Mechanism Of Probiotics Supplementation Ameliorating Intestinal Stress Injury Induced By Sleep Deprivation

Part 1:The Mechanism of Gut Dysbiosis Induced by Sleep Deprivation in Rhesus MonkeysObjective:To explore the possible mechanism of gut dysbiosis induced by sleep deprivation in rhesus monkeys.Methods:We established sleep deprivation rhesus monkey model via sleep rhythm reverse,bright light and noises.The plasma GABA,stress hormones and inflammatory factors was measured by ELISA assays.The relative abundance of gut microbiota and metabolites was measured by 16S rDNA and untargeted LC-MS sequencing,respectively.The potential correlation between plasma GABA and sleep deprivation induced stress responses and gut dysbiosis was analyzed by Pearson correlation analysis.Results:The stimulation,including sleep rhythm reverse,bright light and noises,caused a significant increase of plasma cortisol,norepinephrine and proinflammatory cytokines IL6,IL-8 and TNF-α.In addition,it could lead to a marked reduction of gut microbiota diversity and a significant change in intestinal metabolites.The functional enrichment analysis for differential microbiota and metabolites between sleep deprivation group and control group revealed that the metabolic pathways related to GABA were significantly enriched,including glutamate metabolism and Vitamin B6 metabolism.In addition,we found that the level of plasma GABA in sleep deprivation group was significantly lower than that in control group.Pearman correlation analysis disclosed that plasma GABA was negatively correlated with stress hormones and proinflammatory cytokines IL6,IL-8 and TNF-α,but positively correlated with antiinflammatory cytokine IL-10 and the relative abundance of intestinal Lactobacillus.Conclusion:Sleep deprivation rhesus monkey model could be established via sleep rhythm reverse,bright light and noises stimulation.Sleep deprivation might reduce the inhibitory effect of central nervous system on HPA axis through decreasing the level of plasma GABA,which led to the increase of plasma stress hormones and gut dysbiosis.Part 2:Cortisol Suppressed Intestinal Epithelial Cells Stress Repair Through GR-P21 PathwayObjective:To explore the effect of stress hormones cortisol on the stress repair ability of intestinal epithelial cells NCM460.Methods:NCM460 cells were treated respectively:a.control group:serum-free DMEM+DMSO;b.cortisol intervention group 1:serum-free DMEM+lmmol/L cortisol;c.cortisol intervention group 2:serum-free DMEM+3mmol/L cortisol;d.cortisol intervention group 3:serum-free DMEM+5mmol/L cortisol.The cell viability was examined by CCK8 and colony formation assays.The cell migration was examined by Transwell assays.The western blotting,real-time quantitative PCR,transcriptome sequencing,receptor blocking and lentivirus transfection were used to explore the possible mechanism of cortisol affecting NCM460 proliferation and migration.Results:Cortisol could significantly inhibit NCM460 proliferation in the CCK8 assays and colony formation assays,and suppress NCM460 migration in the Transwell assays.The results of western blot and qRT-PCR demonstrated that cortisol could significantly change the expression of proliferation and migration regulatory genes.The transcriptome sequencing revealed that the level of cyclin-dependent kinase inhibitor P21 was significantly higher in cortisol intervention group than that in control group.In vitro test,cortisol can significantly upregulate the expression of P21 on the level of mRNA and proteins,which can be reversed by glucocorticoid receptor(GR)antagonist RU486.In addition,the inhibition of NCM460 proliferation and migration caused by cortisol could be opposed by the knockdown of P21.Conclusion:Cortisol could significantly inhibit NCM460 proliferation and migration through GR-P21 pathway.Part 3:The Mechanism of GABA-Producing Probiotics Supplementation Improving Stress Responses and Gut Dysbiosis Induced Sleep DeprivationObjective:To explore the possible mechanism of probiotics supplementation improving stress responses and gut dysbiosis induced by sleep deprivation.Methods:Fourteen sleep-deprivation rhesus monkeys were randomized into two groups and treated respectively:a.control group:food and water;b.probiotics intervention group:food and water containing bifid-triple viable capsule.The plasma GABA,stress hormones and inflammatory factors was measured by ELISA assays.The relative abundance of gut microbiota and metabolites was measured by 16S rDNA and untargeted LC-MS sequencing.Results:The results of gut microbiome showed that probiotics supplementation could significantly change the structure of intestinal microbial community,resulting in a significant decrease of Firmicutes,and a marked increase of Bacteroidetes and Lactobacillus.The results of metabolomic analysis showed that probiotics supplementation could change 31.7%(72/227)differential metabolites induced by sleep deprivation.The functional enrichment analysis for differential microbiota and metabolites between probiotics supplementation group and control group revealed that the metabolic pathways related to GABA were significantly enriched,including tricarboxylic acid cycle,glutamate metabolism and Vitamin B6 metabolism.The results of ELISA demonstrated that the level of plasma GABA in probiotics supplementation group was significantly higher than that in control group,but the level of cortisol,norepinephrine,and proinflammatory cytokines IL-8 and TNF-α in probiotics intervention group was significantly lower than that in control group.Conclusion:Probiotics supplementation could significantly increase the relative abundance of intestinal Lactobacillus and the absorption of dietary GABA,which could enhance the inhibitory effect of central nervous system on HPA axis,reduce the level of plasma stress hormones,and improve gut dysbiosis.