Construction and Evaluation of an Animal Model Combining Insomnia with the Syndrome of Liver Depression and Blood Deficiency
Author：LAN Bijuan, LI Ruting, SHI Qinghuan, CHEN Xinyi, XIAO Zhuoyang, CHU Yiyu, LIU Yan, LI Feng
Unit：School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 102488, China
Quote：引用：兰碧娟，李儒婷，施晴寰，陈心怡，肖卓阳，初怡宇，刘燕，李峰.失眠肝郁血虚证病证结合动物模型构建与评价[J].中医药导报,2026,32(2):59-66,79.
DOI：10.13862/j.cn43-1446/r.2026.02.010
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Abstract：
Objective: To establish and evaluate an
animal model combining insomnia with the syndrome of liver depression and blood
deficiency using intraperitoneal injection of p-chlorophenylalanine (PCPA)
combined with chronic tail-clamping stimulation and tail bleeding. Methods:
Totally 30 male specific pathogen-free (SPF) Sprague-Dawley (SD) rats were
randomly divided into a blank control group and a model group. The blank
control group received no intervention, while the model group was subjected to
modeling via intraperitoneal injection of PCPA combined with chronic
tail-clamping stimulation and tail bleeding. After 21 days, the general
condition of the rats was observed, and their syndrome characteristics, sleep
rhythm, and behavior were evaluated. Sleep latency and sleep duration were
recorded using the sodium pentobarbital righting reflex test. Arterial blood
counts of red blood cells (RBC), hemoglobin (HGB), white blood cells (WBC), and
platelets (PLT), as well as serum levels of aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and blood urea nitrogen (BUN) were measured.
Hematoxylin-eosin (HE) and Nissl staining were used to observe the
histopathology of the CA1 region of the hippocampus. Enzyme-linked
immunosorbent assay (ELISA) was used to detect the levels of serotonin (5-HT)
and dopamine (DA) in the hippocampal tissue and serum. Results: Compared with
the blank control group, rats in the model group showed increased daytime
activity, nocturnal lethargy with a preference for lying down, mental fatigue,
dry and yellowish fur, and sensitivity to external sound and light stimuli.
After modeling, the model group exhibited significantly prolonged sleep latency
and shortened sleep duration; the 24-hour average sleep time percentage
decreased, while the average sleep time percentage during the light phase
decreased and that during the dark phase increased; in the open field test, the
total movement distance, total number of grid crossings, number of central zone
crossings, time spent in the central zone, rearing time, and grooming time were
all significantly reduced; in the elevated plus maze test, both the percentage
of entries into the open arms and the percentage of time spent in the open arms
were decreased; grip strength was reduced; in the Morris water maze test,
escape latency was increased, whereas the number of platform crossings and the
time spent swimming in the target quadrant were decreased; arterial RBC, HGB,
and PLT levels were decreased; serum ALT, AST, and BUN levels were increased.
Rats in the model group showed certain pathological changes in the hippocampal
CA1 region; 5-HT levels in the hippocampal tissue and serum were decreased,
while DA levels were increased. Conclusion: The animal model constructed using
the multifactorial method involving intraperitoneal injection of PCPA combined
with chronic tail-clamping stimulation and tail bleeding correlates well with
the syndrome characteristics and clinical reality of insomnia with liver
depression and blood deficiency syndrome, providing an experimental basis for
subsequent new drug development and research on mechanisms of action.
Key words：insomnia; syndrome of liver depression and blood deficiency; rats; disease-syndrome combination; animal model
摘要：
目的：应用腹腔注射对氯苯丙氨酸（PCPA）复合慢性夹尾刺激及尾部放血法，构建失眠肝郁血虚证病证结合动物模型及其评价体系。方法：将30只雄性无特定病原体（SPF）级SD大鼠随机分为空白对照组和模型组。空白对照组不干预，模型组采用腹腔注射PCPA复合慢性夹尾刺激及尾部放血法进行造模。21 d后观察大鼠一般情况，评价大鼠证候特点、睡眠节律、行为学，采用戊巴比妥钠翻正实验记录大鼠睡眠潜伏期与睡眠持续时间，检测大鼠动脉血红细胞计数（RBC）、血红蛋白（HGB）、白细胞计数（WBC）和血小板计数（PLT）以及血清中天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、血尿素氮（BUN）的含量，苏木精-伊红（HE）染色和尼氏（Nissl）染色观察大鼠海马组织CA1区组织病理，酶联免疫吸附试验（ELISA）检测海马组织和血清中5-羟色胺（5-HT）、多巴胺（DA）的含量。结果：与空白对照组比较，模型组大鼠白天活动增加，夜间慵懒喜卧，精神倦怠，毛发干枯色黄，对外界声、光刺激敏感。造模结束后模型组大鼠睡眠潜伏期显著延长，睡眠持续时间显著缩短；24 h平均睡眠时间百分比下降、光照期的平均睡眠时间百分比减少、黑暗期的平均睡眠时间百分比增加；旷场实验中总运动距离、总穿格次数、中央区穿格次数、中央区停留时间、直立时间、修饰时间均显著减少；高架十字迷宫实验中开放臂进入次数和开放臂停留时间百分比均降低；抓握力降低；Morris水迷宫实验中逃逸潜伏期增加，穿越平台次数、目标象限游泳时间均降低，动脉血中RBC、HGB、PLT降低；血清中ALT、AST、BUN含量升高。模型组大鼠海马CA1区存在一定病理改变或异常表现；海马组织和血清中的5-HT含量降低，DA水平升高。结论：腹腔注射PCPA复合慢性夹尾刺激以及尾部放血的多因素复合造模法构建的动物模型较为符合失眠肝郁血虚证的证候特点与临床实际，可为后续的新药开发及作用机制研究提供实验基础。
关键词：失眠；肝郁血虚证；大鼠；病证结合；动物模型
Release time：2026-03-05
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