A Preliminary Microbiota Modulating Behavior And The Molecular Mechanism In Hypothalamic-Pituitary-Adrenal Axis

BackgroundIntestinal microbes are a huge system with about 10¹³-10¹⁴ microbes,which are 10 times the number of organismal cells and 150 times of the genome in the human body.There are more than one thousand species in the adult intestinal tract,which have a great influence on animal behavior.An increasing body of research indicates that intestinal microbes affect brain function and neurogenesis,including sensitivity to stress response.Although the body could generally adapt to changes in external stress,chronic persistent stress could cause gastrointestinal and mental disorders.In fact,several studies have shown that stress is associated with functional digestive diseases,peptic ulcer disease or inflammatory bowel disease.Clinical studies have found that the role of intestinal microbes is existent in CNS function.Bacterial infections in intestinal tract could induce anxiety-like behavior in rats,which may be due to stimulation of brain regions,then stimulation of visceral sensory information and mood through the vagus nerve.In addition,germ-free?GF?mice usually have higher responses in hypothalamic-pituitary-adrenal?HPA?axis and altered serotonin and norepinephrine levels compared with specific pathogen-free?SPF?mice.Maternal separation is more likely to cause dysfunction in intestinal mucosa and visceral hyperalgesia in adult rats under stress.However,the study about the effect of intestinal micro-organisms on behavior and the HPA axis under external stress is less.ObjectiveTo investigate the effects of microbial colonization on behavior,we examined behavioral changes associated with hormones and hormone receptors in the HPA axis under stress.This study may provide a new approach for treating stress-related diseases through intervention into intestinal microflora.MethodsWe tested GF mice and SPF mice,divided into four groups.A chronic restraint stress?CRS?protocol was utilized to induce external pressure in two stress groups by restraining mice in a conical centrifuge tube for 4hours per day for 21 days.And then we tested behavior,hormones and receptors.All data were analyzed with two-way ANOVA to detect significant differences among groups..Results1.After CRS model,in the open field test?OFT?,SPF stressed group moved a shorter total distance in the OFT compared with the GF stressed group.GF restraint-stressed mice explored more time than SPF restraint-stressed mice in the center of the OFT.GF stressed animals moved a greater distance than GF non-stressed mice in the center of the OFT.Mice in the SPF CRS group moved a greater distance in the center than those in the SPF non-CRS group.GF stressed mice spent more time exploring than GF control animals in the center of the OFT.GF non-stressed control animals moved a significantly greater distance than SPF non-stressed mice in the center of the OFT,and also we found in time.2.The CRH,ACTH,CORT and ALD levels of SPF restraint-stressed mice exhibited a significantly greater decrease than those of GF restraint-stressed mice in the hypothalamus tissue.The concentrations of ACTH and ALD in GF restraint-stressed mice homogenates were higher than in the GF control group.Moreover,the Crhr1 mRNA levels of SPF CRS mice were significantly decreased compared with GF CRS mice,but the Nr3c2 mRNA levels of those were significantly increased compared with GF CRS mice.The ratio of MR to GR?MR/GR?significantly increased in SPF CRS mice compared with GF CRS mice.Similarly,after CRS,Crhr1 mRNA expression in GF CRS mice was increased compared with GF control mice,but a significant decrease was also found in MR/GR.ConclusionAll results revealed that SPF mice exhibited more anxiety-like behavior than GF mice under the same external stress.Moreover,we also found that GF mice exhibited significant differences in hormones and hormone receptors compared with SPF mice.In conclusion,Imbalances of the HPA axis caused by intestinal microbes could affect the neuroendocrine system in the brain,eventually resulting in an anxiety-like behavioral phenotype.