背景:巨噬细胞在炎症性肠病(IBD)中表现出不同的表型,并根据其极化状态促进炎症或组织修复。酒精是药物制剂中广泛使用的溶剂,和它的消费与结肠炎的风险增加有关;然而,其对IBD中巨噬细胞的影响尚不清楚。
目的:本研究旨在研究酒精对葡聚糖硫酸钠(DSS)诱导的结肠炎巨噬细胞的影响,并了解其作用机制。
方法:将DSS处理的C57BL/6小鼠暴露于不同浓度的酒精,瞬时受体电位香草素1(TRPV1)拮抗剂,和5-氨基水杨酸。远端结肠被切除,固定,染色,并进行了组织学分析,通过苏木精和伊红(H&E)染色和免疫荧光染色。比率[Ca2+]i测量值,西方印迹,定量聚合酶链反应,细胞因子测量,和RNA测序分析也进行了。腹膜巨噬细胞和RAW264.7细胞用于体外实验,并进行了各种试验来评估细胞反应,基因表达,和信号通路。
结果:酒精加剧了DSS治疗的小鼠结肠炎,并促进了结肠巨噬细胞分泌各种炎性细胞因子。酒精增强腹膜巨噬细胞中脂多糖(LPS)诱导的钙离子内流,而TRPV1拮抗剂卡萨西平(CPZ)抑制LPS和/或酒精诱导的巨噬细胞钙内流。酒精和LPS激活MAPK/P38,MAPK/ERK,和NF-κB信号通路,并诱导巨噬细胞M2b极化,导致炎症细胞因子如Tnf的表达水平增加,Il1b,和Il10。此外,CPZ可以抑制酒精或LPS对上述通路和炎症因子的促进作用,逆转巨噬细胞M2b极化和促进酒精诱导的结肠炎。含有2(NOD2)的核苷酸结合寡聚化结构域的抑制部分地抑制了醇和LPS对巨噬细胞的作用。
结论:酒精加重实验性结肠炎,并通过TRPV1-MAPK/NF-κB诱导巨噬细胞M2b极化。我们的研究通过阐明TRPV1在酒精加剧性结肠炎中的作用,为IBD治疗的潜在治疗靶点提供了新的见解。使用CPZ作为潜在的治疗选择。识别瞬时受体电位锚蛋白亚型1(TRPA1)作为治疗靶标扩大了未来的研究范围。
BACKGROUND: Macrophages exhibit different phenotypes in inflammatory bowel disease (IBD) and promote inflammation or tissue repair depending on their polarization state. Alcohol is a widely used solvent in pharmaceutical formulations, and its consumption is associated with an increased risk of colitis; however, its effects on macrophages in IBD remain poorly understood.
OBJECTIVE: This study aimed to investigate the effect of alcohol on macrophages in dextran sodium sulfate (DSS)-induced colitis and understand the underlying mechanisms.
METHODS: DSS-treated C57BL/6 mice were exposed to varying concentrations of alcohol, transient receptor potential vanilloid 1 (TRPV1) antagonist, and 5-aminosalicylic acid. The distal colon was resected, fixed, stained, and histologically analyzed, through hematoxylin and eosin (H&E) staining and immunofluorescence staining. Ratio [Ca2+]i measurements, western blotting, quantitative polymerase chain reaction, cytokine measurements, and RNA sequencing analyses were also performed. Peritoneal macrophages and RAW264.7 cells were used for in vitro experiments, and various assays were performed to evaluate cellular responses, gene expression, and signaling pathways.
RESULTS: Alcohol exacerbated DSS-treated mice colitis and promoted the secretion of various inflammatory cytokines from colonic macrophages. Alcohol enhances the calcium ion influx induced by lipopolysaccharide (LPS) in peritoneal macrophages, while the TRPV1 antagonist capsazepine (CPZ) inhibits LPS- and/or alcohol- induced calcium influx in macrophages. Alcohol and LPS activate the MAPK/P38, MAPK/ERK, and NF-κB signaling pathways and induce the macrophage M2b polarization, resulting in the increased expression level of inflammatory cytokines such as Tnf, Il1b, and Il10. Additionally, CPZ can inhibit the facilitatory effects of alcohol or LPS on the abovementioned pathways and inflammatory factors, reversing macrophage M2b polarization and promoting alcohol-induced colitis. The inhibition of nucleotide binding oligomerization domain containing 2 (NOD2) partially suppressed the alcohol and LPS effects on macrophages.
CONCLUSIONS: Alcohol exacerbates experimental colitis and induces M2b polarization of macrophage via TRPV1-MAPK/NF-κB. Our study provides new insights into the potential therapeutic targets for IBD treatment by elucidating the role of TRPV1 in alcohol-exacerbated colitis, using CPZ as a potential therapeutic option. The identification of transient receptor potential ankyrin subtype 1 (TRPA1) as a therapeutic target expands the scope of future research.