Study On The Improvement And Mechanism Of Water Extract Of Eucommia Ulmoides Oliver On Insomnia In Mice

Insomnia is a sleep disorder caused by a variety of causes.It is characterized by difficulty falling or staying asleep despite adequate opportunities for sleep,which interferes with the body’s normal daytime activities.Insomnia not only causes metabolic disorders and mental dysfunction in the body,but also increases the risk of diseases.Eucommia ulmoides Oliver is a traditional Chinese medicine(TCM),which is slightly warm in nature and pungent and sweet in flavour.It has such pharmacological effects as nourishing the liver and kidney,strengthening the muscles and bones and enhancing the will.At present,TCM practitioners often use Eucommia ulmoides Oliver with other herbs in the clinical treatment of insomnia.However,the improvement effect and molecular mechanism of Eucommia ulmoides Oliver on insomnia are not clear.Therefore,in this study,eszopiclone was chosen as a positive control and the mouse models of insomnia were established by using DL-4-chlorophenylalanine(PCPA).The improvement effect and mechanism of water extract of Eucommia ulmoides Oliver on insomnia in mice were studied by using network pharmacology,molecular docking,behavioral assay,fluorescence quantitative PCR,immunohistochemistry,Enzyme immunosorbent and Western blot.Results were as follows:(1)Network pharmacological analysis and molecular docking validation of the mechanism of Eucommia ulmoides Oliver in improving insomnia.TCMSP,OMIM and Gene Cards databases were used to obtain 89 targets for Eucommia ulmoides Oliver and 557 targets for insomnia.Then the intersection was taken to construct the protein interaction network to screen the core targets(HTR3A,DRD2,GABRA1,GABRA2and HTR2A).Gene ontology(GO)analysis and enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG)were performed by using the DAVID database.A total of 57 biological processes,15 cellular components,15 molecular functions and 7KEGG signaling pathways were obtained.By constructing a network of Eucommia ulmoides Oliver constituents-insomnia targets-KEGG pathways,main water-soluble active components of Eucommia ulmoides Oliver for improving insomnia were screened as(-)-Tabernemontanine and Epiquinidine.The molecular docking results showed that(-)-Tabernemontanine and Epiquinidine had good docking activities with DRD2,GABRA1 and HTR2A.(2)Watre extract of Eucommia ulmoides Oliver has improving effects on insomniac mice.A suprathreshold dose of pentobarbital sodium(50 mg/kg)induced sleep experiment was performed in mice.The results showed a significant increase in sleep latency and a decrease in sleep duration when mice were intraperitoneally injected with PCPA.Insomnia model established successfully.And the water extract of Eucommia ulmoides Oliver could effectively increase the sleep rate,shorten the sleep latency and prolong the sleep time in PCPA insomniac mice.The results of behavioral tests showed that water extract of Eucommia ulmoides Oliver also reduced excitatory and anxiety-like behaviors in PCPA insomniac mice in the open field and elevated cross maze.The mRNA expression levels of the hypothalamic Clock genes in mice were further examined by using fluorescent quantitative PCR.The results showed that water extract of Eucommia ulmoides Oliver could up-regulate the mRNA expression levels of Clock and Bmal1 genes and down-regulate the mRNA expression levels of Per1 gene in hypothalamus of insomniac mice.Immunofluorescence staining results showed that water extract of Eucommia ulmoides Oliver could reduce the density of c-Fos~+cells in the hippocampus CA3 region of insomniac mice and inhibit stress in these neurons.(3)Regulation of 5-HT,GABA and DA in insomniac mice by water extract of Eucommia ulmoides Oliver.The results of Enzyme immunosorbent showed that water extract of Eucommia ulmoides Oliver could inhibit the decrease of hydroxy tryptamine(5-HT),dopamine(DA)andγ-aminobutyric acid(GABA)levels in the serum of insomniac mice.Combined with the results of network pharmacology,the predicted targets were validated by using fluorescence quantitative PCR,and it was found that water extract of Eucommia ulmoides Oliver could up-regulate the mRNA expression levels of HTR1A,GABRA1 and GABRA2 genes and down-regulate the mRNA expression levels of HTR2A and DRD2 genes in the hypothalamus of insomniac mice.Western blot was used to assess the expression of relevant receptors and proteins on the DAergic synaptic signaling pathway.The results showed that water extract of Eucommia ulmoides Oliver could inhibit the expression of DRD2 receptors,promote the expression of dopamine transporter(DAT)and phosphorylation of protein kinase B(Akt)in the hypothalamus of insomniac mice.In conclusion,water extract of Eucommia ulmoides Oliver can inhibit the decrease of 5-HT,GABA and DA levels in insomnia mice by regulating the expression of relevant synaptic receptors and proteins in Serotonergic,GABAergic and Dopaminergic synaptic signal pathways,and thus perform its effects of improving insomnia and relieving anxiety.