| With in-depth studies in the field of intestinal flora,there is much evidence that the intestinal flora is highly correlated with chronic diseases such as obesity,hypertension,and diabetes in the host,and these chronic diseases are being studied from the perspective of previously neglected intestinal flora.The origin,development and prevention and control strategies are expected to bring new ideas for the prevention and control of chronic diseases.Based on these studies at home and abroad and on the basis of self-verification,our laboratory proposed the scientific hypothesis that“Hunger Sensation Comes from Gut Flora”and through a large number of studies to develop the“Flexible Fasting”technology,through the continuous 7-14 days of taking the composite prebiotics to reduce and eliminate the hunger of the human body,allowing the body to enter the simulated fasting state,helping the human body to restore the intestinal flora to a normal state,providing a reference for improving chronic diseases from the etiology.Since the development of this technology,we have successively cooperated with many hospitals to complete relevant clinical studies,and achieved good results in physiological weight loss and improvement of various chronic diseases.In order to further study the effect of flexible fasting technology on the improvement of chronic disease indicators such as hyperglycemia of obesity people,this paper firstly cooperates with the Nutrition Department of the Second People's Hospital of Taiyuan City to recruit volunteers to experience the one-week flexible fasting technology to improve chronic diseases such as hyperglycemia of obesity people.The physiological and biochemical indexes of volunteers were detected and recorded before and after the intervention.Statistical analysis was performed using statistical software SPSS 16.0,and the figure was performed using GraphPad Prism.The results showed that volunteers'body weight,BMI,visceral fat mass and body fat decreased significantly(P<0.01),diastolic blood pressure(P=0.003),systolic blood pressure(P=0.045),and fasting blood glucose(P=0.001),fasting insulin levels(P=0.000)also decreased significantly,while renal function,liver function and electrolytes showed a slight fluctuation in the normal range,indicating that flexible fasting technology is not only benificial to hyperglycemia and other chronic diseases,but has a great safety.According to the improvement of the fasting blood glucose and fasting blood insulin,combined with the previous research of the flexible fasting technology in the laboratory(improving the intestinal microecology of volunteers,specifically promoting the growth of beneficial bacteria such as lactobacilli,bifidobacteria.),it could speculate that the improvement of blood glucose metabolism had some relationship of the improvement of intestinal microecology.To further illustrate the correlation between intestinal flora and blood glucose metabolism,we designed the use of antibiotics cocktails to establish a mice intestinal microecological disorder model to observe the effects of intestinal microecology on blood glucose metabolism in mice.On the basis of this hypothesis,firstly,we selected four widely used strains of mice such as C57,BALB/c,CD1 and KM for oral glucose tolerance test(OGTT)(15%glucose solution,0.2 mL per mice),and then use Origin 8.0 to fit glucose metabolism curve of mice with different time points(0,5,10,15,30,60,120min).The result shows all the glucose curve come to the peak in 15 mins and back to the normal level in 120 mins,and fitted to one model which name is Giddsings model(?),which means the four strains of mice have barely no difference in OGTT.After that,we choose C57 mice to be the object for the next research because it is the most sensitive mice to glucose solution among the four strains.The plan is randomly divided 90 males C57 mice(which weight is between 18 and 22gram)into 6 groups,which respectively are“saline-control group”,“antibiotic-control group”,“saline-high fat group”,“antibiotic-high fat group”,“saline-high glucose group”and“antibiotic-high glucose group”.All mice were pre-fed for one week(normal diet,drinking water)in the conventional environment before the start of the experiment to adapt to the environment,and then be intervened by antibiotic cocktails(reference texts were penicillin,vancomycin,metronidazole,neomycin).and different diet as the former designment for 4 weeks.These mice body weight and blood glucose are measured by routine methods.Serum insulin,serum histamine and serum adrenaline concentration are measured by enzyme-linked immunosorbent assay(ELISA).The glucose tolerance of the mice is measured by OGTT.Curve fitting is performed by Origin 8.0 to figure out the characteristic of the glucose metabolism of mice.The results show mice blood glucose is dramatically decrease(P=0.000),and the change trend of glucose tolerance in mice also deviated from the Giddings model(R~2<0.95),while the mice which divided by different diet(control,high fat,high glucose)have no impact on glucose level and OGTT curve.Multivariate analysis of variance shows that there is no interaction between antibiotics and diet intervention in the weight,serum insulin and serum histamine levels of the mice.There is no statistical difference between the antibiotics and the saline group,but there is statistically different among different diet group.Compared with the control diet group,serum insulin level and serum histamine level are significantly lower in the high-fat diet group(P=0.000,P=0.033),while there is no significant difference in body weight.Compared with the control diet group,the weight of the high glucose diet group is significantly lower(P=0.012),serum histamine level is significantly increased(P=0.002),while serum insulin level has no statistically different.Compared with the high fat diet group,the weight of the high glucose diet group was significantly lower(P=0.000),serum insulin level and serum histamine level are significantly increased(P=0.000,P=0.000).Antibiotics and diet interventions have an interaction with serum adrenaline levels(P<0.05).Antibiotic and diet have intervention in the serum epinephrine,there are difference between antibiotic group and saline group in each diet group,antibiotic group mice have lower serum epinephrine level in control and high-glucose group(P=0.008,P=0.013),while have higher serum epinephrine level in high-fat group(P=0.007).In order to study the different of intestinal flora between before and after antibiotic-induced change of glucose metabolism,we used 16S rRNA sequencing technology to analyze the fecal samples of each group of mice,and cut the filtered information to perform OTUs(Operational Taxonomic Units).Clustering,species annotation and abundance analysis reflect changes in the distribution characteristics of intestinal flora in each group of mice.We can see the antibiotic group show lower flora abundance than saline group from two indicators which name is respectively rarefaction curve and rank abundance.We can also find antibiotic induce the species diversity and microbe abundance decrease from two indicators observed species and shannon index.The result of principal coordinate analysis(PCoA)and principal component analysis(PCA)show there are clustering difference between antibiotic group and saline group.As the result of t-test and MetaStat method show at the phylum level,antibiotic induce the relative abundance of proteobacteria which containing pathogens and opportunistic pathogens increase(P<0.01),and induce the relative abundance of dominant bacteria in normal intestinal flora such as mycobacterium,mycobacterium,and actinomycetes decrease(P<0.01).At the genus level,there are statistical differences between“antibiotic-control diet group”and“saline-control diet group”among all bacteria except of lactobacillus and roseburia.Among them,antibiotic induce the relative abundance of klebsiella,parasutterella,and akkermansia increase,and the relative abundance of the remaining species decreased.The result of comparation of the“saline-high fat diet group”and“antibiotic-high fat group”show except of akkermansia,dubosiella,alloprevotella,mucispirillum,other genus all has statistically difference(P<0.05 or P<0.01).Antibiotic induce relative abundance of parasutterella increase and others decrease.The“antibiotic-high glucose diet group”compared with“saline-high glucose diet group”showed that except for bifidobacterium and roseburia,the other species are statistically different(P<0.05 or P<0.01),among them,antibiotic intervention causing an increase in the relative abundance of klebsiella,parasutterella,and akkermansia,the relative abundance of the remaining species is reduced.In addition,there is a statistically significant difference in the abundance of certain species between each diet groups without antibiotic intervention(saline group).At the level of the phylum,the abundance of the proteobacteria of the“saline high-fat diet group”is significantly higher than that of the“saline-control diet group”(P=0.021).Compared with the“saline-control diet group”,the unidentified or unclassified flora(unidentified_Bacteria)and the typhimurium abundance are significantly increased in the“saline high glucose diet group”(P=0.037,P=0.005).Compared with the“saline-high fat diet group”,the abundance of bacteroides,unidentified_bacteria and flexobacteria in the“saline-high glucose diet group”is significantly increased(P=0.024,P=0.035,P=0.004).At the genus level,dubosiella,odoribacter,butryicimonas,oscillibacter,and parabacteroides in the“saline-high fat diet group”are significantly lower than those in the“saline-control diet group”(P<0.05),and stenotrophomonas is significantly higher than“saline-control diet group”(P=0.038).Compared with the“saline-control diet group”,intesinimonas,odoribacter,anaerotuncus,candidatus_saccharimonas,bilophila,enterorhabdus in the“saline-high glucose group”are significantly increase(P<0.05),and dubosiella is significantly decrease(P=0.023).Compared with“saline-control diet group”,dubosiella,unidentified_ruminococcaceae,alistipes,intestinimonas,odoribacter,candidatus_saccharimonas,butyricimonas,oscillibacter,ruminiclostridium,and tyzzerella of“saline-high glucose group”are significantly increase.Thus,the use of antibiotic cocktail resulted in a decrease in random blood glucose level of mice,which altered the glucose tolerance curve model of C57 mice(R~2<0.95),probably due to antibiotic cocktail(penicillin,vancomycin,metronidazole,neomycin)non-specially,non-selectively removal of the intestinal flora,reducing the diversity and relative abundance of the flora,thereby reducing the absorption and conversion utilization of the intestinal flora to food.At the same time,the akkermansia discovered in this study showed a growth change after the intervention of compound antibiotics.Combined with the existing literature,akkermansia will improve IFN-?by negative regulation.The glucose tolerance of the host reduces the blood glucose level of the host.In this experiment,the use of compound antibiotics as a negative regulator of intestinal flora changed the intestinal microecology,suggesting that there is a certain correlation between intestinal flora and host glucose metabolism,but the specific relationship needs further study.In summary,this study further demonstrates that flexible fasting technology can safely and effectively reduce the fasting blood glucose and fasting insulin levels of volunteers,and improve chronic diseases such as obesity and hyperglycemia.Combining the literature with previous studies,this result may be related to the improved microecology of the host intestines.In order to study the correlation between intestinal flora and host blood glucose metabolism,oral glucose tolerance experiments were performed by selecting four common strains of mice,and it was found that the blood glucose metabolism of each strain of mice accorded with the Giddings curve model C57,which has high sensitivity to blood glucose,was selected as the experimental object.The antibiotic cocktail be used to intervene in the intestinal flora and combined with different diet such as high fat and high glucose to feed the animals.The abundance of intestinal bacteria in the antibiotic-treated group was found.Significant changes in diversity,intestinal micro-ecology,etc.(including a significant increase in some intestinal flora),accompanied by a decrease in blood glucose in animals,deviated from the previous Giddings curve model,suggesting that the intestinal flora is related to host glucose metabolism provides reference information for the subsequent study of host glucose metabolism regulation from the perspective of intestinal flora. |
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