Alcohol promotes macrophage M2b polarization in colitis by modulating the TRPV1-MAPK/NF-kB pathways

Macrophages are key immune cells in inflammatory bowel disease (IBD) that can exhibit different phenotypes and promote inflammation. Alcohol consumption is associated with increased risk and severity of IBD. We performed in vitro and in vivo experiments with dextran sodium sulfate-induced mice colitis model, peritoneal macrophages and RAW264.7 cell line to investigate the effect and mechanism of alcohol on macrophages in colitis. Our study has found that alcohol exacerbates colitis in mice, increases the aggregation of colonic macrophages, and promotes the production of various inflammatory factors. Alcohol increases lipopolysaccharides (LPS)-induced calcium influx in macrophages, which is inhibited by the TRPV1 antagonist Capsazepine (CPZ). Alcohol and LPS together induce M2b polarization of macrophages and promote the phosphorylation and nuclear translocation of P38, ERK1/2 and NF-?B, and could be inhibited by CPZ and NOD2 inhibitor. Thus, alcohol can aggravate experimental colitis in mice and promote macrophage M2b polarization through the TRPV1-MAPK/NF-kB pathways. Overall design: Peritoneal macrophages used for RNA-seq were cultured with RPMI 1640 with 1% streptomycin and penicillin for 1 h to keep the cell state consistent and stimulated by LPS 1µg/mL, LPS 1µg/mL with 5% EtOH, LPS 1µg/mL with CPZ 100 µM for 4 h.

巨噬细胞（Macrophages）是炎症性肠病（inflammatory bowel disease, IBD）中的关键免疫细胞，可呈现不同表型并促进炎症发生。饮酒与IBD的发病风险升高及病情严重程度增加相关。本研究采用葡聚糖硫酸钠诱导的小鼠结肠炎模型、腹腔巨噬细胞及RAW264.7细胞系开展体外与体内实验，以探究酒精对结肠炎中巨噬细胞的作用及机制。

本研究发现，酒精可加重小鼠结肠炎，增加结肠巨噬细胞的聚集，并促进多种炎性细胞因子的产生。酒精可增强脂多糖（lipopolysaccharides, LPS）诱导的巨噬细胞钙内流，该效应可被TRPV1拮抗剂辣椒平（Capsazepine, CPZ）抑制。酒精与LPS共同诱导巨噬细胞发生M2b极化，并促进P38、ERK1/2及NF-κB的磷酸化与核转位，上述效应可被CPZ及NOD2抑制剂阻断。综上，酒精可加重小鼠实验性结肠炎，并通过TRPV1-MAPK/NF-κB通路促进巨噬细胞M2b极化。

实验设计：用于RNA测序的腹腔巨噬细胞采用含1%链霉素与青霉素的RPMI 1640培养基培养1小时以维持细胞状态一致性，随后分别用1μg/mL LPS、1μg/mL LPS联合5%乙醇（EtOH）、1μg/mL LPS联合100μM CPZ刺激4小时。