背景:慢性前列腺炎/慢性盆腔疼痛综合征(CP/CPPS)在世界范围内非常普遍,饮酒是一个显著的促成因素。研究表明,肠道菌群可以受到饮酒的影响,并且是调节Th17细胞免疫的重要介质。然而,目前尚不清楚酒精加剧CP/CPPS的确切机制以及肠道菌群在这一过程中的作用。
方法:我们首先构建了最常用的CP/CPPS动物模型,实验性自身免疫性前列腺炎(EAP)模型,通过免疫测定。基于此,将小鼠分为EAP组和饮酒EAP组。通过16SrRNA测序和非靶向代谢组学分析,确定了两组之间不同的肠道微生物群及其代谢产物。随后,我们进行了针对胆固醇的代谢组学检测,以确定胆固醇的确切差异.此外,采用流式细胞术和免疫组织化学等多种方法检测Th17细胞的分化状态和27-羟胆固醇(差异胆固醇)及其上游调节因子-甾醇调节元件结合蛋白2(SREBP2)治疗前列腺炎的严重程度.最后,进行粪便移植以初步研究酒精摄入是否会加剧免疫受体小鼠的EAP。
结果:酒精摄入增加了Th17细胞的比例和相关炎症因子的水平。它还导致改变的肠道细菌丰富度和增加的肠道通透性。进一步的代谢组学分析显示,EAP与酒精喂养的EAP小鼠在多种代谢产物上存在显著差异。代谢途径富集分析表明,与胆固醇合成和代谢相关的途径显著富集,随后通过检测代谢酶的表达来证实。通过靶向胆固醇合成,酒精喂养的EAP小鼠中27-羟基胆固醇显着增加。随后的机制研究表明,补充27-羟基胆固醇可以在体内和体外加重EAP并促进Th17细胞分化,它由SREBP2调节。此外,我们观察到,酒精摄入小鼠的粪便移植会加重正常饮食的免疫受体小鼠的EAP。
结论:我们的研究首次表明酒精摄入促进Th17细胞分化,并通过微生物来源的胆固醇生物合成加剧EAP。
BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is very common worldwide, and alcohol consumption is a notable contributing factor. Researches have shown that gut microbiota can be influenced by alcohol consumption and is an important mediator in regulating Th17 cell immunity. However, it is still unclear the exact mechanism by which alcohol exacerbates the CP/CPPS and the role of gut microbiota in this process.
METHODS: We first constructed the most-commonly used animal model for CP/CPPS, the experimental autoimmune prostatitis (EAP) model, through immunoassay. Based on this, mice were divided into EAP group and alcohol-consuming EAP group. By 16S rRNA sequencing and non-targeted metabolomics analysis, differential gut microbiota and their metabolites between the two groups were identified. Subsequently, metabolomics detection targeting cholesterols was carried out to identify the exact difference in cholesterol. Furthermore, multiple methods such as flow cytometry and immunohistochemistry were used to detect the differentiation status of Th17 cells and severity of prostatitis treated with 27-hydroxycholesterol (the differential cholesterol) and its upstream regulatory factor-sterol regulatory element-binding protein 2 (SREBP2). Lastly, fecal transplantation was conducted to preliminary study on whether alcohol intake exacerbates EAP in immune receptor mice.
RESULTS: Alcohol intake increased the proportion of Th17 cells and levels of related inflammatory factors. It also led to an altered gut bacterial richness and increased gut permeability. Further metabolomic analysis showed that there were significant differences in a variety of metabolites between EAP and alcohol-fed EAP mice. Metabolic pathway enrichment analysis showed that the pathways related to cholesterol synthesis and metabolism were significantly enriched, which was subsequently confirmed by detecting the expression of metabolic enzymes. By targeting cholesterol synthesis, 27-hydroxycholesterol was significantly increased in alcohol-fed EAP mice. Subsequent mechanistic research showed that supplementation with 27-hydroxycholesterol could aggravate EAP and promote Th17 cell differentiation both in vivo and in vitro, which is regulated by SREBP2. In addition, we observed that fecal transplantation from mice with alcohol intake aggravated EAP in immunized recipient mice fed a normal diet.
CONCLUSIONS: Our study is the first to show that alcohol intake promotes Th17 cell differentiation and exacerbates EAP through microbiota-derived cholesterol biosynthesis.