基于肠道菌群变化研究大黄附子汤对实验性重症胰腺炎防治作用的机理

Severe acute pancreatitis (SAP) is a common clinical emergency, with a passive therapeutic method and high mortality. Its occurrence and development may be related to the intestinal flora imbalance. In clinical we have successfully applied Dahuangfuzi decoction to the treatment of SAP, and previous studies have proved that Dahuangfuzi can prevent ALI induced by SAP, but the underlying mechanism is not fully understood. In this study, on a rat model of SAP and with the intervention treatment of Dahuangfuzi, we dynamically detect the intestinal flora presentation in the occurrence and developing process of SAP, and changes in species and quantity of flora in the serum and intestinal mucosa during different stage by high throughput sequencing analysis of Illumina MiSeq platform. To isolate and identify the key strains with bioinformatics analysis, and verify the damage and correlation among proteins of Occludin, ZO-1, p-VASP, cICC and mSCF in the target organs such as pancreas, lung and intestine in parallel by immunohistochemistry, PCR and Western blot. To explore the relationship between intestinal barrier and main molecules in the proinflammatory pathway of TLR4－NF－κB during the occurrence and development of SAP, and reveal the deep mechanism and target of Dahuangfuzi in the treatment of SAP. To clarify: 1. changes of intestinal flora and its significance in the occurrence and development of SAP; 2. the target of Dahuangfuzi on SAP at the level of intestinal flora. We want to study the mechanism of SAP from a new perspective and to provide ideas for the modern research of traditional Chinese medicine in the field of emergency and critical care.

重症急性胰腺炎（SAP）是常见的急危重症，治疗方法被动，死亡率高。其发生与发展可能与肠道菌群失调有关。我们应用大黄附子汤临床治疗SAP有效，研究证明大黄附子汤能够防治SAP所致ALI，但机理未完全明了。本研究以大鼠SAP为模型，用大黄附子汤治疗，利用Illumina MiSeq平台微生物高通量测序技术动态检测SAP发生发展过程中以及大黄附子汤作用后不同时段肠道菌群表达种类和数量的变化，结合生物信息学，分离鉴定关键菌种，并通过免疫组化等方法验证胰腺、肺、肠等靶器官Occludin、ICC 等蛋白损害关联。探寻SAP肠屏障与致炎通路TLR4－NF-κB主要分子的相互关系，揭示大黄附子汤治疗SAP的深层次机理及作用靶点。阐明1、SAP发生发展过程中肠道菌群变化及意义；2、大黄附子汤对SAP在肠道菌群层面作用的靶点。从新的角度对SAP发生机制进行探讨，为中医药在急危重症领域应用的现代研究提供思路。

重症急性胰腺炎（SAP）是常见的急危重症，死亡率高。有研究表明应用中药复方通过调整肠道菌群结构治疗疾病。多年临床应用证实大黄附子汤治疗SAP有效。大黄附子汤是否能通过调节肠道菌群变化达到防治SAP及其并发症，尚不清楚。为此，课题组历时四年研究，取得了系列成果：（1）SAP时肠道乳杆菌属（Lactobacillus）相对丰度明显下降，大黄附子汤治疗可增加Lactobacillus等益生菌含量，改变SAP肠道菌群丰度和菌群多样性，降低血清内毒素、IL-1β水平，有效抑制炎症因子的瀑布式激活，缓解SAP病情；（2）大鼠肠道菌群受季节因素影响；（3）SAP后肠道结构破坏，血浆胃动素（MTL）浓度降低，回肠 Cajal间质细胞（ICC）数量减少，分布不均匀，平滑肌和ICC细胞膜上胃动素受体（MTL-R）减少，肠蠕动节律紊乱，出现肠道功能障碍。大黄附子汤通过调节MTL的分泌，恢复ICC细胞及MTL-R的数量，影响肠神经系统（ENS）重塑，改善SAP大鼠肠道动力障碍; （4）大黄附子汤可能诱导肝Kuppfer细胞向M2型分化，促使抗炎因子IL-10表达分泌增加，减轻SAP时全身炎症反应；（5）大黄附子汤可通过增加肠微褶皱细胞（M细胞）转运蛋白GP2表达，增加肠上皮内淋巴细胞（IELs）数量，增强肠道黏膜免疫功能功能，阻止肠道菌群及内毒素移位，减轻SAP肠损伤；（6）采用中药整合药理学平台筛选出大黄附子汤158种药效成分和659个靶标，大黄附子汤可能通过调节多种炎性因子和细胞因子的释放、并调节内分泌系统和免疫活性发挥治疗SAP的作用。