Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis

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. 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 γ-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. 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. 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.