The Effect Of Chronic Stress On The Intestinal Microecology Of Colorectal Cancer And The Mechanism Of Xiaoyao San

Objectives:1.To establish a chronic stress colorectal cancer animal model by chronic restraint stress（which was abbreviated as CRS）modeling combined with behavioral tests and study the effects of chronic stress on tumor growth and metastasis.2.To study the effects of chronic stress on the intestinal barrier,gut microbiota and its metabolites.3.To study the mechanisms of Xiaoyao Powder in inhibiting the growth of chronic stress colorectal cancer by improving the intestinal barrier and regulating gut microbiota and its metabolites.Methods:1.Chronic stress animal model was established by using CRS method（self-made perforated tube,8 hours per day,continuous for 14 days）,combined with behavioral tests methods such as sucrose preference test and tail suspension test.2.By continuously monitoring the body mass,food intake,fecal traits（including constipation and blood in the stool）and rectal prolapse of CRS during modeling,the disease activity index（DAI）was calculated to evaluate the effects of CRS on the general condition of nude mice.3.A subcutaneous xenograft model was established by subcutaneous injection of HCT116 tumor cells,and a CRS lung metastasis model was established by injecting HCT116_luc tumor cells into the tail vein.The volume of the tumor,the mass of nude mice,and the small animal living imaging system were used to observe the effects of CRS on the growth and metastasis of colorectal cancer.4.The effects of CRS on the intestinal structure of nude mice were observed by HE staining,immunohistochemistry,Tunel detection,transmission electron microscopy.5.The levels of neuroendocrine hormones such as 5-HT,ADR and NADR in the serum of nude mice and the expression of inflammatory factors such as IL-6 and TNF-αand s Ig A in intestinal mucosa were detected by ELISA kits.6.To study the effect of Xiaoyao Powder on subcutaneous transplantation of CRS colorectal cancer,we used the CRS colorectal cancer subcutaneous xenograft model.The medium dose of Xiaoyao Powder（0.2ml/20g/d）,high dose of Xiaoyao Powder（0.4ml/20g/d）,and positive control drug fluoxetine（10mg/kg/d）were continuous intragastric administrated for 21 days.7.The effect of CRS on the species and quantity of gut microbiota and the intervention of Xiaoyao Powder were detected by bacterial 16S r RNA sequencing technology.8.The differential metabolites of gut microbiota were screened by LC-MS non-targeted metabolomics technology,and the biological information of differential metabolism was further explored by path analysis,association analysis and cluster analysis.Results:1.Chronic restraint stress can reduce food intake,body mass and spleen index in nude mice and increase DAI.After drug intervention,Xiaoyao Powder medium dose,Xiaoyao Powder high dose and fluoxetine could improve food intake,body mass and spleen index,and decreased DAI（P<0.05）,but Xiaoyao Powder medium dose was much better（P<0.01）.2.In terms of behavioral evaluation,chronic stress can reduce the preference of sucrose in nude mice（P?0.05）,and significantly increase the time of suspension test（P?0.01）.However,the results of open field test and elevated plus maze test are not significant（P>0.05）.After drug intervention,the medium dose of Xiaoyao Powder,the high dose of Xiaoyao Powder and fluoxetine could increase the sucrose preference and shorten the immobility time during the suspension test（P<0.05）,but the effect of Xiaoyao Powder was much better（P<0.01）.3.The volume of subcutaneous xenografts in the CRS group（P<0.01）and tumor weight（P<0.05）were higher than that in the Control group,and the lung metastasis rate of colorectal cancer in the CRS group was higher（P<0.05）,the volume of lung metastases in the CRS group was larger than that in the Control group（P<0.05）.4.Compared with the Control group,the intestinal permeability of the CRS group increased significantly（P<0.05）,the secretion of s Ig A in the intestinal mucosa decreased（P<0.05）,and the expression of ZO-1 and Occludin in the large intestine tissue decreased（P<0.05）.Among the three groups of drug intervention,the medium dose of Xiaoyao Powder was significant（P<0.05）,but the high dose of Xiaoyao Powder and fluoxetine group was not significant（P>0.05）.5.Compared with the Control group,the expression of 5-HT in the serum of the CRS group was decreased（P<0.05）,while the expression of NADR,ADR,IL-6 and TNF-αwas increased（P<0.05）.After drug intervention,the concentration of 5-HT in serum increased（P<0.05）,the concentration of NADR,ADR,IL-6 and TNF-αin serum decreased（P<0.05）,and the decrease of the medium dose of Xiaoyao Powder was more significant（P<0.01）.6.Compared with the Control group,the abundance of Bacteroidetes and Proteobacteria in the intestinal flora of the CRS group was significantly reduced（P<0.05）,and the abundance of Firmicutes and Saccharibacteria was significantly elevated（P<0.05）.After drug intervention,the abundance of Bacteroides and Proteus increased（P<0.05）,and the increase of the medium dose of Xiaoyao Powder was more significant（P<0.01）.The abundances of Bacteroidetes and Proteobacteria were decreased（P<0.05）,and the medium dose of Xiaoyao Powder was more significant（P<0.01）.7.Compared with the Control group,Carpaine and DG（15:0/20:4（5Z,8Z,11Z,14Z）/0:0）were significantly reduced in the CRS group（P<0.01）,PE（14:0/0:0）,PE（14:0/0:0）,Lyso PE（15:0/0:0）and Lyso PE（0:0/22:0）were significantly elevated（P<0.01）.After drug intervention,Carpaine and DG（15:0/20:4（5Z,8Z,11Z,14Z）/0:0）increased significantly（P<0.01）,PE（16:0/0:0）,PE（14:0/0:0）,Lyso PE（15:0/0:0）and Lyso PE（0:0/22:0）decreased significantly（P<0.01）,but there were no significant differences among the medium dose of Xiaoyao Powder,the high dose of Xiaoyao Powder and fluoxetine（P>0.05）.Conclusions:1.The chronic restraint stress modeling method combined with behavioral test and evaluation is a relatively reliable and reproducible method for chronic stress animal models.2.Chronic stress can promote the growth and metastasis of colorectal cancer,and its mechanisms were related to the activation of inflammatory response and HPA axis.3.Chronic stress can damage the intestinal barrier by increasing intestinal permeability,reducing intestinal tight junction proteins and inhibiting the secretion of s Ig A in the intestinal mucosa.4.Chronic stress can lead to imbalance of gut microbiota and abnormalities of gut microbiota metabolites,increasing the risk of intestinal pathogens and their metabolic toxins ectopic.5.Xiaoyao Powder can inhibit chronic stress colorectal cancer growth by improving the general state of nude mice,reducing disease activity index,reducing the expression of inflammatory factors and neuroendocrine hormones,improving intestinal barrier function and regulating the imbalance of gut microbiota and its metabolites.