OBJECTIVE Based on network pharmacology and experimental validation to reveal the mechanism of action of Shuangxia Decoction in the treatment of insomnia and to provide data support for clinical use.

METHODS The effective active ingredients and targets of Shuangxia Decoction were screened out by ETCM database, the targets associated with insomnia were screened out by CTD database, the meeting points between insomnia and Shuangxia Decoction were obtained by Venn diagram. In order to acquire the core targets and perform visual analysis, the STRING database’s PPI was imported into Cytoscape 3.9.1. DAVID’s database was used to analyze the role of GO and the pathway enrichment of KEGG, and preliminarily predicting the mechanism of action of Shuangxia Decoction in treating insomnia. Used 4-chlorophenylacetic acid(PCPA) insomnia model mice as the primary research subject, conducted behavioral experiments on the elevated cross maze, 5-hydroxytryptamine(5-HT), 5-hydroxyindole acetic acid(5-HIAA) and 5-hydroxytryptophan(5-HTP) content was detected by LC-MS. Interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), 5-hydroxytryptamine 1A receptor(5-HT1AR), 5-hydroxytryptamine 2A receptor(5-HT2AR), 5-hydroxytryptamine transporter protein(5-HTT) and tryptophan hydroxylase 2(TPH2) gene expression were detected by qRT-PCR. Nissl staining was used to observe neuronal cell morphology in brain tissue, as well as protein expression of 5-HT1AR, 5-HT2AR, 5-HTT and TPH2 detected by immunohistochemical.

RESULTS The network pharmacological analysis obtained 49 kinds of chemically active ingredients, 11 kinds of main acting ingredients, 107 drug-disease intersection targets and 17 core targets of Shuangxia Decoction for the treatment of insomnia. It was hypothesized that the main active ingredient in Shuangxia Decoction may achieve sedative and tranquilizing effects through anti-inflammatory, protection of nervous cells and that the key targets were mainly in the signaling pathways of serotonergic synapse, amphetamine addiction, cAMP signaling pathway, and neuroactive ligand-receptor interactions. The results of the animal experiments showed that, compared with the normal group, the distance, dwell time and number of entries in the open arm of the elevated cross maze were significantly lower in the model group(P<0.05 or P<0.01); the gene expression of IL-1β, IL-6, and TNF-α were significantly higher(P<0.01), Nissl staining showed oedema of neuronal cells, vacuolar degeneration, Nissl's bodies were lightly stained and fewer in number and unevenly distributed; the contents of 5-HT, 5-HTP and 5-HIAA were significantly lower in the prefrontal cortex(PFC) of mice(P<0.01); the gene and protein expression of 5-HT2AR was significantly higher in the PFC of mice(P<0.05 or P<0.01), as well as the gene and protein expression of 5-HT1AR and 5-HTT were significantly decreased(P<0.01); the gene and protein expression of TPH2 in the dorsal raphe nucleus(DRN) were significantly decreased(P<0.01). Compared with the model group, the distance, residence time and number of entries in the open arm of the elevated cross maze were significantly higher in the Shuangxia Decoction groups(P<0.05 or P<0.01); the gene expression of IL-1β, IL-6, and TNF-α were significantly decreased(P<0.01), brain tissue neuronal cell damage had improved to varying degrees, with relatively clear structures and relatively regular Nissl's bodies; the contents of 5-HT, 5-HTP and 5-HIAA in the PFC, as well as the gene and protein expression of TPH2 in the DRN were significantly increased in both the positive and Shuangxia Decoction groups(P<0.05 or P<0.01). In the positive group, the gene expression of 5-HTT and the protein expression of 5-HT1AR in the PFC were significantly increased, while the gene expression of 5-HT2AR was significantly decreased(P<0.05). In the Shuangxia Decoction group, the gene and protein expression of 5-HT1AR and 5-HTT in the PFC were significantly increased(P<0.05 or P<0.01), while the gene and protein expression of 5-HT2AR were significantly decreased(P<0.01). Compared with model group, in the Pinelliae Rhizoma and Prunellae Spica groups, the protein expression of 5-HT2AR was significantly lower(P<0.05 or P<0.01) and the protein expression of TPH2 in DRN was significantly increased(P<0.05).

CONCLUSION Treatment of PCPA insomnia model mice by Shuangxia Decoction may be achieved by anti-inflammation, protection of neurons and increasing the level of 5-HTP and promoting the synthesis, reuptake and metabolism of 5-HT.