Effect And Mechanism Of Knockdown Of Escherichia Coli On Blood Glucose In Mice

Diabetes is a chronic metabolic disease.With the modernization of city and the aging of the population,the morbidity has been increasing in recent years,which has seriously affected people’s lives and health.As the largest digestive organ of the human body,the intestinal tract plays an important role in the body.There are a huge number of microorganisms in the intestine,which are closely related to the physiological functions of the human body such as nutrition,immunity and metabolism.Bacteriophage is a kind of virus that can infect bacteria.Its biggest characteristic is host specificity.It can target host bacterium in a complex microbial environment without affecting other bacterial communities,which has incomparable advantages over broad-spectrum antibiotics.Escherichia coli is abundant in the environment and has been widely studied as a model strain.However,its physiological function in animal intestinal tract is not clear,and currently there is a lack of method to study its function in situ in the intestinal tract.In this project,we intend to use a variety of bacteriophages to target E.coli in the mouse intestine to explore the physiological functions of E.coli in the body.The results of the study are as follows:（1）Two strains of bacteriophage Md1 and Fc1 was selected from the sewage with Escherichia coli in the mouse intestine as the host.The plaque were all circular,with diameters of 7-6 mm,and the optimal multiplicity of infection is 0.01.The incubation period of Md1 is 10 minutes,and the lysis period lasts about90 minutes.The incubation period of Fc1 is 20 minutes and the lysis period lasts about 60 minutes.Md1 is still active in 100℃for 20 minutes,and it was completely inactivated in 121℃for 20 minutes.The titer of Fc1 decreases sharply when the temperature is higher than 60℃,it was completely inactivated at 90℃for 20 minutes.We got a mixed phage with a titer of 10¹² PFU/m L by mixing Md1,Fc1,T4 and T7 following the ratio of 1:1:1:1.（2）The 630 strains of E.coli have been isolated from the mouse intestines,while 585 of them can be infected by one or more phages.By using the 16S sequencing technology,the results show that five of the strains were not E.coli,and the five strains were derived from strains that were insensitive to all four phages,it is known that 585 of the 625 strains of E.coli can be infected by one kind or two more kinds phages.The p H sensitivity test showed that all four phages were able to tolerate acidic environment,and the titer could be maintained above 10~6 PFU/m L at p H 1.（3）After Gavaging mice with mixed phage,the number of Escherichia coli in the intestines of the mice was reduced by 2-3 orders of magnitude compared with the control group.The blood glucose level,body weight,weight gain,average oxygen consumption and basal metabolic rate of the mice were reduced.Using mixed phage gavage on type 2 diabetes model mice,the number of Escherichia coli in the intestine of the mice in this group was 1-2 orders of magnitude lower than that in the control group,but there was no significant difference in blood glucose between the inactivated phage treatment group.The serum levels of insulin（INS）,glucagon-like peptide-1（GLP-1）,glucokinase（GCK）and pyruvate kinase（PK）in each group of mice were determined.The GLP-1 level of mice in the treatment group is higher than the control group and has a significant difference;there is a significant difference that the GCK level of the inactivated phage group is higher than the other two groups;The level of PK in the healthy control group was significantly higher than that in the model mixed phage treatment,and other groups did not show a significant difference.（4）The level of Phocaeicola,Borkfalkia,Akkermansia,Fusobacterium,Erysipelatoclostridium,Butyrivibrio and Kineothrix in phage treated group were higher than those in control group（p<0.05）.Compared with the phage treated group,the mice in the control group was higher in energy production and conversion,post-translational modification,protein conversion,coenzyme transportation and metabolism, nucleotide transportation and metabolism,lipid transportation and metabolism functions than the phage treatment group（p<0.05）.There are significant differences between the control group and the phage treatment group in glycoside hydrolase,glycosyltransferase polysaccharide lyase,and carbohydrate lipase（p<0.05）.From ipath pathway,the phage treatment group has a unique way in fatty acid metabolism and amino acid synthesis.The conclusion from metabolomics approach provides that the metabolic level of L-arginine, dopaquinone,glycitein A,paraxanthine,LPC（18:1）,Lyso PC（P-18:0）,vitamin D₃,arachidonic Acid,20- hydroxy leukotriene E4,7,10,13,16,19-docosapentaenoic acid,Lyso PC（18:0）,Lyso PC（P-18:1（9Z））and amygdalin in the phage treatment group was higher than that in the control group（p<0.05）.Besides,it showed significant differences between the experimental group and control group in linolenic acid metabolism,N-polysaccharide biosynthesis and choline metabolism（p<0.05）.