疏肝和胃汤调节应激大鼠肠道菌群紊乱诱导的肠黏膜NLRP3 炎症小体变化的机制研究

Chronic stress has become an important cause of mental and gastrointestinal diseases. Studies have shown that chronic stress can cause gut microbiome disturbance, which is related to the NLRP3 inflammasome of intestinal mucosa.TCM regards that chronic liver-qi depression can influence stomach, which in turn develops syndrome of liver-stomach disharmony induced by liver-qi depression. It is similar to the gastrointestinal dysfunction caused by chronic stress in the aspects of etiology, clinical symptoms and treatment. It has been shown in our previous work that Shugan hewei Decoction can relieve stress symptoms and regulate gastrointestinal function by regulating behavioristics and brain-gut peptides. The pre-experiment results showed that this prescription also had a regulatory effect on gut microbiome of stressed rats, whether Shuganhewei Decoction can play a role in regulating the changes of intestinal mucosa NLRP3 inflammasome induced by gut microbiome disturbance remains to be further studied. As a breakthrough point, the research is to observe the changes of structure of gut microbiome, and the expressions of NLRP3, ASC, Caspase-1,downstream inflammatory factors(IL-1β、IL-18), as well as associated proteins(TLRs、p-NF-κB、P2X7R、NF-κB) in serum and intestinal mucosa, and the effect of Shuganhewei Decoction, applied advanced methods, including behaviorstics,16s-RNA,ELISA,IF,Western Blot, RT-PCR, adopted the model rats which were made by the method of chronic unpredictable mild stress (CUMS)and separation. The purpose of this research is to reveal the mechanisms of Shuganhewei Decoction in regulating gastrointestinal function from the perspective of chronic stress- gut microbiome -inflammasome, laying a foundation for further research on syndrome of stomach disorder induced by liver-qi depression.

慢性应激已成为精神及胃肠功能疾病的重要病因。研究发现慢性应激可引起肠道菌群紊乱，且与肠黏膜NLRP3炎症小体互为影响。中医认为肝郁日久，影响及胃，与慢性应激引起的胃肠功能障碍在病因、症状及治疗方面具有相似性。课题组前期研究显示疏肝和胃汤通过调节应激大鼠行为学及脑肠肽缓解应激与胃肠症状，预实验结果表明本方对应激大鼠肠道菌群有调节作用，但该方是否通过调节菌群紊乱诱导的肠黏膜NLRP3炎症小体变化发挥作用有待进一步研究。本课题采用慢性应激加孤养法造模，应用16s-RNA测序观察肠道菌群变化，ELISA、IF、RT-PCR、WB法观察血清及肠黏膜NLRP3、ASC、Caspase-1，下游炎症因子IL-1β、IL-18及炎症小体相关蛋白TLRs、p-NF-κB、P2X7R、NF-κB的表达，从“慢性应激-肠道菌群-炎症小体”的角度，揭示疏肝和胃汤调节胃肠功能的作用机制，为深入研究肝郁犯胃奠定基础。

目的：研究疏肝和胃汤（SHD）对盲肠菌群和盲肠黏膜NLRP3炎症小体的调节作用，探讨SHD改善胃肠功能的作用机制。方法：将雄性SD大鼠随机分为6组，空白组、模型组、SHD低剂量组、SHD高剂量组、四逆散组（SNS）和低聚果糖组（FOS）。采用慢性不可预知性应激（CUS）结合孤养法造模，在第21d给予低、高两种不同浓度SHD（生药量分别为0.56g/ml、1.12g/ml），四逆散（生药量为0.28g/ml）及低聚果糖灌胃干预。模型制作成功后，应用16SrRNA测序观察盲肠菌群变化，ELISA、IF、RT-qPCR、WB法观察血清与盲肠黏膜NLRP3、ASC、Caspase-1，下游炎症因子IL-1β、IL-18及TLR4/NF-κB信号通路中关键蛋白TLR4、p-NF-κB、NF-κB、P2X7R的表达。结果：与模型组比较，①行为学与肠功能评价：SHD高剂量组能够显著改善大鼠的应激状态与肠道功能，具体表现为体质量增长、行为学改变、粪便性状Bristol评分与粪便含水率明显上升。②盲肠组织病理学观察：SHD高剂量组能够显著减轻盲肠黏膜损伤。③肠道菌群检测：SHD高、低剂量组，SNS和FOS组可不同程度逆转慢性应激引起的盲肠菌群紊乱，恢复门水平Firmicutes、Bacteroidetes与Proteobacteria丰度比例，降低属水平Lactobacillus、Lachnospiraceae_NK4A136_group丰度，增加Roseburia、Blautia、Prevotella_9、Prevotella_1丰度，其中SHD高剂量调节作用最为明显。④炎症小体相关指标的检测：SHD高剂量组可调节CUS诱导的血清和盲肠黏膜NLRP3、ASC、Caspase-1、IL-1β和IL-18的过度表达，以及TLR4/NF-κB信号通路的激活。⑤肠道菌群与炎症小体相关性分析：Spearman相关分析显示，血清和盲肠黏膜NLRP3炎症小体及下游炎症因子IL-1β、IL-18与盲肠生物标志物菌群显著相关。结论：SHD调节胃肠功能的潜在机制之一，可能是通过调节盲肠菌群及其紊乱诱导的盲肠黏膜NLRP3炎症小体激活通路中的多个靶点实现的。其中，SHD高剂量具有更好的效果。本研究可为中医药防治应激致胃肠功能障碍相关的“心理-躯体”共病提供新思路。