Study On The Protective Effect Of Melatonin-mediated Mouse Gut Microbiome On Escherichia Coli Meningitis Model

Avian pathogenic Escherichia coli(Avian pathogenic Escherichia coli,APEC)is one of the main extraintestinal pathogenic Escherichia coli,which can cause a variety of diseases in poultry,including myocarditis,septicemia and meningitis.In recent years,it has been found that APEC has the potential of zoonosis,and seriously threatens the health and development of poultry industry and human health.Escherichia coli meningitis is mostly caused by bacteremia or septicemia with high mortality and survivors have severe nervous system injury sequelae.Bacterial infection can disorder the host intestinal flora and destroy the intestinal barrier,while healthy intestinal microflora can alleviate bacterial septicemia and meningitis.Melatonin has a variety of biological functions,including anti-inflammation,anti-oxidation,anti apoptosis and so on to prevent or treat the occurrence and development of diseases.At present,melatonin is used as an adjuvant in the treatment of bacterial meninigits,but it has not been reported in the prevention of bacterial meningitis and its related mechanism.Avian meningitis Escherichia coli APEC TW-XM is isolated from the brain tissue of duck with septicemia/meningitis.It can infect weaned mice and chickens with severe acute septicemia and meningitis,threatening the health of poultry and the development of poultry industry.The purpose of this study is to explore whether melatonin can prevent the infection of meninigitis APEC in mice,and to explore the preventive effect mechanism of melatonin on Escherichia coli meningitis in mice from the point of intestinal flora.The specific research contents are as follows:1.Effect of melatonin on meningitis-induced APEC TW-XM infection in mice.Based on the mouse model of Escherichia coli meningitis.Different concentrations of melatonin(10 mg/kg,30 mg/kg,60 mg/kg)intraperitoneally were supplemented.One week later,APEC TW-XM(1×107 cfu)was injected intraperitoneally.The results of neurological symptom score and mortality showed that there was no significant difference between 10 mg/kg melatonin group and APEC TW-XM-infection group;while the neurological symptom score and mortality of mice in 30 mg/kg melatonin group and 60 mg/kg melatonin group significantly decreased,there was no signigicantly difference between 30 mg/kg melatonin group and 60 mg/kg melatonin group.Thus,30 mg/kg was selected as the dose of melatonin pretreatment group for follow-up analysis.Furthermore,the effect of melatonin on the prevention of Escherichia coli meningitis were analyzed by bacterial count,Evans blue,qPCR,ELISA,and HE staining.The bacterial load results showed that melatonin could significantly reduce the bacterial load of APEC TW-XM in various tissues and blood of mice(p<0 01).And melatonin could significantly inhibit the increase of bloodbrain barrier permeability induced by APEC TW-XM infection(p<0.001),and decreased the levels of IL-1?,TNF-? and IL-6 in brain tissue and serum,and neutrophil infiltration in brain tissue.These results suggest that melatonin can effectively inhibit the colonization of APEC TW-XM in mice,protect the integrity of the blood-brain barrier,inhibit inflammation and neutrophil infiltration in brain tissue,and prevent meningitis induced by APEC TW-XM infection.2.Effect of melatonin on intestinal barrier integrity and gut microbiome in APEC TW-XM-infected mice.Diamine oxygenase(Diamine Oxidase,DAO),fluorescein isothiocyanate-dextran(Fluorescein isothiocyanate-dextran,FITC-dextran),D-lactic acid(D-lac)and Hematoxylin-Eosin(HE)staining and Limulus amebocyte lysate kit of endotoxin were used to detect gut barrier integrity.Intestinal inflammatory factors were detected by qPCR and ELISA.The gut microbiome composition and abudance were analyzed by 16S rRNA sequencing.The results showed that the contents of DAO,FITC-dextran and D-lac in TW-XM group were significantly increased.At the same time,the colonic HE pathologyical confirmed that APEC TW-XM infection seriously damaged the structure of gut barrier.However,the contents of DAO,FITC-dextran and D-lac in TW-XM+MT group(APEC TW-XM+Melatonin group)were signigicantly lower than those in TW-XM group.At the same time,colonic HE pathological analysis showed that melatonin could protect the integrity of intestinal barrier.The results of colonic inflammatory showed that melatonin could signigicantly reduce the expression of factors IL-1?,TNF-? and IL-6 compared with TW-XM group.The results of endotoxin test showed that melatonin significantly reduced the content of endotoxin in blood and brain tissue(p<0.05)compared with TW-XM group.The diversity and composition abundance analysis in colon by16S rRNA sequencing showed that APEC TW-XM infection decreased the ratio of Firmicutes to Bacteroides,suggesting that it disturbed the composition and abundance of gut microbiome in mice.Melatonin pretreatment could signigicantly improve the composition and abundance of harmful bacteria and alleviate the decrease of the abundance of beneficial bacteria.These results suggest that melatonin can effectively prevent and protect the integrity of intestinal barrier and maintain the composition and relative abundance of healthy gut microbiome in APEC TW-XM-induced meningitis mice,thus reducing the content of endotoxin in meningitis mice and alleviating the occurrence of septicemia.3.Melatonin prevents APEC TW-XM-induced meningitis in mice by mediating gut microbiome.The mouse model of gut microbiome dysbiosis was established by adding four antibiotics(streptomycin(1 g/L),ampicillin(1 g/L),gentamicin(1 g/L)and vancomycin(0.5 g/L))to the drinking water of mice.Mice were co-treated with melatonin for one week,and then infected with APEC TW-XM.At the same time,the mice were treated with melatonin and co-treated with melatonin for one week,and then infected with APEC TW-XM.The results showed that the neurological symptom score and mortality rate of meningitis in TW-XM+Antibiotic group(APEC TWXM+Antibiotic group)was higher than those of TW-XM+MT group,while the neurological symptoms and survival rate of TW-XM+MT+Antibiotic group(APEC TW-XM+Melatonin+antibiotic group)were significantly lower than those of TWXM+MT+Antibiotic group.The results of bacterial load in tissues and blood showed that the bacterial load in various tissues and blood in TW-XM+Antibiotic group and TW-XM+MT+Antibiotic group was significantly higher than those in TW-XM+MT group,and the blood-brain barrier permeability in TW-XM+MT+Antibiotic group was significantly higher than that in TW-XM+MT group(p<0.05).The results of inflammatory factors showed that the expression of IL-1?,TNF-? and IL-6 in brain and serum of TW-XM+Antibiotic and TW-XM+MT+Antibiotic groups were significantly higher than those of TW-XM+MT group(p<0.05),and there was lots of neutrophils infiltration in brain tissue.These results suggest that melatonin needs to mediate healthy gut microbiome to prevent APEC TW-XM-induced meningitis.4.Melatonin can prevent the gut barrier disruption and the changes of gut microbiome in mice induced by APEC TW-XM via mediating gut microbiome.Compared with TW-XM+MT+Antibiotic group,the levels of DAO,FITC-dextran and D-lac in TW-XM+Antibiotic group and TW-XM+MT+Antibiotic group was significantly increased,and the colonic structure and morphology of TWXM+MT+Antibiotic group were seriously damaged.Similarly,the expressions of inflammatory factors IL-1?,TNF-? and IL-6 in TW-XM+MT+Antibiotic group were significantly higher than those in TW-XM+MT group.The results of endotoxin test showed that the content of endotoxin in TW-XM+Antibiotic group and TWXM+MT+Antibiotic group was significantly higher than that in TW-XM+MT group.The results of 16S rRNA sequencing showed that compared with TW-XM group,there was no significant difference in a diversity except Shannon index,but Simpson and Chao1 index in TW-XM-MT+Antibiotic group decreased significantly(p<0.05).The detection results of ? diversity(NMDS,PCA and PCoA)showed that there were significant differences in gut microbiome in TW-XM+MT+Antibiotic group.The results of different flora analysis and gut microbiome composition and relative abundance analysis at the phylum level that Proteobacteria was the dominant bacteria in TW-XM+MT+Antibiotic group,and the composition and relative abundance of other gut microbiomes was significantly decreased compared with TW-XM+MT group.At the order level,Xanthomonadales was significantly higher than that in TW-XM group,while the relative abundance of Clostridiales,Bacteroidales and Lactobacillales in TWXM+Antibiotic group was significantly lower than that in TW-XM group and TW-XM group.At the genus level,the dominant bacteria in TW-XM+MT+Antibiotic group was Stenotrophomonas,while the relative abundance of Lactobacillus,Bacteroides and Alloprevotella was significantly lower than that in TW-XM group.At the species level,the relative abundance of Bacteroides-vulgatus,Lactobacillus_reuteri and Lactobacillus_intestinalis in TW-XM+MT+Antibiotic group was significantly lower than that in TW-XM+MT group,although there was no significant difference compared TW-XM group,but the abundance was still significantly decreased.These results suggest that melatonin attenuates intestinal injury mediated by APEC TW-XMinfection and protects mice from APEC TW-XM-induced meningitis by mediating gut microbiome.To sum up,melatonin can effectively prevent meningitis induced by APEC TW-XM infection in mice,and its effect may be to protect the integrity of intestinal barrier and reduce the content of endotoxin in meningitis mice by mediating gut microbiome,to alleviate the occurrence of septicemia and play a role in prevention and protection.