Intervention Of Compound Fermentation On PCPA-induced Sleep Deprivation Mice

Sleep is a key determinant of physical and mental health.With the rapid development of society,such as circadian rhythm,diet and life stress will affect sleep quality.Lack of sleep will also lead to low immunity,mental or emotional disorders etc.,which may induce many diseases.Long-term medication for insomnia can have side effects such as addiction,memory or movement disorder and tolerance.The research progress of Chinese medicine and modern biomedicine shows that cereal buds and Zizyphus jujuba,which are homologous to medicine and food,have a good intervention effect on insomnia,while probiotics can improve the immunity of the body and promote sleep.In this study,Kombucha,Acetic acid bacteria,Yeast and Lactobacillus plantarum were used to ferment with Zizyphus jujuba,cereal buds and grape seed to explore the improvement and mechanism of natural drugs on insomnia.Objective: To explore the ameliorative effect and potential mechanism of probiotic transformation products such as cereal bud and wild jujube on P-chloro-phenylalanine(PCPA)sleep deprivation mice,and to provide theoretical and experimental basis for developing natural drugs to relieve insomnia.Methods:(1)Sensory identification method was used to optimize the formula of fermentation raw materials and the composition and ratio of fermentation strains;(2)The contents of γ-aminobutyric acid(GABA)and L-theanine were detected by ELISA and HPLC;(3)Sleep deprivation model was established by intraperitoneal injection of PCPA in C57BL/6 mice,and whether the model was successful or not was judged by pentobarbital sodium sleep synergy experiment;(4)The sleep behavior of normal Control group,Model group,compound fermentation product(KFY)group and positive Control Diazepam(DZP)group were analyzed by pentobarbital sodium sleep synergistic experiment;(5)Analyze the changes of emotion and behavior of mice before and after intervention by open field experiment;(6)The changes of 5-hydroxytryptamine(5-HT),GABA and Glutamic acid(Glu)in hypothalamus were analyzed by ELISA before and after the intervention;(7)Using ELISA technique to analyze the changes of serum levels of Interleukin-6(IL-6),Interleukin-6(IL-1 β)and Tumor necrosis factor-α(TNF-α)before and after the intervention;(8)GC-MS was used to analyze the difference of intestinal short-chain fatty acid content of mice before and after intervention,and to analyze the effect of compound fermentation products on intestinal short-chain fatty acid synthesis and its possible mechanism;(9)16S r DNA sequencing was used to analyze the effect of compound fermentation products on intestinal flora of mice before and after intervention and its possible mechanism.Results:(1)1:30 was the best material-liquid ratio of cereal medium,and the strains of Angel yeast(SY)0.02 g,Angel yeast(RW)0.02 g,Acetobacter 0.02 g,Lactobacillus plantarum L9 4% and Tea Fungus 6% were used as the combined fermentation formula;(2)The contents of GABA and L-theanine in 7 L fermentation products were 12.555μ mol/L(1.295 mg/L)and 0.471 mg/m L respectively by ELISA;(3)Sleep deprivation mice model was successfully established by intraperitoneal injection of 400 mg/kg PCPA for 2 days,and the sleep duration of each mouse was significantly reduced compared with that before the model was established;(4)The sleep behavior of mice induced by PCPA showed that the sleep incubation period increased and the sleep duration decreased;After 14 days of administration,the sleep incubation period of mice in KFY group decreased(P > 0.05)and the sleep duration increased(P < 0.0001)compared with that in Model group;(5)Mice induced by PCPA showed that the frequency of crossing the central compartment decreased,while the frequency of defecation and grooming increased;After14 days of administration,compared with Model Group,the times of crossing the central compartment in KFY Group increased(P < 0.05),while the times of defecation and grooming decreased(P > 0.05 and P < 0.05);(6)The levels of 5-HT and GABA in hypothalamus of mice induced by PCPA decreased,while the levels of Glu and Glu/GABA increased;The levels of 5-HT,GABA,Glu and Glu/GABA were improved after 14 days of administration;(7)The levels of TNF-α and IL-6 increased and the level of IL-1 β decreased in mice induced by PCPA;After 14 days of administration,the levels of TNF-α in KFY group were higher than those in Model group(P > 0.05),while the levels of IL-6 and IL-1 β were lower than those in Model group(P < 0.01 and P > 0.05);(8)Propionic acid level increased most obviously in mice induced by PCPA;After14 days of administration,all seven short-chain fats changed;(9)The composition and diversity of intestinal flora in mice induced by PCPA changed.Conclusion:(1)Compound fermentation can improve PCPA-induced sleep deprivation by shortening sleep incubation period and prolonging sleep duration;(2)Compound fermentation can alleviate anxiety induced by PCPA;(3)By supplementing GABA and 5-HT in hypothalamus,the compound fermentation can reduce the sleep-wake imbalance induced by PCPA regulated by Glu;(4)The low sleep quality induced by PCPA was related to the high propionic acid concentration,and the compound fermentation products had influence on the microflora producing short chain fatty acids;(5)Sleep deprivation induced by PCPA can affect the changes of intestinal flora composition.Compound fermentation can intervene the flora imbalance caused by sleep deprivation by increasing the abundance of beneficial bacteria Lactobacillus,reducing Bacteroides,and increasing the ratio of Sclerenchyma/Bacteroides.