CATH-B1抑制肠外致病性大肠杆菌诱导小胶质细胞炎性反应的作用研究

CATH-B1 inhibits extraintestinal pathogenic Escherichia coli-induced inflammatory response in microglia

摘要:

【目的】探究抗菌肽CATH-B1对肠外致病性大肠杆菌RS218感染诱导小胶质细胞(BV2细胞)炎症反应的影响及作用机制。【方法】使用RS218感染小胶质细胞作为炎症模型，试验分为Mock组、RS218感染组和CATH-B1预处理+RS218感染组。采用Cell Counting Kit-8 (CCK8)试剂盒检测细胞活力，通过菌落计数法检测CATH-B1对细菌生长的影响和细菌的黏附入侵，酶联免疫吸附测定法检测炎症因子白细胞介素(interleukin, IL)-1β、IL-6、IL-12和肿瘤坏死因子-α (tumor necrosis factor α, TNF-α)含量，实时荧光定量PCR (quantitative real-time PCR, RT-PCR)检测IL-1β和IL-6 mRNA表达水平，蛋白质印迹分析(Western blotting)法检测细胞中转录因子蛋白家族核因子κB (nuclear factor kappa-B, NF-κB) P65和P-P65、丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK) ERK和P-ERK的蛋白表达水平。【结果】CATH-B1显著降低了RS218诱导的促炎细胞因子IL-1β、IL-6和IL-12的水平，同时显著抑制IL-1β和IL-6 mRNA表达，尽管CATH-B1对细菌的黏附入侵无显著影响，但CATH-B1能够显著抑制P65和ERK磷酸化蛋白的表达。【结论】CATH-B1通过抑制NF-κB和MAPK信号通路的活化来发挥抑制炎症作用，为阐明抗菌肽抗神经炎症机制提供了重要依据。

Abstract:

[Objective] To explore the effect and mechanism of the antimicrobial peptide CATH-B1 on extraintestinal pathogenic Escherichia coli (RS218)-induced inflammatory response in microglia. [Methods] We used RS218-infected mouse microglial BV2 cells as the inflammation model in vitro and set three groups: Mock, RS218 infection, and CATH-B1 pretreatment+RS218 infection. The Cell Counting Kit-8 (CCK-8) was used to determine cell viability. The colony counting assay was used to examine the growth, adhesion, and invasion of bacterial cells. Enzyme linked immunosorbent assay (ELISA) was employed to determine the concentrations of interleukin (IL)-1β, IL-6, IL-12, and tumor necrosis factor (TNF)-α in the supernatant of cell culture. Quantitative real-time PCR (RT-PCR) was performed to determine the mRNA levels of IL-1β and IL-6. Western blotting was employed to determine the protein levels of nuclear factor-kappa B (NF-κB) P65, the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), and their phosphorylated forms. [Results] CATH-B1 inhibited the RS218-induced secretion of IL-1β, IL-6, and IL-12 and mRNA expression of IL-1β and IL-6. However, CATH-B1 did not affect bacterial adhesion or invasion. In addition, CATH-B1 inhibited the expression of phosphorylated P65 and ERK. [Conclusion] CATH-B1 plays a vital role in reducing inflammation by inhibiting the activation of NF-κB and MAPK signaling pathways. The finding provides a basis for elucidating the mechanism of antimicrobial peptides against neuroinflammation.