A Research Of Probiotics In Relieving Intestinal Injury Induced By H1N1 Influenza Virus Infection In Mice

Studies at home and abroad have shown that influenza virus may cause intestinal damage as well as lung damage in mice.The status of intestinal microbes is closely related to the immune regulation of the body after influenza infection.However,most of the current studies focus on the regulatory role of intestinal flora in lung injury after influenza infection,and gastrointestinal symptoms are often ignored,which may aggravate the illness and even threaten the life of the patients.At present,the prevention and treatment of influenza mainly relies on chemical drugs,influenza vaccines or antibiotics,while the application of prebiotics or probiotics in the prevention and treatment of influenza is less studied.In order to explore the role of prebiotics in alleviating intestinal damage induced by influenza virus infection in mice,this study started from the changes of intestinal flora,based on fecal metabolomics,combined with morphological,molecular biology,immunology and other technical means.This study can provide theoretical support for the prevention and treatment of influenza in production practice.It also provides a theoretical basis for the search for beneficial microorganisms or metabolites that play an active role in preventing intestinal damage induced by H1N1 infection.My research mainly includes the following three parts:Experiment first:Construction and validation of a model of "Intestinal injury induced by H1N1 influenza virus infection in mice"After inoculation of H1N1 influenza virus in mice,both the viral group and the prebiotics group showed different degrees of body weight loss,hair disorder and weight loss,while compared with the virus group,the prebiotics group showed certain relief of these symptoms.The results of HE staining of the intestinal tissue of mice showed that compared with the control group,the intestinal tissue of the virus group and the prebiotic group had different degrees of damage,but the intestinal tissue of the virus group was more severely damaged,with obvious shortening of intestinal villi,infiltration of lymphocytes,and small V/C(height of intestinal villi/depth of crypt)ratio.Fluorescence quantitative PCR results showed that TNF-α,IL-6,IL-1β gene expression level significantly increased after the H1N1 influenza virus infection,the expression of IL-10 trend contrary to the former.It was shown that H1N1 infection caused an increased level of intestinal inflammation,while the probiotic intervention group significantly reduced the level of intestinal inflammation.The results of normal PCR detection of M protein gene of influenza virus in lung and intestinal tissues showed that influenza virus only replicated in the lungs in this experiment and did not enter the intestinal tissues.The above results verified the success of the establishment of "animal model of intestinal injury induced by influenza virus infection" and preliminarily showed that prebiotics could relieve gastrointestinal symptoms induced by influenza virus infection.Experiment two:Metabolomics analysis of mouse fecesCollect feces on days 0,21,and 28 of four groups of mice for fecal metabolomics based on gas chromatography-mass spectrometry(GC-MS)platform,combined with microbiome results,analyze the metabolites of influenza virus infection on the body and Effects of metabolic pathways.At the same time,comparing the virus and virus+prebiotic group,to explore the effect of viral infection on the metabolic level in the case of prebiotics to improve the intestinal flora.H1N1 virus infection caused a significant change in the levels of various metabolites in mice.According to VIP>1,P<0.05,screening including tryptophan,glutamic acid,indoleacetic acid,indolelactic acid,glycine,citrulline,canine 39 metabolites such as urine,arginine,glutamine,alanine,ornithine,threonine,and valine;the pathways affected mainly involve amino acid metabolism,lipid metabolism,and tricarboxylic acid cycle,Energy metabolism processes such as sugar metabolism,and nucleic acid metabolism processes such as purine metabolism.Compared with the influenza virus infection group,some of the key markers in the prebiotic treatment group,such as glutamine,arginine,glutamic acid,kynurenine,tryptophan,indoleacetic acid,indolelactic acid,leucine,and inferior Xanthine,lactic acid,threonine,putrescine,arachidonic acid,deoxycholic acid,etc.have a tendency to callback to the control group.Compared with influenza virus infection group,prebiotics treatment group of some key markers such as glutarnine,arginine,glutarnic acid,dog urine amino acid,tryptophan,indole acetic acid,lactic acid,leucine,indole,had a tendency to callback to the control group,and the metabolite usually and maintaining the normal physiological condition(e.g.,promote the growth of intestinal villus,ISCs proliferation and differentiation,as the membrane composition,provide energy for intestinal cell growth,etc.)and induce intestinal dysbacteriosis or intestinal inflammation.Experiment three:Effect of prebiotic intervention on proliferation,differentiation and tight connection of intestinal stem cells in micemmunohistochemistry,RT-PCR and Western-blot were used to explore the effects of prebiotics treatment on the proliferation and differentiation related pathways of intestinal stem cells and the formation of tight-junction proteins at the three levels of morphology,gene and protein,respectively.The results show that H1N1 infected mice could destroy the intestinal epithelial cell tight junction protein and inhibit the proliferation and differentiation of ISCs,but prebiotic treatment can promote the proliferation and differentiation of intestinal stem cells and the formation of tight junction proteins by regulating Wnt3a/β-Catenin signaling pathway.This effect is related to the regulation of intestinal health by amino acids,lipids,and indole derivative substances in the fecal metabolites compared with the H1N1 group after prebiotic treatment.In summary,viral nasal infection of mice can not only induce intestinal immune damage,but also cause epithelial barrier damage,inhibit epithelial cell proliferation and differentiation,slow epithelial cell replacement,and increase intestinal damage;and pre-feeding prebiotics can improve bacteria Group environment,increase beneficial bacteria,increase metabolites glutamine,arginine,glutamic acid,kynurenine,tryptophan,indoleacetic acid,indolelactic acid,leucine and other small molecules to promote epithelial cells Proliferate and differentiate,promote the formation of tight junction proteins,resist influenza-induced bowel injury,and maintain the integrity of the epithelium.