Changes Of The Intestinal Genera In Rats With Metabolic Disorders Induced By High-fructose-diet And Effects Of Bifidobacterium Intervention

Objective:1.To explore the changes of the intestinal microbiota in rats with metabolic disorders induced by the high fructose diet,and the mechanism of intestinal dysbiosis in metabolic disorders.2.To explore the effects of bifidobacterium on intestinal dysbiosis and the abnormal metabolism of rats.Method:30 male SD rats were randomly and evenly divided into normal control group（group NC）,high fructose diet group（group HFD）and bifidobacterium group（group Bif）.Group NC were fed with standard diet plus tap water,group HFD were fed with standard diet and10%fructose water.Group Bif were fed with standard diet,10%fructose water and bifidobacterium water（1ml/d,1 x 10⁹cfu/ml）was given through intragastric administration.The weight of rats were monitored weekly.The feces of rats were collected at the end of16^th week.The feces were placed in a test tube of aseptic specimens in a freezer at-80℃.On the next day of 16^thh weekend,rats were fasted for 12h.Blood was drawn from rat tail vein before the rats were anaesthetized with aether and fixed tightly.Blood was collected from abdominal aorta,centrifuged at 4 degree centrifugation 10min（3500rpm/min）.Then the plasma was separated into 1.5ml centrifuge tubes and cryopreserved at-40℃.Part of the liver tissues were stripped and fixed at 4%paraformaldehyde for 24h.The tissues were paraffin embedded and stained with hematoxylin eosin and made into the liver pathological section.1.Measurement of body weight and fasting blood glucoseThe weight changes in rats were monitored and recorded weekly.The rats were fasted for 12h on the 16^th weekend.The blood samples were collected from rat tail vein and the levels of fasting blood glucose were detected repeatedly by the dynamic Roche rapid glucose meter.2.Measurement of plasma biochemical indexesThe plasma levels of hepatic enzymes（ALT,AST）and TG were measured by enzyme method.The levels of plasma endotoxin（LPS）in rats were detected by limulus amebocyte lysate.The levels of TNF-alpha were determined by ELISA.3.Measurement of glycometabolism indexesThe levels of the fasting insulin in plasma were measured by ELISA,and the insulin resistance indexes（HOMA-IR）were calculated.The formula as follows:HOMA-IR=FINS（fasting insulin,EU/ml）?FBG（fasting plasma glucose,mmol/l）/22.5.4.Pathological examination of the liver tissueThe liver tissue was stripped and placed in the 4%paraformaldehyde overnight.The residual liquid was removed.The liver tissues were made into 3μm thickness slice.HE staining was performed and 10 times light microscope was used to observe the changes of liver histology.5.Detection of the abundance of the three generaThe total DNA of animal feces were extracted and the Real Time PCR technology was used to detect the content of total bacteria and the three genera which included bacteroides,lactobacillus and fusobacterium.The relative expressions of the three genera to the total bacteria were accounted according to the formula as follows:Relative quantitation=2^-^△△Ct.Result:1.The changes of body weightDuring the experiment,all animals were in good condition except one of group NC.Compared with group NC,the weight increased significantly in group HFD and group Bif on the seventh week（P<0.05）.And from the thirteenth week,the rates of weight increasing in group Bif were lower than that of group HFD（P<0.05）.2.Changes of plasma biochemical indexesCompared with group NC,the levels of plasma liver enzymes,TG,LPS and TNF-alpha in the group HFD were increased significantly（P<0.01）.Compared with group HFD,the levels of plasma liver enzymes,TG,LPS and serum TNF-αin group Bif were significantly decreased（P<0.01）.3.Changes of glycometabolism indexesCompared with group NC,the levels of FBG,FINS and HOMA-IR in group HFD were increased significantly（P<0.01）.Compared with group HFD,the levels of FBG,FINS and HOMA-IR in group Bif were decreased significantly（P<0.01）.The levels of LPS were positively correlated with HOMA-IR（r=0.897,P<0.01）.4.The pathological changes of liver tissueThe margin of liver tissue in the group NC is marroon without greasy feeling.And the radial arrangement of the hepatic cord can be observed under the optical microscope.The volume and the quantities of lipid droplets under the microscope were increased in the group HFD and the edges of tissues were rounded with visible pale yellow fat.Compared with the group HFD,the volume and the number of lipid droplets in group Bif were decreased.5.The changes of relative expressions of the three generaCompared with group NC,the content of bacteroides and lactobacillus were decreased in group HFD,while the abundance of fusobacterium was increased significantly（P<0.01）.Compared with group HFD,the abundance of bacteroides,lactobacillus in group Bif were increased,and the abundance of fusobacterium was decreased significantly（P<0.01）.The levels of LPS were not directly correlated with the content of bacteroides（r=-0.053,P>0.05）,the levels of LPS were negatively correlated with the content of lactobacillus（r=-0.381,P<0.05）,while were positively correlated with the content of fusobacterium（r=0.882,P<0.01）.Conclusion:1.Intestinal dysbiosis of rats can be induced by high fructose diet,which participates in the pathogenesis of metabolic disorders through the mechanism of inflammation.2.Appropriate amount of supplementary bifidobacterium can ameliorate the metabolic disorders by regulating the intestinal dysbiosis.