Study On Mechanism Of Antidepressant And Anti-insomnia Based On Combined Use Of The Magnoflorine And Spinosin From ZSS

Objective:Using network pharmacology and molecular docking technology to explore the potential targets and mechanisms of Magnoflorine and Spinosin to exert the synergistic effect of antidepressant insomnia;To study the effect of p H on the apparent oil-water partition coefficient of Magnoflorine and Spinosin from Zizyphi Spinosae Semen after combination of two chemical compounds,and the effect of the pharmacokinetics of Magnoflorine and Spinosin after combination;To investigate the antidepressant effect of Magnoflorine-Spinosin combination and antidepressant mechanism through Akt-mTOR pathway.Methods:1.The network pharmacology was used to predict the action targets and pathways of Magnoflorine and Spinosin and using Autoduck software to dock Magnoflorine and Spinosin with depressed Targets,the synergistic mechanism of Magnoflorine and Spinosin through Akt-mTOR pathway was demonstrated by calculating the binding energy of receptor and ligand and analyzing docking sites.2.Using shaking flask method to simulate human environment,HPLC was used to detect the concentration of Magnoflorine and Spinosin under different p H conditions,and to determine the effect of combination on oil-water partition coefficient of Magnoflorine and Spinosin.3.HPLC was used to detect the blood concentration of each mouse at different time points in Magnoflorine group(17.0 mg·kg-1),)Spinosin group(23.7 mg·kg-1)and Magnoflorine-Spinosin group(17.0 mg·kg-1+23.7 mg·kg-1),and to determine the effect of combined use on the pharmacokinetics of Magnoflorine and Spinosin in vivo.4.A model of depression and insomnia in mice was constructed using Chronic Unpredictable Mild Stress(CUMS)stimulation combined with horizontal table sleep deprivation,the antidepressant effect of high-dose Magnoflorine-Spinoincombination was determined by detecting behavioral indicators such as weight growth rate,suspension immobility time,forced swimming time,pentobarbital sodium synergistic sleep time,and number of autonomous activities in mice.5.Quantitative Real-time PCR(q PCR)were used to detect the m RNA expression of IDO、IL-1β in the hippocampus of high Magnoflorine group,low Magnoflorine group,high Spinosin group and high Magnoflorine-Spinosin group,and mmunoblotting(Western Blot)were used to detect the protein expression of IDO 、 IL-1β 、 p-m TOR and p-Akt in each group to study effects of Magnoflorine-Spinosin combination on Akt-mTOR pathway.Results:1.Network pharmacology predicts that Magnoflorine and Spinosin exert synergistic antidepressant effects through the co-acting target protein monoamine oxidase B(MAO-B)and Metabolic pathways pathways.Molecular docking results showed that Magnoflorine and Spinosin had low binding energy with Akt、m TOR and IDO and could act on the same active sites and different active sites.Therefore,the combination of Magnoflorine and Spinosin may exert synergistic antidepressant insomnia efficacy through the Akt-mTOR pathway.2.The experimental results of oil-water distribution coefficient showed that in neutral environment,the combination of drugs increased the water solubility of Spinosin and increased the fat solubility of Magnoflorine at the same time.3.Results of pharmacokinetic experiments showed that the combination of drugs could increase the highest blood concentration(Cmax)of Magnoflorine and Spinosin,slow down its elimination rate(CL),increase the area under the curve of blood drug concentration(AUC),showing better drug synergy.4.The combination of high and low doses of Magnoflorine and Spinosin significantly improved the symptoms of depression and insomnia in CUMS mice according to the behavioral results of mice with CUMS : in the tail suspension test(TST)and forced swimming test(FST),the TST、FST immobility time was decreased in the high-dose Magnoflorine-Spinosin groups compared with the high-dose Magnoflorine and the high-dose Spinosin groups,and in the supraliminal and subliminal pentobarbital dose sodium hypnosis experiment,the sleep percentage increased and the sleep duration time did not change significantly in the low-dose Magnoflorine and Spinosin groups compared with the low-dose Magnoflorine group and the low-dose Spinosin group.5.The q PCR results showed that the expression of IDO、IL-1β in the hippocampus of mice in the low-dose Magnoflorine-Spinosin group decreased and showed a synergistic effect compared with the low-dose Magnoflorine groups and the low-dose Spinosin groups.Compared with the high-dose Magnoflorine-Spinosin groups,the expression of IDO 、 IL-1β in the hippocampus decreased in the low-dose Magnoflorine-Spinosin groups.The Western Blot results showed that the combination of high-dose Magnoflorine-Spinosin significantly increased the expression of IDO、p-Akt、p-m TOR in hippocampus of CUMS mice.Conclusion:Magnoflorine-Spinosin exert synergistic anti-depressant insomnia effect through common targets and pathways,and the two components have the same binding sites for IDO、Akt and m TOR,and also have different binding sites,so they show some synergy.At the same time,it is found that the two-component combination increases the liposolubility of Magnoflorine and the water solubility of Spinosin in neutral,After intravenous injection of two components,Cmax was increased,CL was slowed down,AUC was increased for two components in mice,and the effect was enhanced.The pharmacodynamic experiments of the combination of Magnoflorine and Spinosin in vivo showed that the combination could significantly improve the symptoms of CUMS depressed insomnia mice,and the mechanism of antidepressant insomnia may be associated with the reduction of IDO 、IL-1β and the increased phosphorylation of Akt、m TOR.