幽门螺杆菌感染对胃内微环境（胃酸-硝酸盐还原菌-亚硝基化合物）的影响及其在胃黏膜病变中的作用

It has been documented that Helicobacter pylori (Hp) infection alters gastric microbiota compositions; this dysbiosis of gastric microbiota further promotes the pathogenicity of Hp. However, the critical bacterial species or group that influences Hp-induced gastric mucosal lesion and the underlying mechanisms are unclear. Previously, we found that the relative abundance of the nitrate-reducing bacteria (NBs) was increased in patients with Hp infection. Moreover, the NBs as well as the nitrate reductase functions were significantly enriched in patients with gastric cancer compared to chronic gastritis. Thus we hypothesize that Hp infection disturbs the balance of intrinsic microbial community structures in the stomach, especially the increase of opportunistic pathogens NBs. Over the duration of Hp infection, the Hp-associated lower gastric acid secretion promotes the overgrowth of NBs which in turn increase production of their metabolites, namely N-nitroso compounds (NOC). Accumulation of NOC could then impair the DNA damage repair system and subsequently lead to the malignant transformation of gastric mucosa. To test this hypothesis, this project will be designed to examine the changes of gastric microenvironment (NBs, gastric acid and NOC) across different stages of gastric lesions to define the association between microenvironment perturbation and the progression of gastric lesions. Moreover, we will plan to elucidate a possible role of the micro-environmental factors in modulating Hp-induced gastric mucosal pathology and the underlying mechanisms in vivo. This project, which is distinct from previous studies focusing on the virulence factors of Hp, will harness the gastric microenvironment as a breakthrough point to explore new insights into Hp-induced gastric carcinogenesis.

新近发现幽门螺杆菌（Hp）感染可影响胃内其他共栖菌，胃内菌群失衡又进一步促进Hp诱导的胃黏膜病变，但发挥关键作用的菌群及其致病机制尚不清楚。我们前期研究发现Hp感染患者胃内硝酸盐还原菌（NBs）增加；而且胃癌患者NBs丰度又高于胃炎，其硝酸盐还原功能相关通路活性也明显升高。据此我们推测Hp感染打破胃内固有菌群平衡使潜在致病菌NBs增加，随着感染时间延长，Hp相关低胃酸分泌又促进NBs过度繁殖，使其代谢产物N-亚硝基化合物（NOC）大量蓄积，诱发DNA损伤和修复不全，最终导致胃黏膜恶性转化。本研究拟先观察Hp感染不同病变阶段胃内微环境（胃酸、NBs和NOC）的改变，明确微环境紊乱与病变的相关性；随后通过体内试验深入探讨胃内微环境失衡在病变中的作用及机制。既往Hp致病性研究多集中于Hp本身毒力因子，本课题以Hp感染影响胃内微环境为切入点，从菌群失衡的角度展开，结果将为Hp致病机制提供新思路。

幽门螺杆菌（Hp）是胃内常见的致病菌，新近研究发现除Hp外胃内还存在其他微生物，且与胃癌发生密切相关。本研究收集胃炎、肠化和胃癌患者的胃黏膜和胃液样本，应用16S rRNA高通量测序进行微生物多样性分析，结果发现胃内细菌主要包括厚壁菌门、变形菌门、拟杆菌门等，胃黏膜和胃液菌群结构具有显著差异，黏膜中Helicobacter、Lactococcus、Bacillus丰度明显高于胃液，而Neisseria、Haemophilus、Streptococcus则明显低于胃液。Hp感染组胃内菌群结构明显不同于未感染组，且Helicobacter丰度与物种多样性呈显著负相关。胃癌患者Helicobacter丰度明显低于胃炎组。不同病变阶段患者胃内菌群结构具有显著差异，随机森林法筛选胃癌相关标志物，鉴定的7个黏膜细菌（Lactobacillus, Gemella, Enterococcus等）和13个胃液细菌（Lactobacillus, Filifactor, Staphylococcus等）的ROC预测值分别为94%和83%。进一步应用来自青岛市立医院外部数据验证，结果显示其AUC分别为84%和89%，提示其对胃癌具有良好的预测价值。配对分析显示，黏膜和胃液菌群差异在胃癌组较胃炎组明显减小，尤其是黏膜中产亚硝酸盐菌丰度明显升高，接近于胃液水平。进一步通过构建Hp感染动物模型，观察补充益生菌（唾液乳杆菌和鼠李糖乳杆菌）对胃肠道菌群的调节作用及Hp感染诱导胃黏膜病变的影响。结果显示，Hp感染16周后益生菌组较对照组肠化和异型增生的发生率明显降低，转录组测序共筛选到942个差异基因，包括324个上调基因和618个下调基因。GSEA分析显示，差异基因主要富集在炎症相关信号通路如NF-kappa B、Toll-like receptor signaling pathway、TNF signaling pathway。RT2 Profiler PCR Array分析Hp感染组较未感染组上调基因如Il17a、Cxcl3、Ifng等在益生菌组明显下调。益生菌组胃内菌群结构与对照组具有显著差异。本研究提示胃液中的致病菌可能通过移位至黏膜从而促进胃癌发生，调节胃肠道菌群的益生菌可减轻胃黏膜病变。