通便汤恢复肠道菌群并激活5-羟色胺信号:在慢传输型便秘中的作用

Tongbian decoction restores intestinal microbiota and activates 5-hydroxytryptamine signaling: implication in slow transit constipation.

作者信息Hongjia Li, Na Lv, Dongna Li, Yunzhi Qian, Xianghuan Si, Yuanqing Hua, Yujuan Wang, Xiaojuan Han, Tianshu Xu
PMID38287961
发布时间2024-01-15
DOI10.3389/fmicb.2023.1296163

实验完整度

高

包含伪无菌大鼠模型、STC大鼠模型、FMT、16S rRNA测序、Western blot、免疫荧光、免疫组化等多层级实验,并进行了功能验证(体重、粪便含水量、碳末推进率)和机制验证(5-HT信号通路)。

主要模型

SD大鼠伪无菌模型 洛哌丁胺诱导的STC大鼠模型 FMT受体大鼠模型

重点核对

抗生素处理(bacitracin, neomycin, streptomycin,各200 mg/kg)6天构建伪无菌大鼠 STC模型诱导:洛哌丁胺3 mg/kg/d,持续7天 FMT:供体粪便悬液1 mL/100 g体重灌胃,持续6天 TBD给药剂量:24 g/kg,持续14天(第8-21天) 检测指标:体重、粪便含水量、碳末推进率、5-HT信号通路蛋白表达

摘要

Introduction: Slow transit constipation (STC) is a type of functional constipation. The detailed mechanism of STC, for which there is currently no effective treatment, is unknown as of yet. Tongbian decoction (TBD), a traditional Chinese medicinal formula, is commonly used to treat STC in clinical settings. However, the potential impact of TBD on the management of STC via modulation of the gut microbiota remains unclear.Methods: Pseudo-germ-free rats were constructed after 6 days of treatment with bacitracin, neomycin, and streptomycin (abbreviated as ABX forthwith). Based on the successful construction of pseudo-germ-free rats, the STC model (ABX + STC) was induced using loperamide hydrochloride. After successful modeling, based on the different sources of donor rat microbiota, the ABX + STC rats were randomly divided into three groups: Control → ABX + STC, STC → ABX + STC, and STC + TBD → ABX + STC for fecal microbiota transplant (FMT). Body weight, fecal water content, and charcoal power propelling rate of the rats were recorded. Intestinal microbiota was detected by 16S rRNA sequencing, and the 5-hydroxytryptamine (5-HT) signaling pathway was examined by western blots, immunofluorescence, and immunohistochemical analysis.Results: After treatment with fecal bacterial solutions derived from rats treated with Tongbian decoction (TBD), there was an increase in body weight, fecal water content, and the rate of charcoal propulsion in the rats. Additionally, activation of the 5-hydroxytryptamine (5-HT) signaling pathway was observed. The 16S rRNA sequencing results showed that the fecal bacterial solution from TBD-treated rats affected the intestinal microbiota of STC rats by increasing the proliferation of beneficial bacteria and suppressing the expansion of harmful bacteria.Conclusion: Our study showed that TBD alleviated constipation in STC rats by modulating the structure of the intestinal microbiota.

实验结论

提炼研究问题、关键发现与证据,快速把握文章的核心贡献。

研究问题
通便汤是否通过调节肠道菌群缓解慢传输型便秘?
核心机制
通便汤通过调节肠道菌群结构,增加Bacteroidota丰度、降低Firmicutes丰度,并激活5-HT信号通路(升高TPH1、5-HT、5-HT4R表达),从而促进肠道蠕动。
主要证据
在STC大鼠模型中,接受TBD处理大鼠粪菌悬液移植后,大鼠体重、粪便含水量和碳末推进率显著增加;结肠组织病理损伤改善;5-HT、TPH1、5-HT4R表达升高;16S rRNA测序显示菌群结构趋于正常。
研究意义
该研究为通便汤治疗STC提供了肠道菌群和5-HT信号通路的新证据,并促进了中药的临床应用。

研究路径

按研究推进顺序梳理实验设计、验证步骤与关键观察。

1

构建伪无菌大鼠模型

消除肠道菌群,研究菌群对肠道运动的影响

使用广谱抗生素(bacitracin、neomycin、streptomycin,各200 mg/kg)连续处理6天,通过16S rRNA测序验证菌群清除效果。

2

构建STC大鼠模型及分组

建立慢传输型便秘模型并制备不同来源的粪菌悬液

在伪无菌大鼠基础上,使用洛哌丁胺腹腔注射7天诱导STC模型;另外制备对照组、STC组、STC+TBD组供体大鼠粪菌悬液。

3

粪菌移植(FMT)

明确TBD调节的肠道菌群对STC的治疗作用

通过灌胃将供体粪菌悬液移植至STC受体大鼠,持续6天。

4

评估肠道运动功能

评价各组大鼠便秘表型的改善情况

检测体重、粪便含水量和碳末推进率。

5

结肠组织病理学评估

评估结肠组织损伤程度及TBD粪菌移植的改善作用

HE染色观察结肠组织形态学变化并进行组织学评分。

6

5-HT信号通路检测

验证粪菌移植对5-HT信号通路的影响

通过免疫组化、免疫荧光和Western blot检测5-HT、TPH1、5-HT4R的表达及EC细胞数量。

7

肠道菌群分析

分析粪菌移植后肠道菌群结构的变化

收集粪便进行16S rRNA V3-V4区测序,分析菌群多样性和组成。

研究方法

按研究目的归类文中使用的方法,便于定位所需技术。

产品清单

实验环节名称品牌货号
异氟烷----
盐酸洛哌丁胺Xi'anJansen Co., Ltd.LLJO007
杆菌肽SolarbioB8181
新霉素SolarbioN8090
链霉素SolarbioS8290
5-HT一抗SigmaS5545
二抗ServicebioGB23303
DAB辣根过氧化物酶显色试剂盒ServicebioG1212
红色荧光二抗BiyuntianA0516
嗜铬粒蛋白A一抗Proteintech60135
Alexa Fluor 488标记二抗BiyuntianA0428
DAPIBiossC0204002
RIRA裂解液ServicebioG2002
BCA试剂盒ServicebioG2026
SDS-PAGE凝胶ServicebioG2037
TPH1抗体CST12339S
5-HT4R抗体ABclonalA2802
GAPDH抗体ServicebioGB15004
二抗ServicebioGB23303
ECL发光液----
DNA提取试剂盒TianGen--
NEB Next® Ultra™ II FS DNA PCR-free文库制备试剂盒New England BiolabsE7430L
Illumina测序平台Illumina--
电热恒温干燥箱Shanghai Jinghong Experimental Equipment Co., Ltd.DHC-9030A
阿拉伯胶Suzhou Yijing Food Additives Co., Ltd.--
高岭土Tianjin Beilian Fine Chemicals Development Co., Ltd.--
光学显微镜DM500LeicaDM500
荧光显微镜DMi8LeicaDMi8
ImageJ软件----

关键环节

汇总复现实验时建议重点确认的条件及原文阅读提示。

环节核对要点
伪无菌大鼠模型构建
抗生素种类及剂量、给药时间、菌群清除效果验证
阅读提示:见2.4节 Materials and methods
STC模型构建
洛哌丁胺剂量、给药途径、造模周期
阅读提示:见2.4节 Materials and methods
粪菌移植实验
供体分组、粪菌悬液制备、移植剂量和频次
阅读提示:见2.3和2.4节
5-HT信号通路检测
抗体来源及稀释比例、组织样本处理
阅读提示:见2.8-2.10节
肠道菌群分析
16S rRNA测序区域、数据分析流程
阅读提示:见2.11节