目的 基于TLR4/NF-κB/MLCK 通路研究百合地黄汤对对氯苯丙氨酸（PCPA）及多因素刺激所致失眠伴 肠道菌群失调小鼠的治疗作用及机制，为临床用药提供理论依据。方法 将84 只KM 小鼠随机分为正常组、 模型组、阳性药物组（地西泮，1.38 mg·kg-1）、百合组（2.25 g·kg-1）、地黄组（2.25 g·kg-1）、百合地黄汤组 （4.5 g·kg-1）。采用夹尾2 min、昼夜颠倒24 h、垫料潮湿24 h、45°鼠笼倾斜24 h、交替鼠笼24 h、禁食不禁 水24 h、冷水浴3 min 等多因素刺激4 周结合PCPA 腹腔注射2 d 共同制备失眠小鼠模型。模型复制成功后， 给予相应的药物治疗。采用高架十字迷宫系统观察小鼠的焦虑样行为；通过翻正反射实验观察睡眠潜伏期和睡 眠持续时间；16sRNA 测序法分析小鼠肠道菌群组成结构；液相色谱-质谱联用（LC-MS）检测脑和结肠γ-氨 基丁酸（GABA）、谷氨酸（Glu）、色氨酸（Trp）、5-羟色氨酸（5-HTP）、5-羟色胺（5-HT）的浓度变化；免疫组织 化学法检测结肠中闭锁小带蛋白1（ZO-1）、闭合蛋白（Occludin）的表达情况；实时荧光定量PCR 法（qRTPCR） 检测结肠组织中基因白细胞介素1β（IL-1β）、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）、ZO-1、 Occludin、Toll 样受体4（TLR4）、核因子κB（NF-κB）、肌球蛋白轻链激酶（MLCK）的表达情况；蛋白免疫印迹 法（Western Blot）检测结肠上皮组织中蛋白TLR4、NF-κB、磷酸化-核因子κB（p-NF-κB）、MLCK、肌球蛋白 轻链（MLC）和磷酸化-肌球蛋白轻链（p-MLC）表达情况。结果 与正常组小鼠比较，模型组小鼠在高架十字迷 宫中进入开臂区域的距离、在开臂区域停留的时间明显缩短（P＜0.05）；睡眠潜伏期明显延长、睡眠持续时间 明显缩短（P＜0.05）；肠道菌群的丰富度、均匀度降低（P＜0.01）；脑内神经递质GABA、Trp、5-HTP、5-HT 浓 度明显降低（P＜0.01），Glu 浓度明显升高（P＜0.01）；结肠中GABA、Glu 浓度明显降低（P＜0.01），Trp、5-HTP、 5-HT 浓度明显升高（P＜0.01）；结肠组织炎性因子IL-1β、IL-6 以及TNF-α 基因表达水平明显升高（P＜ 0.01），ZO-1、Occludin 基因和蛋白表达水平明显降低（P＜0.01），通路中TLR4、NF-κB、MLCK 基因表达水 平明显升高（P＜0.01），TLR4、p-NF-κB、MLCK、p-MLC 蛋白表达水平明显升高（P＜0.01）。与模型组小鼠比 较，百合地黄汤能够明显延长失眠小鼠在高架十字迷宫中进入开臂区域距离、在开臂区域停留的时间（P＜ 0.05）；明显缩短睡眠潜伏期、增加睡眠持续时间（P＜0.05）；肠道菌群的丰富度、均匀度升高（P＜0.01）；升高 脑内神经递质GABA、Trp、5-HTP、5-HT 浓度（P＜0.05，P＜0.01），降低Glu 浓度（P＜0.01）；升高结肠中 GABA、Glu 浓度（P＜0.01），降低Trp、5-HTP、5-HT 浓度（P＜0.05，P＜0.01）；下调IL-1β、IL-6、TNF-α 基因表达水平（P＜0.01），上调ZO-1、Occludin 基因和蛋白表达水平（P＜0.01），下调通路中TLR4、NF-κB、 MLCK 基因表达水平和TLR4、p-NF-κB、MLCK、p-MLC 蛋白表达水平（P＜0.01）。结论 百合地黄汤能够 有效治疗失眠伴肠道菌群失调症状，其作用机制可能与调节脑和肠内神经递质紊乱、下调TLR4/NF-κB/MLCK 信 号通路表达、上调紧密连接蛋白表达、降低炎症反应，进而修复肠黏膜机械屏障有关。

Abstract： Objective Based on TLR4/NF- κB/MLCK pathway， the therapeutic effect and mechanism of Baihe Dihuang Decoction on insomnia with intestinal flora disturbance in mice induced by p-chlorophenlalanine （PCPA） and multi-factor stimulation were studied. The aim is to provide theoretical basis for clinical use. Methods Eightyfour KM mice were randomly divided into normal group，model group，positive group （Diazepam，1.38 mg·kg-1）， Baihe group （2.25 g·kg-1）， Dihuang group （2.25 g·kg-1） and Baihe Dihuang Decoction group （4.5 g·kg-1）. Insomnia mouse model was established by intraperitoneal injection of PCPA for 2 days combined with 4 weeks of multi-factor stimulation，including stimulating the tail with forceps clip for 2 minutes，reversing day and night for 24 hours，wetting the padding for 24 hours，tilting the cage at 45° for 24 hours，alternating the cages for 24 hours， fasting food for 24 hours，and getting cold bath for 3 minutes，etc. After successfully modeling，corresponding drug treatment was given. The anxiety-like behavior of mice was observed by elevated cross maze system. The latency and duration of sleep were observed by righting reflex experiment. 16sRNA sequencing was used to analyze the composition and structure of intestinal flora in mice. The concentration changes of γ-aminobutyric acid （GABA）， glutamic acid （Glu）， tryptophan （Trp）， 5-hydroxytryptophan （5-HTP） and 5-hydroxytryptamine （5-HT） in brain and colon were detected by liquid chromatography-mass spectrometry （LC-MS） . Immunohistochemical method was used to detect the expressions of zona atresia protein 1 （ZO-1） and Occludin in colon. Real-time fluorescence quantitative PCR （qRT-PCR） was used to detect the genes including interleukin-1β （IL-1β）， interleukin-6（IL-6），tumor necrosis factor-α（TNF-α），ZO-1，Occludin，Toll-like receptor 4 （TLR4），nuclear factor κB （NF-κB） and myosin light chain kinase（MLCK） in colonic tissue. Western Blot was used to detect the expressions of TLR4，NF-κB，phosphorylated nuclear factor κB （p-NF-κB），MLCK，myosin light chain （MLC） and phosphorylated myosin light chain （p-MLC） in colonic epithelial tissue. Results Compared with the normal group，the distance of entering the open arm and the duration of stay in the open arm of model group in the elevated cross maze were significantly shortened （P＜0.05）. The sleep latency was significantly prolonged， and the sleep duration was significantly shortened （P＜0.05）. The richness and uniformity of intestinal flora were decreased （P＜ 0.05， P＜0.01）. The concentration of neurotransmitters GABA， Trp， 5-HTP and 5-HT in the brain decreased significantly （P＜0.01）， while the concentration of Glu increased significantly （P＜0.01）. The concentration of GABA and Glu in colon decreased significantly （P＜0.01）， while the concentration of Trp， 5-HTP and 5-HT increased significantly （P＜0.01）. The expression levels of inflammatory factors IL-1β，IL-6 and TNF-α in colonic tissue were significantly increased （P＜0.01），and the expression levels of ZO-1 and Occludin genes and proteins were significantly decreased （P＜0.01）. The gene expression levels of TLR4，NF-κB and MLCK were significantly increased （P＜0.01），and the protein expression levels of TLR4，p-NF-κB，MLCK and p-MLC were significantly increased （P＜0.01）. Compared with the model group， Baihe Dihuang Decoction could significantly prolong the distance of entering the open arm and the duration of stay in the open arm of insomnia mice in the elevated cross maze （P＜0.05）. The sleep latency was significantly shortened，and the sleep duration was significantly increased （P＜0.05）. The richness and uniformity of intestinal flora were increased （P＜0.05，P＜0.01）. The concentration of neurotransmitters GABA， Trp， 5-HTP and 5-HT in brain was increased （P＜0.05， P＜0.01）， and the concentration of Glu was decreased （P＜0.01）. The concentration of GABA and Glu in colon was increased （P＜ 0.01）， while the concentration of Trp， 5-HTP and 5-HT was decreased （P＜0.05， P＜0.01）. The expression levels of IL-1β， IL-6 and TNF-α genes were down-regulated （P＜0.01）， the expression levels of ZO-1 and Occludin genes and proteins were up-regulated （P＜0.01）， TLR4， NF-κB， MLCK gene expression levels and TLR4， p-NF- κB， MLCK， p-MLC protein expression levels were down-regulated in the pathway （P＜0.01）. Conclusion Baihe Dihuang Decoction can effectively treat insomnia with intestinal flora disorders. Its mechanism of action may be related to the regulation of brain and intestinal neurotransmitter disorders，down-regulating TLR4/NF- κB/MLCK signaling pathway，and up-regulating tight junction proteins expression，reducing inflammatory responses， and then repairing the mechanical barrier of intestinal mucosa.

R285.5

河北省中医药管理局科研计划项目（2022081）。