| Objective: 1 To establish the model of nonalcoholic fatty liver disease(NAFLD) in mice. 2 To observe changes of histopathology, biochemical indices, expression of protein, immunology and intestinal bacteria after gavage of saccharomyces boulardii, so that to explore the effects and mechanism of saccharomyces boulardii on mice of NAFLD.Methods:1 38 male mice of C57BL/6, 6~8 weeks, were fed AIN93 control diet for 7 days, then divided into two groups randomly, the Control group(16 mice) and the Western diet(WD) group(22 mice), two groups of mice were fed Control diet and WD diet(high fat diet with high fructose and sucrose in drinking water of 42g/L) respectively for 33 weeks duration. Then twelve mice of WD were divided into TB group and TD group(6 mice per group) randomly, which were given saccharomyces boulardi(5.98×108 CFU/kg /day) or fructose(284mg/kg/day) + lactose(19mg/kg/day) by gavage once a day for 6 weeks, then sacrificed at the end of 6 weeks.2 The general conditions of the mice were observed,such as hair gloss, appetite and so on. The body weight was measured weekly.3 The changes of liver morphology, liver weight, the hepatosomatic indices were detected in each group.4 The changes of liver histopathology were observed with stain of hematoxylin and eosin(HE).5 The changes of Triglyceride(TG), Aspartate aminotransferase(AST), Alanine aminotransferase(ALT), Interleukin-17(IL-17) and endotoxin were investigated in each group.6 The expressions of α-SMA m RNA, TGF-β m RNA, TNF-α m RNA were observed by Real-time fluorescent quantitation PCR.7 The lymphocytes in the liver and spleen from each group were isolated, the ratio of CD4+/CD8+ and the expressions of CD4+T IL-17 and CD4+T RORγt cells were identified by fluorescence activated cell sorting.8 The expressions of α-SMA and TNF-α in liver were identified by immunohistochemistry.9 The changes of intestinal flora in mice were observed with method of 16 s r RNA macro genome sequencing.Results:1 The histopathology revealed normal in the Control group, while the WD group showed diffuse hepatic steatosis and inflammatory cell infiltration. Compared with TD group, hepatic steatosis and inflammation of the TB group were reduced.2 Compared with Control group, the WD group revealed higher serum levels of ALT, AST and TG(P<0.05). Compared with TD group, the TB group had lower serum levels of ALT, AST and TG(P<0.05).3 Real time QPCR: Compared with Control group, the WD group showed significantly increased expressions of α-SMA, TGF-β and TNF-α m RNA(P < 0.05). Compared with TD group, the TB group had lower expressions of TNF-α m RNA(P<0.05), while TGF-β m RNA and α-SMA m RNA showed no differences(P>0.05).4 Serum level of IL-17: Compared with Control group, the WD group displayed higher serum level of IL-17(P<0.05), Compared with TD group, the TB group had lower serum level of IL-17(P<0.05).5 Serum level of endotoxin: Compared with Control group, the WD group displayed higher serum level of endotoxin(P<0.05), compared with TD group, the TB group had lower serum level of endotoxin(P<0.05).6 Flow cytometry results:Liver: Compared with Control group, the ratio of CD4+/CD8+ in WD group was elevated(P<0.05). Compared with Control group, the expression of CD4+T RORγt in WD group was increased(P<0.05), however, there showed no differences in the expression of CD4+T IL-17 between the two groups(P>0.05). Compared with TD group, the TB group showed lower ratio of CD4+/CD8+ and lower expression of CD4+T RORγt(P<0.05), while there showed no differences between TB and TD group in the expression of CD4+T IL-17 in liver(P > 0.05). Spleen: The ratio of CD4+/CD8+, the expression of CD4+T RORγt and CD4+T IL-17 in spleen showed no significant differences between two groups before and after intervention(P>0.05).7 Immunohistochemistry: Compared with Control group, the WD group showed increased expressions of α-SMA and TNF-α(P<0.05). Compared with TD group, the TB group showed lower expression of TNF-α(P<0.05), while there showed no differences of α-SMA between TB and TD groups(P>0.05).8 16 s r RNA macro genome sequencing: Compared with TD group, there showed increases of the expressions of Firmicutes, Verrucomicrobia and Actinobacteria in TB group, while the expressions of Bacteroidetes and Proteobacteria decreased in TB group(P<0.05). Among the top fifteen kinds of intestinal flora, there showed no differences in the rest of the flora between two groups(P>0.05).Conclusions:1 NAFLD model was established successfully with classical Western diet.2 Saccharomyces boulardii may play a role in the treatment of NAFLD by regulating the intestinal flora.3 Saccharomyces boulardii may play an important role in NAFLD by down-regulating the the ratio of CD4+/CD8+ in lymphocytes of liver.4 Saccharomyces boulardii may play an important role in NAFLD by down-regulating the expression of CD4+T RORγt in lymphocytes of liver.5 Saccharomyces boulardii may play an important role in NAFLD by inhibiting the expressions of endotoxin, TNF-α and IL-17. |
PDF Full Text Request