基于“脑-肠-菌”轴理论探讨顺胃降逆颗粒对功能性消化不良大鼠脑肠肽及肠道菌群的影响

Effects of Shunwei Jiangni granule on brain-gut peptides and gut microbiota of functional dyspepsia rats based on the “Brain-Gut-Microbiota Axis” theory

摘要:

目的 基于“脑-肠-菌”轴理论，深入探究顺胃降逆颗粒对功能性消化不良大鼠模型的作用机制。方法 采用夹尾打斗结合隔日禁食与冷热交替饮水的方法复制功能性消化不良大鼠模型，以顺胃降逆颗粒作为治疗药物，同时设置多潘立酮片作为阳性对照药物，持续14 d。进行体重称量、检测小肠推进率、胃排空率。HE染色检测功能性消化不良大鼠胃组织和十二指肠组织病理情况。酶联免疫吸附试验（ELISA）检测血清脑肠肽胃动素（MTL）、胃泌素（GAS）、5-羟色胺（5-HT）水平。采用实时荧光定量聚合酶链反应（qRT-PCR）和Western blotting检测大鼠胃窦、十二指肠、下丘脑组织血管活性肠肽（VIP）、降钙素基因相关肽（CGRP） mRNA和蛋白表达。采用16S rDNA扩增子测序法进行功能性消化不良大鼠的肠道菌群分析。结果 与对照组比较，模型组大鼠胃排空率、小肠推进率下降（P <0.05）；与模型组比较，顺胃降逆颗粒高剂量组和阳性药物组胃排空率、小肠推进率升高（P <0.05）。胃组织和十二指肠组织病理检测结果显示，与对照组相比，模型组胃黏膜及十二指肠组织病变程度、炎症细胞浸润和增生增加；与模型组相比，顺胃降逆颗粒治疗后大鼠胃黏膜及十二指肠组织病变程度明显减轻，炎症细胞浸润和增生减少。与对照组相比，模型组大鼠血清MTL、GAS、5-HT水平降低（P <0.05）；与模型组相比，顺胃降逆颗粒低、中、高剂量组和阳性药物组大鼠血清MTL、GAS、5-HT水平升高（P <0.05）。qRT-PCR结果显示，与对照组比较，模型组大鼠胃窦、十二指肠和下丘脑CGRP和VIP mRNA相对表达量升高（P <0.05）；与模型组比较，顺胃降逆颗粒低、中、高组与阳性药物组VIP、CGRP mRNA相对表达量降低（P <0.05）。Western blotting结果显示，与对照组比较，模型组大鼠胃窦、十二指肠和下丘脑组织中VIP、CGRP蛋白相对表达量水平升高（P <0.05）；与模型组相比，顺胃降逆颗粒低、中、高组与阳性药物组VIP、CGRP蛋白相对表达量水平降低（P <0.05）。在肠道菌群方面，顺胃降逆颗粒能够调节功能性消化不良大鼠肠道菌群结构和组成，增加有益菌群如阿克曼氏菌属、袋熊杆菌属等的相对丰度，降低潜在有害菌群如粪杆菌属等的丰度。结论 顺胃降逆颗粒能够提升功能性消化不良大鼠的胃排空率及小肠推进率，并有效改善胃组织与十二指肠组织的病理形态学损伤，能上调血清MTL、GAS、5-HT的表达，同时下调胃窦、十二指肠、下丘脑的VIP和CGRP表达。此外，顺胃降逆颗粒还显著调节了功能性消化不良大鼠肠道菌群的结构与组成，增加了有益菌群如阿克曼氏菌属、袋熊杆菌属的相对丰度，降低了潜在有害菌群如粪杆菌属的丰度。

Abstract:

Objective To investigate the mechanism of Shunwei Jiangni granule (SWJN) on functional dyspepsia (FD) in a rat model, focusing on the brain-gut-microbiota axis.Methods A rat model of FD was established by tail-clamping combined with irregular feeding and alternate hot/cold water gavage for 14 days. The rats were randomly divided into the following groups: control, model, SWJN (low, medium, and high-dose), and domperidone (positive control). Body weight was measured weekly. Gastric emptying rate and small intestinal propulsion rate were determined. Pathological changes in gastric and duodenal tissues were examined by hematoxylin and eosin (HE) staining. Serum levels of motilin (MTL), gastrin (GAS), and 5-hydroxytryptamine (5-HT) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression levels of vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) in the gastric antrum, duodenum, and hypothalamus were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Gut microbiota composition was analyzed by 16S rDNA amplicon sequencing.Results Compared with the control group, the model group showed significantly decreased gastric emptying and small intestinal propulsion rates (P < 0.05), aggravated histopathological injuries with increased inflammatory cell infiltration and hyperplasia in gastric and duodenal tissues, lower serum levels of MTL, GAS, and 5-HT (P < 0.05), and higher mRNA and protein expression levels of VIP and CGRP in the gastric antrum, duodenum, and hypothalamus (P < 0.05). Compared with the model group, the high-dose SWJN group and the domperidone group showed increased gastric emptying and small intestinal propulsion rates (P < 0.05). All SWJN-treated groups and the domperidone group exhibited alleviated gastric and duodenal tissue damage, reduced inflammatory cell infiltration, elevated serum levels of MTL, GAS, and 5-HT (P < 0.05), and downregulated mRNA and protein expression of VIP and CGRP in the aforementioned tissues (P < 0.05). Additionally, SWJN treatment modulated the structure and composition of the gut microbiota in FD rats, increasing the relative abundance of beneficial bacteria such as Akkermansiaand Phascolarctobacterium, while decreasing that of potentially harmful bacteria such as Faecalibacterium.Conclusion SWJN can improve gastrointestinal motility (increasing gastric emptying and small intestinal propulsion rates), alleviate histopathological damage in the stomach and duodenum, upregulate serum levels of MTL, GAS, and 5-HT, downregulate the expression of VIP and CGRP in the gastric antrum, duodenum, and hypothalamus, and beneficially modulate the gut microbiota structure in FD rats. These findings suggest that its therapeutic effects are mediated, at least in part, through the regulation of the brain-gut-microbiota axis.