| 1.BackgroundDepressive disorder(MDD)has the highest disability rate in the world and is the main cause of disability among adolescents.Adolescence is the peak period of depression.Stressful experiences in adolescence not only lead to depression-like behaviors,but also change the structure of intestinal flora of host through bidirectional signal communication of microbialenteric-brain axis.Studies have shown that the microbial-gut-brain axis plays an important role in the pathogenesis of depression.As an important component of microbial-gut-brain,regulating the structure of intestinal flora can improve the emotional state of the host.Therefore,intestinal flora may be a new target for the treatment of depression.However,due to the rich diversity of intestinal flora and the complex relationship with intestinal immune and endocrine systems,the mechanism of its improvement of depression symptoms remains unclear.Short chain fatty acids(SCFAs)are small metabolites produced by the fermentation of indigestible carbohydrates by intestinal microorganisms,and their main components include acetate,propionate and butyrate.SCFAs can play an important role in maintaining the integrity of the intestinal barrier and regulating the immune and inflammatory cascade by binding to natural receptors on the cell surface.Therefore,SCFAs are important mediators of intestinal microorganisms in regulating body functions.Microglia are important immune cells in the brain,which secrete inflammatory factors and neurotrophic factors to maintain the homeostasis of the intracerebral environment and participate in neurogenesis and synaptic pruning.The balance between proinflammatory and anti-inflammatory functions of microglia plays an important role in the occurrence and treatment of depression.When the host encounters stress,pathogen invasion,infection and other stressful events,microglia in the brain are activated and release pro-inflammatory factors which causes neuroinflammatory response,and then induces neurotoxicity.Blocking the early activation of microglia can improve depression-like behavior to some extent.The study on the role and mechanisms of microglia in the pathogenesis of depression is important to explore new treatments for depression.Adolescence is a critical period for brain development and maturation.Therefore,studies on adolescent depression must consider the mechanisms by which stress affects neural development.The hippocampus is an important brain region related to learning and memory function and also an important region of neurogenesis.The processes of proliferation,migration,differentiation and maturation of hippocampal neurons are highly regulated by environmental and biological factors.Rodent studies have shown that sustained psychosocial stress leads to impairment of synaptic plasticity,neurodevelopmental disorders,and depressive behavior.The number of granulosa cells in the anterior and middle dentate gyrus is reduced in depressive patients,while antidepressants increase the number of new hippocampal neurons in depressed patients.Therefore,neurogenesis disorder in dentate gyrus(DG)is one of the important pathogenesis of depression.Rifaximin is a non-systemic antibiotic with high safety due to its low systemic absorption rate.Rifaximin treatment promotes the growth of probiotics such as lactobacillus and bifidobacterium,and positively regulates the proportion of intestinal probiotics and potential pathogenic bacteria.In addition,unlike traditional antibiotics,which tend to cause structural imbalance of intestinal flora,rifaximin treatment maintains the stability of the overall structure of intestinal flora and regulates the metabolic function of bacteria.Therefore,we speculate that rifaximin is an effective means to regulate the structure and function of intestinal flora.This study focuses on the effects of chronic unpredictable mild stress and rifaximin on intestinal microflora,short-chain fatty acids,microglial function and hippocampal neurogenesis in adolescent rats.The aims of the study are to clarify the role of intestinal microflora in the occurrence and development of depression,and investigate whether rifaximin can regulate intestinal microflora and its metabolite short-chain fatty acids to exert antidepressant effect,and explore its possible mechanism.2.Objectives(1)To observe the effects of CUMS and rifaximin on intestinal flora of adolescent’s rats,verify whether rifaximin can exert antidepressant effects by regulating intestinal flora,and search for key microbiota.(2)SCFA of intestinal flora metabolites is detected by gas chromatography-mass spectrometry to explore the effects of CUMS and rifaximin on different types of SCFA,and explores the key mediators of rifaximin regulating the function of microbial-gut-brain axis.(3)To investigate whether rifaximin can improve depression-like symptoms by regulating microglia function and neurogenesis in the hippocampal DG region,and explores its mechanism.3.Methods(1)Experimental animals and groupingFifty-six 3-week-old male Wistar rats were randomly divided into four groups:control group(CON),rifaximin treatment group(CON+R),chronic unpredictable mild stress treatment group(CUMS),chronic unpredictable mild stress and rifaximin treatment group(CUMS+R).CUMS modeling was conducted according to standard procedures.During modeling,CON+R group and CUMS+R group were given rifaximin solution 150mg/kg intragastric treatment.Subsequently,all rats underwent behavioral tests.After the behavioral test,feces,serum and brain tissues were collected for molecular biological experiments according to experimental requirements.(2)Experimental methods①Sugar water preference test was performed to determine the baseline of sugar water consumption in the four groups,and then random stimulation was used for 4 weeks to establish the CUMS rat model.After the modeling was completed,the sugar water preference experiment was conducted again to test the rat anhedonia to verify whether the depression model was successfully established.②Open field experiment was used to test the autonomous activity,inquiry behavior and anxiety-like behavior of rats in the new and exotic environment.③ Morris water maze test was used to test the spatial learning and memory ability of rats.④The changes of intestinal flora in rats were detected by 16S rDNA sequencing.⑤The short-chain fatty acids in serum and brain tissues of rats was determined by gas chromatography-mass spectrometry.⑥The intestinal barrier integrity of rats was tested by hematoxylin-eosin staining and immunofluorescence staining.⑦The expression of pro-inflammatory factors and anti-inflammatory factors in hippocampus of rats was detected by elisa.⑧Immunofluorescence staining was used to detect the morphology,number,phenotype,phagocytic function and neurogenesis and migration of hippocampal microglia in rats.⑨The morphology and density of dendritic spines were detected by Golgi staining.⑩The effect of sodium butyrate on the inflammatory function and phagocytosis of primary microglia was verified by in vitro cell culture.4.Results(1)Rifaximin improved depression-like behavior and impairment of learning and memory function caused by CUMS in adolescent rats.(2)Rifaximin improved the intestinal flora disorder caused by CUMS.(3)Rifaximin increased the butyric acid in serum and hippocampus,and this change was positively correlated with the increase in relative abundance of Ruminococcus bromii and Lachnospiraceae caused by rifaximin.(4)Rifaximin suppressed the pro-inflammatory function and phagocytosis of microglia in the hippocampus caused by CUMS,and increased the expression of anti-inflammatory factors Interleukin-10 and Interleukin-lra.Butyric acid may be the key mediator of rifaximin regulating microglia function.(5)Rifaximin improved CUMS-induced neurogenesis and migration disorders,and increased the density of dendritic spines in the hippocampal DG region.(6)In vitro experiments,the physiological dose of sodium butyrate(0.3uM)improved the proinflammatory activity and phagocytic activity of microglia caused by lipopolysaccharide,and increased the expression of anti-inflammatory factors IL-10 and IL1ra.5.ConclusionsCUMS enhanced the proinflammatory function and phagocytosis of microglia,and caused Neurogenic disorder in hippocampal DG,leading to depression-like behavior and cognitive dysfunction.Rifaximin regulated microglial inflammation and phagocytic function through intestinal microflora metabolite-butyric acid,improved neurogenesis disorders,and corrected depressive-like behavior and cognitive dysfunction in adolescent rats.Therefore,rifaximin regulation of intestinal microbiota and its metabolite butyric acid may be a new method for the treatment of adolescent depression. |
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