基于肠道菌群的调肝理脾法调节D-IBS内脏高敏感机制的研究

Visceral hypersensitivity is the main cause of pain, discomfort and other IBS symptoms.-The latest research shows that IBS patients generally suffer a certain degree of intestinal flora imbalance; which may be the key factor of causing IBS visceral high sensitivity. Through the effect of harmonizing Liver and Spleen in the treatment of D-IBS is obvious, the specific mechanism is still unclear. Our early research found that Tong-Xie-Yao-Fang(TXYF) can relieve the visceral hypersensitivity in D-IBS by 5-HT signalling pathway, and harmonize the distribution and composition of intestinal flora. Further studies have found that the mechanism of TXYF may be related to the adjustment of microbial -brain-gut axis and improvement of intestinal mucosal immunity. In this study, animal models were used to observe the effects of TXYF on intestinal flora, and explore the relationship between the changes of intestinal flora of D-IBS and TRPV1, 5-HT, mast cells, etc. Secondly, qgp-1 cells and hmc-1 cells were used to investigate TXYF’s effect on 5-HT’s release and mast cell’s release on histamine. At last, the main pathways and target molecules of TXYF were re-investigated by specific inhibitors, RNAi and other intervened methods. Based on researches this study can clarify the signaling pathways involved in TXYF’s regulating process on intestinal flora, revealing intestinal flora’s effect in the treatment of visceral hypersensitivity by TXYF, and provide reference for future researches on treating IBS by traditional Chinese medicine.

内脏高敏感是导致IBS腹痛、腹部不适等症状的主要原因。最新研究表明：IBS患者普遍存在一定程度的肠道菌群失调，这可能是导致IBS内脏高敏感的关键因素。调肝理脾治法在D-IBS的诊疗中疗效确切，但具体作用机制尚不明确。课题组前期围绕5-HT通路探讨痛泻要方降低D-IBS内脏高敏感的机制，发现其对肠道菌群具有调节作用，其作用机制可能与调整微生物-脑=肠轴、改善肠道黏膜免疫有关。课题利用动物模型观察痛泻要方对肠道菌群的影响，探讨D-IBS肠道菌群的改变与TRPV1、5-HT、肥大细胞等分子的关系；利用QGP-1细胞和HMC-1细胞，探讨痛泻要方对5-HT释放和肥大细胞释放组胺的影响；采用特异性抑制剂和RNAi等干预手段，反证痛泻要方作用的主要通路和靶分子。通过研究阐明痛泻要方调节肠道菌群所涉及的信号通路，揭示肠道菌群在痛泻要方调节内脏高敏感信号传导中的作用，为中药治疗IBS的研究提供参考。

肠易激综合征（IBS）是最常见的功能性胃肠病，目前西医治疗可缓解症状，但常发复发作，影响患者工作和生活。中医辨证论治IBS具有独特优势，其中痛泻要方治疗IBS疗效确切。课题组前期对痛泻要方改善IBS腹泻的机制进行了研究，本研究拟对痛泻要方改善内脏高敏感的机制进行探索。研究方法：1、母婴分离联合束缚应激建立IBS-D大鼠模型，利用电子恒压器检测结肠球囊扩张刺激下腹外斜肌电流变化，进行AWR评分、粪便Bristol评分等以综合评价痛泻要方对IBS-D大鼠内脏高敏感和症状的改善。2、运用16S扩增子测序分析痛泻要方对IBS-D大鼠肠道菌群的影响。3、运用RT-PCR检测脑肠轴Msi1、Ngn3、Epac1、TRPV1、SERT、MUC2、PAR2等分子mRNA表达，运用Western bolt检测上述分子的蛋白表达，通过Elisa法检测5-HT、细胞因子含量，特异性染色肥大细胞和杯状细胞，运用免疫组化观察相关分子表达。4、液质联用分析痛泻要方及其含药血清，结合网络药理学分析，在QGP-1和HMC-1细胞中观察痛泻要方及其含药血清对5-HT、组胺等递质释放的影响。研究结果：1、痛泻要方减轻IBS大鼠内脏高敏感，改善稀便、体重增长速度减慢、悬尾不动时间延长；2、IBS大鼠各肠段均存在菌群失调，痛泻要方主要调节结肠和粪便菌群，使其结构趋向于正常菌群；3、痛泻要方减少结肠和血浆5-HT含量，增加SERT表达，减少Epac1、TRPV1表达；4、痛泻要方减少肠道转录因子Ngn3表达，减少MC数量，减少Tryp、IFN-γ、TLR4表达；5、痛泻要方增加杯状细胞数量,增加MUC2、Occludin表达；6、痛泻要方降低PAR2的表达，抑制Ca2+内流，减少MC活化和组胺分泌。研究结论：痛泻要方通过影响肠道菌群，降低Ngn3表达，影响肠道内分泌细胞分化，减少MC数量和活化，增加杯状细胞数量，具有调节肠道黏膜免疫和维护肠道黏膜屏障的作用，从而缓解内脏高敏感。痛泻要方可能通过肠道菌群作用于脑肠轴Epac1-TRPV1-5-HT信号系统，调节5-HT合成和转运，影响内脏感觉的传递，缓解内脏高敏感。.科学意义：本研究为中医药治疗疾病中肠道菌群的作用研究提供了一定的参考，为痛泻要方治疗D-IBS的推广运用提供了一定的科学依据。