Gong Qiming, Jiang Yan, Lu Junling, You Yanwu
Department of Nephrology, Affiliated Hospital, Youjiang Medical University for Nationalities, Baise 533000, China.
Science Laboratory Center, Youjiang Medical University for Nationalities, Baise 533000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Dec 30;40(12):1726-1731. doi: 10.12122/j.issn.1673-4254.2020.12.05.
To explore the mechanism by which fractalkine (CX3CL1; FKN) inhibits lipopolysaccharide (LPS)-induced immunological response in RAW264.7 cells.
A RAW264.7 cell model overexpressing FKN was established by transfection with the lentiviral vector CX3CL1. The effects of LPS, ICG-001 (a Wnt/β-catenin signaling pathway inhibitor), either alone or in combination, on M1 polarization of na?ve and FKN-overexpressing RAW264.7 cells were evaluated by detecting of intereukin-6 (IL-6) and tumor necrosis factor-α (TNF-) using ELISA. The protein expressions of the inflammatory factors (iNOS, TNF-, and IL-6), FKN, Wnt-4, and β-catenin were detected by Western blotting. The subcellular localization of IL-6 in the cells was detected by immunofluorescence assay.
The RAW264.7 cell model of FKN overexpression was successfully established. In na?ve RAW264.7 cells, treatment with both ICG-001 and LPS, as compared with LPS alone, significant promoted TNF- and IL-6 secretions, increased intracellular levels of TNF-, IL-6 and iNOS ( < 0.05), and reduced intracellular FKN, Wnt-4 and β-catenin levels ( < 0.01). In FKN-overexpressing RAW264.7 cells, LPS treatment significantly reduced the secretion of TNF- and IL-6 and intracellular levels of TNF-, IL-6 and iNOS ( < 0.01), increased intracellular FKN, Wnt-4 and β-catenin protein contents ( < 0.01), and inhibited IL-6 localization in the cytoplasm; compared with LPS, the combined treatment with ICG-001 and LPS obviously enhanced IL-6 localization in the cytoplasm of the cells.
FKN overexpression suppresses LPS-induced M1 type polarization of RAW264.7 cells by activating Wnt/β-catenin signaling pathway.
探讨趋化因子（CX3CL1；FKN）抑制脂多糖（LPS）诱导的RAW264.7细胞免疫反应的机制。
通过慢病毒载体CX3CL1转染建立过表达FKN的RAW264.7细胞模型。通过ELISA检测白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α），评估LPS、ICG-001（一种Wnt/β-连环蛋白信号通路抑制剂）单独或联合作用对未处理及过表达FKN的RAW264.7细胞M1极化的影响。通过蛋白质印迹法检测炎症因子（诱导型一氧化氮合酶、TNF-α和IL-6）、FKN、Wnt-4和β-连环蛋白的蛋白表达。通过免疫荧光法检测细胞中IL-6的亚细胞定位。
成功建立了过表达FKN的RAW264.7细胞模型。在未处理的RAW264.7细胞中，与单独使用LPS相比，ICG-001和LPS联合处理显著促进了TNF-α和IL-6的分泌，增加了细胞内TNF-α、IL-6和诱导型一氧化氮合酶的水平（P<0.05），并降低了细胞内FKN、Wnt-4和β-连环蛋白的水平（P<0.01）。在过表达FKN的RAW264.7细胞中，LPS处理显著降低了TNF-α和IL-6的分泌以及细胞内TNF-α、IL-6和诱导型一氧化氮合酶的水平（P<0.01），增加了细胞内FKN、Wnt-4和β-连环蛋白的蛋白含量（P<0.01），并抑制了IL-6在细胞质中的定位；与LPS相比，ICG-001和LPS联合处理明显增强了IL-6在细胞质中的定位。
FKN过表达通过激活Wnt/β-连环蛋白信号通路抑制LPS诱导的RAW264.7细胞M1型极化。
