on the expression of Toll-like receptor 4 (TLR4), IL-6 and IL-8 in EAhy926 cells (The human endothelial hybridoma of human umbilical vein endothelial cells (HUVEC) and the human epithelial cell line A549, characterized by endothelial phenotype and biology). [Methods] EAhy926 cells were treated with different concentrations of the cell wall of Streptococcus mutans. The proliferation of EAhy926 cells was determined by MTT assay. The mRNA expression of TLR4, IL-6 and IL-8 in EAhy926 cells was detected by reverse transcription polymerase chain reaction (RT-PCR). TLR4 was analyzed by flow cytometry. The production of IL-6 and IL-8 in the cultured supernatants was measured by biochemical method and ELISA respectively. TLR4 blocking assay was used to investigate the relationship between IL-6, IL-8 and TLR4 mRNA expression. [Results] The proliferation of EAhy926 cells was significantly enhanced by the cell wall of Streptococcus mutans at 6 h (P<0.05), and inhibited after 12 h (P<0.05), both time- and dose-dependent. The expression of mRNA and protein for TLR4 in EAhy926 cells was increased after stimulated by the cell wall of Streptococcus mutans, peaking at 16 h (P<0.05), and then decreased. The expression of IL-6 and IL-8 mRNA was significantly induced when exposed to the cell wall, reaching the maximal level at 16 h and 24 h, respectively (P<0.05). Meanwhile, the cell wall of Streptococcus mutans induced the production of IL-6 and IL-8 (P<0.05). The expression of IL-6 and IL-8 mRNA in EAhy926 cells was blocked by anti-human TLR4 antibodies significantly. [Conclusion] The cell wall of Streptococcus mutans upregulated the expression of TLR4 and induced the production of inflammatory cytokines IL-6 and IL-8, indicating that the expression of TLR4 of EAhy926 cells may elicit a TLR4-mediated innate immune response and contribute to production of inflammatory cytokines IL-6 and IL-8