Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis
Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis
复制标题
加味酸枣仁汤通过调节Orexin-A和HPA轴稳态对失眠小鼠模型的催眠作用
DOI：
10.1016/j.biopha.2021.112141
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发表时间：
2021-09-10
影响因子：
7.5
通讯作者：
Chen, Su-Hong
中科院分区：
文献类型：
--
作者：
Dong, Ying-Jie;Jiang, Ning-Hua;Chen, Su-Hong
目的：经中药复方酸枣人汤改良而成的加减酸枣人汤（MSZRD），是一种治疗失眠的传统中药方剂。我们之前的研究表明，MSZRD可以通过调节Orexin-A来改善胃肠道不适相关的失眠。本研究首次探讨了MSZRD对对氯苯丙氨酸（PCPA）联合多因素随机刺激致失眠小鼠的作用及其可能机制。方法：对ICR小鼠进行多因素刺激14 d后，连续2 d腹腔注射PCPA混悬液（30 mg/mL），建立失眠模型。给ICR小鼠注射3种不同剂量的MSZRD（3.6、7.2和14.4 g/kg/天），持续24天。测定摄取量和背部体温，进行行为试验和戊巴比妥钠诱发睡眠试验。采用酶联免疫吸附试验（ELISA）试剂盒检测血清中促肾上腺皮质激素（Orexin-A）、促肾上腺皮质激素（CRH）、促肾上腺皮质激素（ACTH）、肾上腺皮质激素（CORT）及下丘脑中5-羟色胺（5-HT）、多巴胺（DA）、去甲肾上腺素（NE）水平。采用高效液相色谱法测定γ -氨基丁酸（GABA）和谷氨酸（Glu）的含量。Western blot （WB）和免疫组化染色（ICH）检测5HT1A受体（5-HTRIA）和食欲素受体2抗体（OX2R）的表达。采用苏木精和伊红（H&E）染色及尼氏染色评价下丘脑组织的组织学变化。结果：相对于安慰剂组，MSZRD可缩短失眠小鼠的睡眠潜伏期（P < 0.05, 0.01），延长小鼠的睡眠时间（P < 0.05, 0.01），改善小鼠的昼夜节律紊乱。此外，MSZRD有效提高失眠小鼠下丘脑5-HT和5-HTR1A蛋白含量（P < 0.05, 0.01），下调DA和NE含量（P < 0.05, 0.01）。重要的是，MSZRD组血清GABA浓度升高（P < 0.05）， Glu/ GABA比值降低（P < 0.05）。此外，MSZRD降低了HPA轴的CORT、ACTH和CRH相关激素水平（P < 0.05, 0.01）。同时，MSZRD显著下调失眠小鼠血清Orexin-A含量（P < 0.05）和下丘脑OX2R表达（P < 0.05）。此外，MSZRD还能改善失眠小鼠下丘脑的组织病理学改变。结论：MSZRD对失眠小鼠模型有改善睡眠作用。其机制可能是调节Orexin-A的表达影响失眠小鼠下丘脑轴的稳态及相关神经递质的释放。
Aim: Modified Suanzaoren Decoction (MSZRD) is obtained by improving Suanzaoren Decoction (SZRT), a traditional Chinese herbal prescription that has been used to treat insomnia for more than thousands of years. Our previous study showed that MSZRD can improve the gastrointestinal discomfort related insomnia by regulating Orexin-A. This study is the first study to evaluate the effects and possible mechanisms of MSZRD in mice with insomnia caused by p-chlorophenylalanine (PCPA) combined with multifactor random stimulation. Methods: After 14 days of multifactor stimulation to ICR mice, a PCPA suspension (30 mg/mL) was injected intraperitoneally for two consecutive days to establish an insomnia model. Three different doses of MSZRD (3.6, 7.2, and 14.4 g/kg/day) were given to ICR mice for 24 days. The food intake and back temperature were measured, and behavioral tests and pentobarbital sodium-induced sleep tests were conducted. The levels of Orexin-A, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and adrenocortical hormones (CORT) in the serum and 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in hypothalamus were measured using enzyme-linked immunosorbent assay (ELISA) kits. The levels of gamma-aminobutyric acid (GABA) and glutamic acid (Glu) were measured by high-performance liquid chromatography (HPLC). The expression of 5HT1A receptor (5-HTRIA) and orexin receptor 2 antibody (OX2R) was measured by Western blot (WB) and immunohistochemical staining (ICH). Hematoxylin and eosin (H&E) staining and Nissl staining were used to assess the histological changes in hypothalamus tissue. Results: Of note, MSZRD can shorten the sleep latency of insomnia mice (P < 0.05, 0.01), prolonged the sleep duration of mice (P < 0.05, 0.01), and improve the circadian rhythm disorder relative to placebo-treated animals. Furthermore, MSZRD effectively increased the content of 5-HT and 5-HTR1A protein in the hypothalamus of insomnia mice (P < 0.05, 0.01), while downregulated the content of DA and NE (P < 0.05, 0.01). Importantly, serum GABA concentration was increased by treatment with MSZRD (P < 0.05), as reflected by a decreased Glu/ GABA ratio (P < 0.05). Moreover, MSZRD decreased the levels of CORT, ACTH, and CRH related hormones in HPA axis (P < 0.05, 0.01). At the same time, MSZRD significantly downregulated the serum Orexin-A content in insomnia mice (P < 0.05), as well as hypothalamic OX2R expression (P < 0.05). In addition, MSZRD also improved the histopathological changes in hypothalamus in insomnia mice. Conclusion: MSZRD has sleep-improvement effect in mice model of insomnia. The mechanism may be that regulating the expression of Orexin-A affects the homeostasis of HPA axis and the release of related neurotransmitters in mice with insomnia.