基于肠道菌群初步探讨半夏秫米汤改善失眠的作用机制

Preliminary exploration of the mechanism by which Banxia Shumi Decoction improves insomnia based on the gut microbiota

关键词:  失眠  半夏秫米汤  肠道菌群  16S rRNA  肠屏障蛋白  炎症因子

Key words:  insomnia  Banxia Shumi Decoction  gut microbiota  16S rRNA  intestinal barrier proteins  inflammatory

摘要:

[目的] 基于16S rRNA测序探讨半夏秫米汤对失眠小鼠肠道菌群的影响，并初步探讨其作用机制。[方法] 将60只SPF级雌性C57BL/6JNifdc小鼠随机分为空白组、模型组、阳性药组、半夏秫米汤低、中、高剂量组。除空白组之外，其他组小鼠腹腔注射对氯苯丙氨酸（PCPA，400 mg/kg）联合水平台造模3 d。给药7 d后，进行旷场实验，之后采集粪便进行16S rRNA测序，分析小鼠的肠道菌群丰富度和物种组成；免疫荧光和蛋白免疫印记法（Western blot）检测肠屏障蛋白表达水平；酶联免疫吸附法（ELISA）检测小鼠下丘脑中5-羟色胺（5-HT）、γ-氨基丁酸（GABA）含量以及结肠中白细胞介素-6（IL-6）含量；利用HE染色观察小鼠下丘脑病理损伤；免疫组化观察小鼠下丘脑中离子钙结合衔接分子-1（Iba-1）的表达。[结果] 半夏秫米汤能够增加小鼠行动的总路程、平均速度、站立次数，并且减少静止时间，增加肠道菌群的丰富度和物种多样性，上调肠屏障蛋白的表达，增加小鼠下丘脑中的5-HT和GABA含量，降低结肠的炎症水平，减轻下丘脑病理损伤，抑制小胶质细胞活化。[结论] 半夏秫米汤能够减轻失眠小鼠下丘脑病理损伤，可通过保护肠屏障、抑制肠和脑中炎症反应、调节肠道菌群而发挥抗失眠作用。

Abstract:

[Objective] To investigate the effects of Banxia Shumi decoction on the gut microbiota in a mouse model of insomnia using 16S rRNA sequencing，and to preliminarily explore its underlying mechanisms. [Methods] 60 SPF-grade female C57BL/6JNifdc mice were randomly divided into a blank group，a model group，a positive drug group，and low-，medium-，and high-dose Banxia Shumi decoction groups. Except for the blank group，mice in other groups received intraperitoneal injections of PCPA（400 mg/kg） combined with a water platform for three days to establish the model. After seven days of treatment，an open field test was conducted. Subsequently，fecal samples were collected for 16S rRNA sequencing to analyze the richness and species composition of the gut microbiota in mice. Immunofluorescence and Western blot were used to detect the expression levels of intestinal barrier proteins. Enzyme-linked immunosorbent assay was employed to measure the contents of 5-Hydroxytryptamine（5-HT） and Gamma-Aminobutyric Acid（GABA） in the hypothalamus and Interleukin-6（IL-6） in the colon. Hematoxylin and eosin（HE） staining was used to observe pathological damage in the mouse hypothalamus. Immunohistochemistry was applied to examine the expression of Ionized calcium-binding adapter molecule-1（Iba-1） in the mouse hypothalamus. [Results] Treatment with Banxia Shumi decoction increased the total distance traveled，average speed，and rearing frequency in mice，while reducing resting time. It also enhanced the richness and diversity of the gut microbiota，up-regulated the expression of intestinal barrier proteins，increased the contents of 5-HT and GABA in the hypothalamus，reduced colonic inflammation levels，alleviated pathological damage in the hypothalamus，and inhibited microglial activation. [Conclusion] Banxia Shumi decoction can alleviate pathological damage in the hypothalamus of mice with insomnia，exerting anti-insomnia effects by protecting the intestinal barrier，inhibiting inflammatory responses in the gut and brain，and modulating the gut microbiota.