| Objective:To explore the role and mechanism of chronic stressinduced gut microbial dysbiosis in stress-related brain injury.Methods:(1)In order to explore the role of gut microbial dysbiosis induced by chronic stress in stress-related brain injury,adult SPF SD rats were randomly divided into control group(control),chronic stress group(stress),gut microbiome transplantation group(FMT)and sham transplantation group(sham).The control group was fed normally for 4weeks;Rats in chronic stress group received restraint stress every night for4 weeks;The gut microbiome transplantation group received cecal content transplantation of rats on the 7th,14 th,21st and 28 th day of chronic stress,once a week,four times in total;Rats in the sham transplantation group received intestinal catheters 4 times,once a week.The above four groups began the following behavioral tests on the day after the last stress: open field experiment(detecting activity ability),new object recognition experiment(detecting learning and memory ability),Barnes maze experiment(detecting learning and spatial memory ability),and forced swimming experiment(detecting anxiety and depression).After the last stress,the peripheral blood of rats was taken to detect glucocorticoid and IL-6;The cecal contents were taken and the microbial were detected by16 S r RNA gene sequencing and analysis.(2)In order to test whether probiotics can improve the brain injury induced by chronic stress,adult SPF SD Rats were randomly divided into control group,chronic stress + probiotics group and chronic stress +placebo group.The rats in chronic stress + probiotic group were supplemented with probiotics from drinking water every day during 4weeks of restraint stress;Rats in chronic stress + placebo group were supplemented with placebo from drinking water every day during 4 weeks of restraint stress.All rats in the above groups began the open field,new object recognition,Barnes maze and forced swimming experiments on the day of the end of the last stress.After the last stress,the peripheral blood of rats was taken to detect glucocorticoid and IL-6;The cecal contents were taken and the microbial were detected by 16 S r RNA gene sequencing and analysis.(3)In order to explore the mechanism of gut microbial dysbiosis affecting brain injury under chronic stress,metabolomics detection and differential metabolite analysis were carried out on the peripheral blood of control group,chronic stress group,gut microbiome transplantation group and sham transplantation group rats in(1)after the end of the last stress.Further,the differential metabolites(such as Vit B6 metabolite product-4-pyridoxic acid)were quantitatively verified by Quantitative liquid chromatography-mass spectrometry(HPLC-MS);The expression of alkaline phosphatase(Vit B6 key metabolic enzymes)in colon of each group were detected by immunohistochemistry.(4)In order to further confirm whether Vit B6 metabolic disorder is the mechanism of gut microbial dysbiosis affecting brain injury under chronic stress,adult SPF SD rats were randomly divided into control group,chronic stress + Vit B6 group(stress + Vit B6)and chronic stress + saline group(stress + saline).The rats in chronic stress + Vit B6 group received restraint stress for 4 weeks and were injected intraperitoneally with 10mg/ kg Vit B6 every day;Rats in the chronic stress + saline group received restraint stress for 4 weeks and were injected with equal volume of saline intraperitoneally every day.All rats in the above groups began the open field,new object recognition,Barnes maze and forced swimming experiments on the day of the end of the last stress.After the last stress,the peripheral blood of rats was taken to detect glucocorticoid,IL-6 and blood cell count;The activation of microglia and the level of neurogenesis in rat hippocampus were detected by immunohistochemistry,the expression of IL-6 in hippocampus was detected by ELISA,and the protein expression of hippocampal synaptic markers SYN and PSD95 were detected by western blot.The expression of ALP in liver and colon was detected by immunohistochemistry,and the morphological changes of ileum were detected by HE staining;Rats peritoneal macrophages were extracted and the expression of related inflammatory pathway signal molecules was detected by q PCR.Results:(1)Compared with the normal group,the level of anxiety and depression increased,the ability of learning and memory decreased,the levels of peripheral blood glucocorticoid and IL-6 increased,and the abundance percentage of five gut microbiotas(g＿lactobacillus,g＿ruminococcus,g＿lachnospiraceae＿nk4a136＿group,g＿norank＿f＿norank＿o＿clostridia＿ucg-014,g＿romboutsia)decreased significantly in the chronic stress group.Compared with the sham transplantation group,the levels of anxiety and depression increased,learning and memory ability decreased,the levels of peripheral blood glucocorticoid and IL-6 increased,and the abundance percentage of the above five gut microbiotas decreased significantly in gut microbiome transplantation group.(2)Compared with the control group,the chronic stress + placebo group had higher levels of anxiety and depression,lower learning and memory ability,higher levels of peripheral blood glucocorticoid and IL-6,and lower abundance percentages of the above five gut microbiotas(g＿lactobacillus,g＿ruminococcus,g＿lachnospiraceae＿nk4a136＿group,g＿norank＿f＿norank＿o＿clostridia＿ucg-014,g＿romboutsia).Compared with the chronic stress + placebo group,the chronic stress + probiotics group significantly reduced the levels of anxiety and depression,significantly enhanced the ability of learning and memory,significantly reduced the levels of peripheral blood glucocorticoid and IL-6,and increased the abundance percentage of the above five gut microbiotas.(3)Compared with the control group,the levels of 249 metabolites in peripheral blood of chronic stress group changed significantly.Compared with the sham transplantation group,the levels of 49 metabolites in peripheral blood of gut microbiome transplantation group changed significantly.The changes of 20 metabolites were similar in chronic stress group and gut microbiome transplantation group.HPLC-MS showed that 4-PA decreased significantly in the plasma of chronic stress group and gut microbiome transplantation group.The expression of ALP in colon epithelial cells decreased in chronic stress group and gut microbiome transplantation group.(4)Compared with the control group,the learning and memory ability decreased,the levels of peripheral blood glucocorticoid and IL-6increased,the activation level of hippocampal microglia and IL-6 increased,the neurogenesis level of hippocampal neurons,weight,the number of peripheral blood cells and the expression of ALP in liver decreased,and the level of IL-6 m RNA in peritoneal macrophages increased in the chronic stress + saline group.Compared with the chronic stress + saline group,the learning and memory ability enhanced,the level of IL-6 in peripheral blood decreased,the activation level of hippocampal microglia and the level of IL-6 in hippocampus decreased,the neurogenesis level of hippocampal neurons,weight and the expression of ALP in liver increased,and the expression level of IL-6 m RNA in peritoneal macrophages decreased in the chronic stress + Vit B6 group,but the level of glucocorticoid in peripheral blood had no significant change.Conclusion:Gut microbial dysbiosis under chronic stress affects the occurrence and development of brain injury,and the metabolic disorder(especially vitamin B6 metabolic disorder)caused by gut microbial dysbiosis is one of the key mechanisms. |
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