| Objective: To establish the model of nonalcoholic fatty liver disease(NAFLD)in mice induced by western diet and then observe the level of endotoxin,early growth response-1(Egr-1)of liver,the changes of intestinal mucosal barrier and their changes after gavage of saccharomyces boulardii,so that to investigate the effect and possible mechanisms of saccharomyces boulardii on mice of NAFLD.Methods:30 male C57BL/6 mice,6-8 weeks old,adaptive fed for one week,then were divided into normal control group(CG,n=12)and model group(MG,n=18)randomly.Normal control group were given normal control diet(AIN93M control diet plus normal drinking water),and model group were given western diet(high fat diet plus high-sugar drinking water).At the end of the twenty-fourth week,6 mice were sacrificed randomly at the normal control group and model group respectively,to collect specimens.Then the model group of 12 mice were divided into saccharomyces boulardii intervention group(TB,n=6)and control intervention group(TD,n=6)randomly.The TB group was given saccharomyces boulardii(5.98×108 CFU/kg/day).The normal control group of 6 mice(NG,n=6)and the TD group were given fructose(284mg/kg/day)+ lactose(19mg/kg/day)by gavage for 6 weeks,then were sacrificed after 6 weeks.Observe hair gloss,behavior,mental status of the mice,and record body weight weekly.After the mice were killed,general situation of liver and ileum were observed,then to resect them and weight the liver.Serum ALT,AST,TC and TG were tested by automatic biochemical analyzer.The serum LPS levels were detected by limulus testing.The histology of liver and ileum were observed microscopically by hematoxylin and eosin(HE)staining.Ultrastructural changes of ileum tissue were observed by transmission electron microscope.The m RNA expression of Egr-1,TNF-α in liver tissue and Occludin,TNF-α in ileum were detected by Real-time quantitation PCR.The protein expression of Egr-1,TNF-α in liver tissue and Occludin,TNF-α in ileum were detected by Western Blot.Results:1 The general conditions of the miceIn normal control group,the fur of mice was smooth,dense and shiny,the mental state was good,and they were flexible,the body weight increased smoothly.In model group,from the beginning of the fourth week,the fur was not smooth,greasy slightly,the weight gained rapidly,they were abdominal obesity gradually.Up to the twenty-fourth week,the mice were depressed,tended to be lazy,sluggish and dull.After given saccharomyces boulardii by gavage,mice of TB group weight decreased,their situation improved.During the experiment,the mice in TB group and TD group died 1 respectively,remaining 5,and the other groups had no injuries and deaths.2 Comparisons of the body weight and the liver weight indexThe body weight and liver weight index of the MG group were significantly higher than CG group(P<0.05).The difference was statistically significant.After intragastric administration,compared with TD group,the body weight and liver weight index of TB group were decreased(P <0.05),but still higher than that of NG group(P<0.05),there were statistically significant difference.3 Histopathology of liver and ileumHistopathology of liver: The CG group revealed the hepatic lobule structure clear and complete,liver cell volume was normal,the liver cells were arranged in order,liver cell cord arranged radially around a central vein,the cell nuclei were large and plump,there wasn’t steatosis and inflammatory infiltration.In the MG group,the arrangement of hepatocytes was loose and disordered,the hepatocytes were filled with a lot of fat vacuoles,ballooning degeneration,and there were varying degrees of inflammatory cells infiltration,punctate necrosis.After intragastric administration,the TB group showed that hepatic steatosis was relieved and inflammatory cells infiltration reduced compared with TD group,however,compared with the NG group,the degree of steatosis and inflammatory infiltration was still obvious in TB group.Histopathology of ileum: In CG group,the terminal ileum mucosa was clear,the structure was complete,the villi and gland were arranged orderly.In MG group,ileum villi was loose,partial atrophy,fracture defect,height variable.Epithelial cells emerged necrosis and shedding,and their gap widened,the separation of epithelium and lamina propria in terminal ileum could be seen.After intragastric administration,Intestinal mucosal injury of TB group was lighter when compared with TD group,but there was still a large gap between the TB group and the NG group.4 Ultrastructural changes of ileum tissue under transmission electron microscopeIn CG group,the intestinal epithelial cells were arranged closely,with abundant microvilli on the surface,regular arrangement.Tight connection was relatively long,small connections.In MG group,the microvilli on the surface of intestinal epithelial cells were scattered,sparse,and even disappeared.Compared with TD group,the microvilli on the surface of epithelial cells in TB group gradually increased,the structure of tight junction was restored,and the gap was reduced after intervention of saccharomyces boulardii,but it wasn’t as good as the NG group.5 Comparison of serum biochemical indexesCompared with CG group,the levels of ALT,AST,TC and TG in MG group were significantly increased(P<0.05).After intragastric administration,compared with TD group,the levels of ALT,AST,TC and TG of TD group were significantly decreased(P<0.05),but still higher than that of NG group(P<0.05),the difference was significant statistically.6 Comparison of serum LPS levelCompared with CG group,the level of LPS in MG group was significantly increased(P<0.05).After intragastric administration,the level of LPS in TB group was lower compared with TD group,but still higher than that of NG group(P<0.05).The difference was statistically significant.7 Expression of Egr-1 and TNF-α in liver tissueApplication of PCR technology and Western Blot technology detected the expression of Egr-1 and TNF-α in liver tissue from the gene level and protein level.As the results showed,the expressions of Egr-1 and TNF-α in MG group were significantly higher than that of CG group(P<0.05).After intragastric administration,the expressions of Egr-1 and TNF-α in TB group were significantly lower than that of TD group(P<0.05),but higher than NG group(P<0.05),the difference was statistically significant.8 Expression of Occludin and TNF-α in terminal ileum tissuePCR and Western Blot results showed: In group MG,the expression of Occludin in ileum tissue was lower than that in CG group(P<0.05),the difference was statistically significant.After intragastric administration,the expression of Occludin in TB group was higher than that in TD group(P<0.05),but was lower than that in NG group(P<0.05),the difference was statistically significant.Compared with CG group,the expression of TNF-α in ileum tissue significantly increased in MG group(P<0.05).After intragastric administration,compared with TD group,the expression of TNF-α in the ileum tissue of TB group was significantly decreased(P<0.05),but still higher than that of NG group(P<0.05),the difference was statistically significant.Conclusions:1 NAFLD mice model was established successfully by taking the western diet,there existed intestinal endotoxemia in NAFLD mice.2 The NAFLD mice have impaired intestinal mucosal mechanical barrier and increased intestinal permeability,which may aggravate the intestinal endotoxemia.3 The expression of Egr-1 in the liver of NAFLD mice was increased,which may be involved in the pathogenesis of NAFLD.4 Saccharomyces boulardii could improve intestinal mucosal mechanical barrier,reduce the intestinal endotoxemia,decrease the expression of Egr-1,thereby improving the severity of liver inflammation and steatosis. |
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