| Objective By observing macro representation,behavioral experiment and sleep detection of chronic stress rats with insomnia,untargeted metabonomics analysis was used to study the anti-insomnia effect and metabolic regulation mechanism of Mongolian medicine Sugmule-4.Method The rats were modelled using a combination of both tail-clamping stimulation and intraperitoneal injection of p-chlorophenylalanine solution.Thirty-six male rats were randomly divided into six groups: normal group,model group,Sugammul-4 high,medium and low(5.2 g/kg,3.6 g/kg,1.8 g/kg)dose administration group,and diazepam(0.92 mg/kg)group.Each group of 6 rats was placed in the same feeding cage,and the rats’ tails were clamped with a gauze-wrapped hemostatic forceps for 45 min,and stimulated to fight with the rats in the same cage to ensure that the tails did not break the skin and bleed for 21 days.On the 16 th and 17 th days of modelling,rats in the model group,the 3 doses of Sugammul-4 administration group,and the diazepam group were injected intraperitoneally with p-chlorophenylalanine solution at 0.4 g/kg body weight.The normal group was administered with an equal volume of carbonate buffer solution.The rats were administered once daily by gavage from day 7 of tail clamping stimulation.The water maze experiment was used to test the learning and memory abilities of the rats,and the spontaneous activity experiment and forced swimming experiment were used to evaluate the mental status of the rats.A non-invasive sleep monitor was used to monitor the sleep duration of the rats for 18 h.The metabolite data were normalized,and the raw peak area of each metabolite was log-transformed(log2)with a base of 2 to reduce the skewed distribution of the values and bring the data closer to a normal distribution,and then normalized using the median.Missing values were then filled in with the smallest value of all samples.The metabolites with significant differences between groups were screened by univariate statistical method of Wilcoxon signed rank sum test,multivariate statistical method of principal component analysis and partial least squares discriminant analysis,and the main metabolic pathways of metabolites with significant differences were obtained based on KEGG pathway enrichment analysis.Result 1.Anti-insomnia effect of Sugammul-4In the model group,rats were observed to have disturbed sleep cycles,depression,and escape after provocation,significantly reduced food intake,dry faeces and greyish colour,and scattered and dull fur.The rats in the model group showed a significant decrease in activity time and number of paw lifts(P<0.01),a significantly longer escape latency(P<0.01),fewer platform crossings(P<0.01)and a significant decrease in sleep time(P<0.01)compared to the normal group,all indicating the success of the experimental model of chronic stress insomnia in rats.Compared with the model group,the rats in the middle and high dose groups of Sugammul-4 basically had a sleep rhythm,a more stable mental state,a neater coat and a normal diet,and their general behavioural status was found to be better than that of the model group,and the behavioural experiments and the monitored sleep time were close to those of the normal group,indicating that the Mongolian drug Sugammul-4 had a therapeutic effect on insomnia.2.Sugammul-4 metabolomic mechanisms include the effects of multiple metabolites as followsTryptophan metabolism and product levels: The serum levels of 5-hydroxytryptamine were significantly lower in the model group compared with the normal group(P<0.05),and the serum levels of 5-hydroxytryptamine and 5-hydroxyindole acetate,a breakdown product of 5-hydroxytryptamine,were significantly higher in the rats treated with diazepam or sugammul-4 compared with the model group(P<0.05).In the model group,kynurenine levels were significantly lower(P<0.05)and kynurenine quinolinic acid was significantly increased(P<0.05)in serum compared to the normal group of rats,whereas kynurenine was significantly higher(P<0.05)and kynurenine quinolinic acid was significantly downregulated(P<0.05)in serum samples after treatment with diazepam and sugammul-4 at moderate and high doses.Histidine metabolism and its product levels: Histidine and most of its downstream metabolites were significantly lower in the hippocampal region of model rats compared to the normal group(P<0.05).Histidine metabolites were increased in the hippocampal region of rats after diazepam and sugammul-4 treatment(P<0.05).In contrast,most of the identified histidine metabolites in serum samples did not change between control and model groups or after drug treatment administrationTyrosine metabolism and its products levels: compared to the normal group,tyrosine metabolism was disturbed in the hippocampus of the model rats,with down-regulated expression of tyrosine and many of its related metabolites(1-carboxyethyl tyrosine,N-acetyl tyrosine,etc.)(P<0.05).These alterations were improved after diazepam and sugammul-4 treatment.Intestinal metabolites: Phenol levels in serum and hippocampal tissue were significantly reduced(P<0.05)and phenol sulfate levels in serum and hippocampal tissue were significantly increased(P<0.05)in model rats after diazepam and sugammul-4treatment;primary bile acid taurine and deoxycholic acid were significantly elevated(P<0.05)in the serum of insomniac rats,and the above substances were significantly These substances were significantly(P<0.05)down-regulated by Sugammul-4 treatment,and secondary bile acid metabolites were changed.Conclusion 1.Sugammul-4 has an anti-insomnia effect,restoring normal sleep rhythms,prolonging sleep time and improving learning and memory in rats with chronic stress insomnia.2.Sugammul-4 plays an important role in the development of insomnia by affecting the amino acid metabolic pathways of tryptophan,histidine and tyrosine,their metabolites and the metabolism of intestinal flora. |
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