Objective To investigate the effect of Hehuan Anshen Prescription on behavior, hypothalamic morphology, and the expression of sleep⁃wake related substances in mice with insomnia induced by para⁃chlorophenylalanine (PCPA). Methods Sixty mice were randomly divided into control group (n=15) or model group (n=45). Mice in the model group were intraperitoneally injected with PCPA for 4 consecutive days to establish an insomnia model, while mice in the control group were intraperitoneally injected with an equal volume of sterile normal saline for 4 consecutive days. Two hours after the last intraperitoneal injection, the pentobarbital sodium righting reflex test was used to validate whether the insomnia mice model was successfully established. The successfully modeled insomnia mice were randomly divided into PCPA model group, Hehuan Anshen Prescription group, or estazolam group (with 15 mice in each group). Intragastric administration of Hehuan Anshen decoction or estazolam suspension was given to the Hehuan Anshen Prescription group or the estazolam group, and the equivalent volume of sterile normal saline was given to the control group and the PCPA model group, for consecutive 7 days. After the end of intragastric administration, the behavioral changes (sleep latency, sleep duration, total area distance, total area average speed, total area immobility time), hypothalamic neuronal morphological changes, and the protein and mRNA expressions of dopamine D1 receptor (D1R), prostaglandin D2 (PGD2), γ⁃aminobutyric acid (GABA), and 5⁃hydroxytryptamine (5⁃HT) in hypothalamic tissue were compared between mice in various groups. Results Two hours after the last intraperitoneal injection, compared with the control group, the sleep latency of the model group was prolonged and the sleep duration was shortened (P<0.05), indicating successful modeling. After the end of intragastric administration, the sleep latency and total area immobility time of the PCPA model group were longer than those of the control group, and the sleep duration, total area distance, and total area average speed were shorter/slower than those of the control group (P<0.05); moreover, the sleep latency and total area immobility time of the Hehuan Anshen Prescription group and the estazolam group were shorter than those of the PCPA model group, and the sleep duration, total area distance, and total area average speed were longer/faster than those of the PCPA model group (P<0.05); however, there was no statistically significant difference in the aforementioned indicators between the Hehuan Anshen Prescription group and the estazolam group (P>0.05). The results of Nissl staining revealed that compared with the control group, the number of Nissl bodies in the hypothalamus of the PCPA model group was significantly reduced, Nissl staining was weakened, some neurons appeared shrunken, cell arrangement was disordered, nuclei were pyknotic, and the structural integrity of some neurons was impaired; in addition, compared with the PCPA model group, the number of Nissl bodies in the hypothalamus of the Hehuan Anshen Prescription group and the estazolam group was increased, Nissl staining was enhanced, neuronal morphology and structural integrity were improved, and the improved effects of both groups were similar. The results of transmission electron microscopy elucidated that compared with the control group, the hypothalamic neuronal nuclear membrane of the PCPA model group was blurred, mitochondria were shrunken, cristae were reduced or disappeared, and the ultrastructural damage was significant; furthermore, compared with the PCPA model group, the improvement of hypothalamic neuronal organelle and ultrastructural damage in the Hehuan Anshen Prescription group and the estazolam group were ameliorated, and the improved effects of both groups were similar. The expressions of D1R protein and mRNA in the hypothalamus of the PCPA model group were higher than those of the control group, while the expressions of PGD2, GABA, and 5⁃HT protein and mRNA were lower than those of the control group (P<0.05). The expressions of D1R protein and mRNA in the hypothalamus of the Hehuan Anshen Prescription group and the estazolam group were lower than those of the PCPA model group, and the expressions of PGD2, GABA, and 5⁃HT protein and mRNA were higher than those of the PCPA model group (P<0.05). The GABA protein and GABA and 5⁃HT mRNA expressions of the estazolam group were lower than those of the Hehuan Anshen Prescription group (P<0.05). Conclusion Hehuan Anshen Prescription may regulate the dynamic balance of hypothalamic sleep⁃wake substances and maintain the sleep state of mice by reducing the expression of the excitatory neurotransmitter D1R, increasing the release of 5⁃HT, and enhancing the expression of the endogenous sleep⁃promoting substance PGD2 and the inhibitory neurotransmitter GABA.

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