大肠湿热证结肠定植菌群和SIgA互作及郁金散的靶向调控机制研究

Large intestine dampness-heat syndrome (LIDHS) is commonly seen in livestock, poultry and human diarrheal diseases. And Yujin Powder (YJP) is one of the most classical prescription for treating LIDHS and has good effects. It has been proved that the homeostasis between intestinal flora and SIgA is significant to the intestinal health. Our previous results showed that the diversity of fecal flora and SIgA secretion were both abnormal in LIDHS. Therefore, this project is focused on the interaction between colonized bacterial flora and SIgA in colon in LIDHS and the target regulation mechanism of Yujin Powder. The change characteristics of colonized bacterial flora in colon mucosa of every group will be accurately analyzed and the functional genes are mined through metagenomics; the characteristics of distribution, density and expression of SIgA, its secretory cell and transport receptor pIgR in colon mucosa of every group are quantitatively analyzed through immunological and molecular biology methods; the network mechanism between the changed bacterial flora and the potential functional genes and SIgA and its regulate factors will be explored based on data mining and interaction network analysis; the target regulation mechanism of YJP on intestinal flora and the secretion of SIgA during the course of treating LIDHS will be analyzed. It is hoped that the interaction relationship between colonized bacterial flora and SIgA in colon in the course of LIDHS and the target regulation mechanism of YJP will be revealed, which will provide evidence for elucidating and abundancing the pathogenesis of LIDHS and guiding and developing the clinical drugs.

大肠湿热证在畜禽及人腹泻性疾病中常见多发。郁金散为治疗该证的经典常用方剂，且疗效较好。当前已证实，肠道菌群与SIgA之间维持稳态对肠道健康具有重要意义。我们前期研究显示，该证中存在粪便菌群多样性与SIgA分泌异常现象。因此，本项目重点研究该证结肠定植菌群和SIgA的互作及郁金散的靶向调控机制。通过宏基因组学精准分析各组结肠黏膜定植菌群的变化特征并挖掘其潜在功能基因；通过免疫及分子生物学技术，定量分析各组结肠黏膜SIgA及分泌细胞和其转运受体pIgR的分布及密度、表达特征；基于数据挖掘和互作网络分析，探索变化菌群及潜在功能基因与SIgA及其调节因子之间的网络机制；分析郁金散在治疗该证时对肠道菌群和SIgA分泌调控的靶向机制。以期揭示结肠定植菌群和SIgA在大肠湿热证发病过程中的相互关系及郁金散对其的靶向调控作用。为阐释与丰富大肠湿热证的发病机制，指导与开发临床治疗用药提供依据。

大肠湿热证是溃疡性结肠炎(UC)等肠道炎症性疾病最主要的证型之一，郁金散为治疗大肠湿热证的经典方剂。本项目按计划完成，主要内容包括：（1）利用响应面法优化了郁金散的醇提工艺，制备条件为：在40℃、30min、40KHZ的超声辅助下，料液比为1：25，回流时间50 min，回流温度50 ℃，乙醇浓度59%；在此条件下制备的郁金散醇提物（YJP-A）经UHPLC-QE-MS/MS方法检测到947种化合物；具有较高的安全性。（2）建立UC小鼠模型，用YJP-A进行干预治疗。其通过对UC小鼠的症状体征、体重降低、疾病活动指数、结肠缩短、组织病理变化、结肠炎症及黏膜屏障的改善作用，表现出较好的治疗作用。（3）研究了YJP-A通过ILC3s-T细胞依赖性IgA-结肠黏液菌群轴缓解DSS诱导的小鼠UC的作用机制：YJP-A可升高UC小鼠结肠肠系膜淋巴结ILC3s数量，促进MHC II的表达，抑制TfH细胞扩增、IL-4的分泌并抑制了Tfh诱导的B细胞向IgA+细胞的类别转换，使IgA分泌减少，恢复结肠黏膜定殖菌群结构。（4）研究了YJP-A对UC小鼠肠肌间神经丛及肌层巨噬细胞的调控作用机制：YJP-A可促进UC小鼠支配肠肌层的儿茶酚胺神经元的修复，而抑制结肠肌层NLRP6的表达，降低了炎症刺激，进而减少肠肌层巨噬细胞释放肠神经保护作用的Arg-1。本项目对传统中兽药方剂郁金散进行了醇提工艺优化，制备了郁金散醇提物，为其颗粒剂的开发奠定了基础；发现了UC新的发病机制，扩宽了治疗UC的策略，为UC发病机制的探索和药物筛选提供了新的重要证据。项目资助下发表论文2篇；授权实用新型专利2项；培养博士研究生1名，硕士研究生2名。项目资助经费24万，支出20.0072万元，剩余3.9928万元，计划用于本项目后续研究和后续论文发表版面费等支出。