| In recent years, nonalcoholic fatty liver disease(NAFLD) has become the most important liver disease gradually in Europe and American and other developed countries. It was suggested that there were 10%-50% adults have liver steatosis worldwide according to epidemiological studies. Some of them can progress to nonalcoholic steatohepatitis(NASH), liver fibrosis, and even liver cirrhosis and hepatocellular carcinoma. It is estimated that NAFLD would likely become a leading cause of end-stage liver diseases and liver transplantation in ten yeas. In the past 20 years, although it was still in controversial for the prevalence and mortality of NAFLD, NAFLD is still recognized as the main causes of non-alcoholic liver cirrhosis. NAFLD is a series of metabolic syndrome presented with high blood pressure, high cholesterol, high blood sugar, obesity, insulin resistance and so on. Its pathophysiology involves in many factors which is still not clear.There is increasing evidence that dysregulation of gut-liver axis is one of the most important factors of NAFLD. Small intestinal bacterial’s overgrowth, intestinal dysfunction and intestinal permeability increase have closely relations to NAFLD. Normally, the intestine contacts with a lot of intestinal bacteria and lipopolysaccharide(LPS) everyday, which can be prevented by intestinal mucosal barrier integrity. When intestinal flora disturbance, impaired intestinal mucosal barrier function and intestinal permeability increase happened, bacteria can pass through the intestinal mucosal barrier, get into the blood circulation and development into intestinal endotoxemia. And by activation of Kupffer cells, it was mediated by LPS-CD14-TLR4 signaling pathway. LPS not only can damage the liver directly, but also increase the expression of TNF-α, IL-6 and other cell factors, which can enhance the action of endotoxin and aggravate liver injury, then developed into a vicious circal. The function of intestinal mucosal barrier plays an important role in the gut-liver axis.Intestinal tract of human contains a large number of microorganisms, and some of bacteria form a symbiotic relationship with human’s body. The definition of probiotics is live microorganisms, which is beneficial to health. It was found that probiotics can not only prevent and treat a variety of gastrointestinal diseases, but also have a beneficial effect on different liver diseases. In recent years, a large number of studies have been performed by researchers to know the beneficial mechanism of probiotics by gut-liver axis. Which was related to regulation of the intestinal microbial composition, anti-bacterial factors secretion, improvement of intestinal epithelial permeability, adjustment of the local and systemic immune function and other factors,while the exact mechanism still needs further research. Saccharomyces boulardii is the only fungi probiotics which is widely used at present, and can not be destroyed by gastric acid, bile and antibiotics. And it can secret some short peptide protein to degrade bacterial toxins, which could regulate the balance of intestinal flora.Objectives: To establish the model of NASH in mice induced by high-fat diet and then investigate the level of endotoxin and the changes of intestinal mucosal barrier to explore the mechanism of intestinal mucosal barrier in NAFLD. It was performed by gavage of Saccharomyces boulardii in NAFLD mice. The purpose is to observe changes of intestinal mucosal barrier and intestinal inflammation by improved intestinal flora, reduced endotoxemia. To investigate the possible mechanisms of the therapeutic effect of the drug in NASH.Methods: ①The model preparation: 6-8 week old C57BL/6 male mice, adaptive fed for one week then weight and marking, then divided into normal control group(NG, n=16) and model group(MG, n=22) randomly. NG group were given control diet and MG group were given high fat diet. At the end of the 33 week, sacrificed five animals of each group randomly to observe the changes of liver histopathology with hematoxylin and eosin(H&E) staining to confirme the successful model of NASH. Then 12 mice were selected from the MG group randomly, and divided into Saccharomyces boulardii intervention group(TB, n=6) and gavage control group(TD, n=6), given high fat diet continuously. TB group were given Saccharomyces boulardii gavage according to 460mg/kg, and the amount of each mouse is 0.5 ml, 1 times per day. The TD group also began to be given accessories(fructose, lactose) of Saccharomyces boulardii, which was dissolved in normal saline. After 8 weeks treatment, all of the mice were sacrificed. ②To observe hair color, activity, eating, drinking and defecation of the mice. And recorded body weight weekly and the intragastric admimistration dose was adjusted according to body weight changes. ③The general situation of the liver and ileum were observed, then to complete resection of the liver and ileum and weight the liver. ④Serum ALT, AST and TG were investigated by Automatic biochemical analyzer. Limulus testing for serum endotoxin level. ⑤The histology of liver was examined microscopically by H&E staining. ⑥The expression of Occludin and IL-17 in ileum tissue of hepatic were detected by immunohistochemistry. ⑦ The m RNA levels of ileum Occludin, IL-17 A, TNF- a, RORγt were detected by real time Q-PCR.Result:1 The body weight and the liver index of mice in each group.At the end of 33 th week, histological pathology by liver H&E staining showed NASH was established. The body weight and liver index in MG group was significantly higher than NG group(body weight: 42.00±3.13 g νs. 28.92±2.62 g, P<0.05; liver index: 8.32±0.68 νs. 5.20±0.42, P<0.05). After intervention treatment, compared with MG group and TD group, TB group were decreased(body weight: 36.02±1.29 g νs. 42.00±3.13 g, P<0.05; 36.02±1.29 g νs. 40.04±2.51 g, P<0.05; liver index: 7.24±0.51 νs. 8.32±0.68, P<0.05; 7.24±0.51 νs. 8.22±0.70, P<0.05), but still higher than that of NG group(body weight: 36.02±1.29 g νs. 28.92±2.62 g, P<0.05; liver index 7.24±0.51 νs. 5.20±0.42, P<0.05). There is no significant changes between TD and MG group(body weight: 40.04±2.51 g νs. 42.00±3.13 g, P>0.05; liver index: 8.22±0.70 νs. 8.32±0.68, P > 0.05). After giving Saccharomyces boulardii treatment to the NASH mice, the body weight decreased, while the gastric perfusion control have no obvious changes.2 Changes of serum biochemical indexes①Compared with NG group, the level of ALT, AST and TG in MG group was significantly increased(ALT: 146.89±7.67 U/L νs. 24.41±5.56 U/L, P<0.05; AST: 124.96±8.79 U/L νs. 53.94±6.34 U/L, P<0.05; TG: 0.80±0.07 mmol/L νs. 0.35±0.08 mmol/L, P<0.05); the TB group’s level of ALT, AST and TG was also increased(ALT: 65.70±4.15 U/L νs. 24.41±5.56 U/L, P<0.05; AST: 110.18±7.19 U/L νs. 53.94±6.34 U/L, P<0.05; TG: 0.50±0.03 mmol/L νs. 0.35±0.08 mmol/L, P<0.05); TD group increased significantly(ALT: 142.66±8.24 U/L νs. 24.41±5.56 U/L, P<0.05; AST: 125.64±0.76 U/L νs. 53.94±6.34 U/L, P<0.05; TG: 0.76±0.07 mmol/L νs. 0.35±0.08 mmol/L, P<0.05); ②Compared with MG group, levels of ALT, AST and TG in TB group were significantly decreased(ALT: 65.17±4.15 U/L νs. 146.89±7.67 U/L, P<0.05; AST: 110.18±7.19 U/L νs. 124.96±8.79 U/L, P<0.05; TG: 0.50±0.03 mmol/L νs. 0.80±0.07 mmol/L, P<0.05); TD group has no significantly change(ALT: 142.66±8.24 U/L νs. 146.89±7.67 U/L, P>0.05; AST: 125.64±0.76 U/L νs. 124.96±8.79 U/L, P>0.05; TG: 0.76±0.07 mmol/L νs. 0.80±0.07 mmol/L, P>0.05). ③Compared with TD group, serum level of ALT, AST and TG in TB group decreased significantly(ALT: 65.17±4.15 U/L νs. 142.66±8.24 U/L, P<0.05; AST: 110.18±7.19 U/L νs. 125.64±0.76 U/L, P<0.05; TG: 0.50±0.03 mmol/L νs. 0.76±0.07 mmol/L, P<0.05).3 Comparison of serum LPS levelsCompared with NG group, the MG group’s level of LPS was significantly increased(0.289±0.031 EU/ml νs. 0.158±0.005 EU/ml, P<0.05). After intervention treatment: ① The level of LPS in group TB was lower compared with MG group and TD group(0.200±0.029 EU/ml νs. 0.289±0.031 EU/ml, P<0.05; 0.200±0.029 EU/ml νs. 0.288±0.031 EU/ml, P<0.05), but still higher than that of NG group(0.200±0.029 EU/ml νs. 0.158±0.005 EU/ml, P<0.05). ② The TD group and MG group’s level of LPS had no significant differences(0.288±0.03 EU/ml νs. 0.289±0.031 EU/ml, P>0.05).4 Liver histological pathology changesNaked eye view: in NG group, the liver has bright color, red brown, sharp edge, soft texture and rich elasticity. In MG group, liver’s diffuse volume increases, pale or sallowish, it’s surface smooth and greasy feeling, edge thick and blunt, the cross section is light yellow or yellow red, less shiny knife surface contamination, fat, slightly hard in quality. The TB group’s liver was yellow, medium texture. But when compared with NG group, the color is still light. TD group and MG group had no obvious change.Under the light microscope: in the NG group, the hepatic lobule structure clear and complete, visible arranged radially around a central vein of liver cell cord. Liver cell volume was normal and boundaries was clear with large and round nucleus and uniform cytoplasm, there wasn’t steatosis and inflammatory infiltration. Liver cells of group MG with a lot of fat vacuoles, ballooning degeneration, inflammatory cells can also be seen in varying degrees of lobular infiltration, inflammatory cell infiltration in portal area and spotty necrosis. After stomach intervention treatment, in the TB group, the liver cell structure still existed around the central vein with mild steatosis of liver cells away from the central vein and a small amount of inflammatory cell infiltration, compare with MG group, there was statistically significant. There were no significant changes between TD and MG group.5 Pathological changes of ileumIn group NG, terminal ileum mucosa structure integrity, villi arranged orderly, neat, not loose, morphology and epithelial cells of normal, were high columnar, no obvious mucosal hyperemia and edema, intestinal glands propria neatly arranged, no infiltration of inflammatory cells; mice in group MG terminal ileum intestinal villi slightly loose, partial atrophy, fracture defect, of different height. Epithelial cell tight junction gap widened, epithelial cell necrosis, epithelium and lamina propria separation, visible edema and inflammatory cell infiltration. Application of Saccharomyces boulardii treatment, TB group’s intestinal mucosal injury is lighter when compared with MG group. TD group and MG group had no obvious change.6 The expression of Occludin protein, IL-17 in mice ileum tissueThe expression of Occludin protein in NG group: the distribution of Occludin protein uniformly at the top junction of intestinal epithelial cells, linear distribution, expression of Tan strong signal; In group MG positive staining of Occludin protein was significantly weakened, continuous decrease, the distribution of expression less than group NG. After intervention, the expression of TB group is higher than MG group, Occludin protein staining deepen, continuous improvement. It had little differences between group TD and group MG. The expression of IL-17: compared with NG group, MG group increased expression;after treatment, TB group decreased expression, but TD group and MG group had no obvious change.7 The expression of Occludin, IL-17 A, TNF- a, RORγtm RNA.Expression of Occludin m RNA in ileum tissue: when compared with NG group, MG group’s expression is lower, the difference was statistically significant(P<0.05); compared with MG group, TB expression increased, but still lower than that in the NG group, the difference was statistically significant(P<0.05); there were no significant differences between TD group and MG group(P>0.05).Expression of IL-17 A m RNA in ileum tissue: when compared with NG group, MG group’s expression is lower, the difference was statistically significant(P<0.05); compared with MG group, TB expression increased, the difference was statistically significant(P<0.05); there was no significant difference between TD group and MG group(P>0.05).Expression of TNF-α m RNA in ileum tissue: when compared with NG group, MG group’s expression is lower, the difference was statistically significant(P<0.05); compared with MG group, TB expression increased, but still lower than that in the NG group, the difference was statistically significant(P<0.05); there were no significant differences between TD group and MG group(P>0.05).Expression of RORγt m RNA in ileum tissue: Compared with NG group, MG group’s expression is lower, the difference was statistically significant(P <0.05); compared with MG group, TB expression increased, the difference was statistically significant(P<0.05); there were no significant differences between TD group and MG group(P>0.05).Conclusion:1 The mice model of NASH could be replicated successfully by high-fat diet, and NASH mice has the disease of intestinal endotoxemia.2 Expression of Occludin protein in NASH mice ileum is reduced, and the damage of mechanical barrier may be an important cause of the intestinal endotoxemia.3 NASH mice intestinal mucosal immune barrier were damaged, and the intestinal mucosal permeability were increased. The Th17 pathway may be involved in the immune response of lymphocytes.4 Saccharomyces boulardii could reduce gut-associated endotoxin and the degree of hepatic steatosis and inflammation and play a role in the treatment of NASH probably by increasing the tight junction Occludin protein expression and regulating Intestinal mucosal immune response. |
PDF Full Text Request