| Objectives: Nonalcoholic steatohepatitis (NASH) is correlated with inheritance-environment-metabolism, which is a clinicopathological syndrome that characterized by hepatic steatosis and lobules inflammation accompanied with focal necrosis. The etiopathogenesis of NASH has not been clearly elucidated. Variety factors such as liver innate immunity, environment-genetic may together involve in the occurrence even progress of NASH.In this study, the rat model of NASH has been established by means of feeding high fat diet, the microbial pharmaceutics-Live Combined Bifidobacterium, Lactobacillus and Streptococcus Thermophilus has been used to intragastric administration. The effect of this drug on such indexes in the liver injury of NASH model that are the expression of CD1d and CXCL16 in the liver tissues and the percentage of hepatic NKT cells as well as serum levels of Th1 type cytokines such as TNF-α, IL-12 has been observed, to investigate the possible therapeutic effect and mechanisms of this drug.Methods: 36 healthy male Wistar rats have been randomly divided into normal control group A, normal control group B, high fat diet for 8 weeks, 12 weeks, 16 weeks groups and Live Combined Bifidobacterium, Lactobacillus and Streptococcus Thermophilus treatment group. The rats have been put to death respectively in modeling 0 weeks, 8 weeks, 12 weeks, and 16 weeks. serum biochemical indexes were detected by automatic biochemical analyzer; Serum levels of IL-12, TNF-αwere detected by enzyme-linked immuno- sorbent assay (ELISA); The changes of liver histology were observed by HE, SudanⅣand Masson staining; The percentage of hepatic NKT cells in T lymphocyte subpopulations was detected by flow cytometry; The expression of CD1d, CXCL16 were detected by RT-PCR and immunohistochemistry method respectively. Results:1 General performance: Normal control rats have shiny fur, good appetite, good state of mind, and their body weight have increased gradually; High fat diet rats have apathetic spirit, reduced activity, loss of appetite, less glossy fur, and their body weight have increased markedly; The spirit, appetite and activity of treatment group rats have been ranged between the normal control group and the high fat diet group.2 Changes of serum indexes2.1 Dynamic changes of serum indexesRats serum cholesterol(TC), triglyceride(TG), alanine aminotransferase (ALT), aspartate aminotransferase(AST) levels in high fat diet groups for 8 weeks, 12 weeks and 16 weeks are increased significantly (P<0.01), compared to those of normal control group A. Those indexes significantly increase with prolonged constructing model time; Serum TNF-αlevels: The rats serum TNF-αlevels of high fat diet groups for 8 weeks, 12 weeks, 16 weeks respectively are 11.39±2.59, 21.2±5.18, 28.23±4.31, which are significantly increased (P<0.01) compared to that of normal control group A (5.92±1.68); Serum IL-12 levels: The rats serum IL-12 levels of high fat diet groups for 8 weeks, 12 weeks, 16 weeks respectively are 49.493±8.024, 68.392±7.302, 78.957±12.434, which are significantly increased (P<0.01) compared to that of normal control group A (34.256±7.518), all the above results show that the serum levels of TNF-α, IL-12 have increased gradually with prolonged constructing model time.2.2 Effect of microbial pharmaceutics on serum indexesThe serum levels of TC, TG, ALT, AST in high fat diet group for 16 weeks respectively are 3.89±0.22, 0.93±0.26, 103.37±20.11, 154.82±17.59, all the above indexes of normal control group B (1.87±0.19, 0.55±0.25, 35.38±12.47, 73.54±10.63) significantly increase (P<0.01); All the above indexes of treat- ment group respectively are 2.12±0.18, 0.81±0.18, 72.74±18.94, 92.56±15.37, are significantly decrease (P <0.05)compared to those of high fat diet group for 16 weeks; Serum TNF-αlevels: serum TNF-αlevel of treatment group are 18.66±3.54, significantly lower (P<0.01) than that of high fat diet 16 group for weeks (28.23±4.31); IL-12 levels: serum IL-12 level of treatment group are 37.562±7.557, significantly lower (P<0.01) than that of high fat diet 16 group for weeks (78.957±12.434).3 Histologic changes of liverIn visual observation, the appearance of rat liver tissue in normal control group is florid, bright and shiny, soft and flexible; the liver volume in high fat diet group rat increases significantly. The color of liver is light yellow, with the surrounding tissue adhesion. The cross section feels greasy and dull; the liver appearance in treatment group rat ranges between normal control group and high fat diet group.In light microscope, the normal hepatocyte arranges in radiation by the center of the central veins in the normal control group, the structure of hepatocyte cord is in good order by HE staining; SudanⅣstaining shows that there is no lipid droplets in hepatocyte and the cytoplasm appears blue; Masson triple-staining shows that there are no obvious deposition of collagen fibers in central vein and periportal. HE, SudanⅣstaining shows that part of hepatocytes are ballooning degeneration, cytoplasm is filled with a large number of pink lipid droplets, hepatocytes steatosis are most evident around the central vein, in which spotty necrosis can be seen in high fat diet 8 weeks group; The swelling degree of hepatocytes increases gradually. The pink lipid droplets increase in cytoplasmic. The central vein is surrounded by infiltration of inflammatory cells, which are mainly mononuclear cells. Focal necrosis can be seen around central vein in high fat diet 12 weeks group; In high fat diet 16 weeks group, there are diffuse steatosis and full of pink lipid droplets in rat hepatocytes. Hepatocyte cord structural is in disorder and intralobular diffuse spotty necrosis and focal necrosis are obvious; While in treatment group, pink lipid droplets in hepatocytes and inflammatory cell infiltration are significantly reduced. There are no significant collagen fibers depositition around central veins as well as portal areas in both high fat 8 weeks group and 12 weeks group rat liver; While in high fat diet 16 weeks group, portal areas expand and collagen fibers deposition around sinusoid can be observed; In treatment group, there are no significant collagen fibers deposition around central veins and portal areas by Masson triple-stain.4 The percentage of hepatic NKT cells in T lymphocyte subpopulations detected by flow cytometry4.1 Dynamic changes of percentage of hepatic NKT cells in T lymph- ocyte subpopulationsThe percentage of hepatic NKT cells in high fat diet 8 weeks group, 12 weeks group and 16 weeks group respectively are (17.48±2.43)%, (15.08±1.81)%, (8.31±3.81)%, which significantly decrease compared to that of normal control group A (25.26±2.56)% (P<0.01), the percentage of hepatic NKT cells decreases significantly with the prolonged constructing model time (P<0.01).4.2 Effect of microbial pharmaceutics on percentage of hepatic NKT cells The percentage of hepatic NKT cells significantly reduces in high fat diet 16 weeks group (8.31±3.81)%, compared to that of normal control B group (22.94±2.65)% (P<0.01); The percentage of hepatic NKT cells (16.90±1.96)% in treatment group significantly increases compared to that of untreated group (8.31±3.81)% (P<0.01).5 CD1d mRNA and CXCL16 mRNA expression in rat liver tissue by RT-PCR detection5.1 Dynamic changes of CXCL16 mRNA and CD1d mRNA in rat liver tissueCD1d mRNA expression in rat liver tissue: the relative content of rat liver CD1d mRNA respectively are 0.640±0.040, 0.579±0.027, 0.507±0.023, 0.447±0.037 in normal control group A, high fat diet 8 weeks group, 12 weeks group and 16 weeks group, which shows that the expression of CD1d mRNA in rat liver tissue decreases significantly with the prolonged constructing model time(P<0.01); The relative content of rat liver tissue CXCL16 mRNA are 0.215±0.031, 0.364±0.032, 0.529±0.046, 0.586±0.016 in normal control group A, high fat diet 8 weeks group, 12 weeks group and 16 weeks group, which shows that the expression of CXCL16 mRNA in rat liver tissue increases significantly with the prolonged constructing model time(P<0.01).5.2 Effect of microbial pharmaceutics on rat liver tissue CD1d mRNA and CXCL16 mRNA expressionThe relative content of rat liver tissue CD1d mRNA significantly reduces in high fat diet 16 weeks group (0.447±0.037), compared to that of normal control B group (0.666±0.027) (P<0.01); The relative content of rat liver tissue CD1d mRNA in treatment group (0.559±0.044) significantly increases compared to that of untreated group (0.447±0.037) (P<0.01).The relative content of rat liver tissue CXCL16 mRNA significantly increases in high fat diet 16 weeks group(0.586±0.016), compared to that of normal control B group (0.217±0.034) (P<0.01); The relative content of rat liver tissue CXCL16 mRNA in treatment group (0.375±0.035) significantly reduces compared to that of untreated group (0.586±0.016) (P<0.01).6 CXCL16 expression in rat liver tissues by immunohistochemistry detection6.1 Dynamic changes of CXCL16 in rat liver tissueThere is weak CXCL16 expression within cytoplasm of rat liver in normal control group (0.5±0.837); Expression of CXCL16 in rat liver tissue of high fat diet groups significantly increases. The number of positive cells increases and the cytoplasmic of positive cells are in dark color. The relative content of rat liver tissue CXCL16 are 10.67±1.862, 21.0±2.898, 25.67±4.844 in high fat diet 8 weeks group, 12 weeks group and 16 weeks group, which shows that the expression of CXCL16 increases significantly compared to that of normal control group A(0.5±0.837) (P<0.01); The expression of CXCL16 in rat liver increases gradually from high fat 0 week to high fat diet 12 weeks(P<0.01), but after 12 weeks of high-fat diet the expression of CXCL16 without significantly increases.6.2 Effect of microbial pharmaceutics on CXCL16 expression in rat liver tissueThe relative content of CXCL16 in rat liver tissue significantly increases in high fat diet 16 weeks group (25.6±4.844), compared to that of normal control B group (0.67±1.211) (P<0.01); The number of positive cells reduces and the relative content of CXCL16 is 8.67±2.805 in treatment group, which significantly reduces compared to that of untreated group (25.6±4.844) (P<0.01).7 Correlation analysis shows that there is linear correlation beteen liver tissue CD1d expression and percentage of hepatic NKT cells and r value is 0.774, P values is less than 0.01; There are negative correlation with percentage of hepatic NKT cells and serum TNF-α, IL-12 levels, percentage of hepatic NKT cells and CXCL16 expression in liver tissue, r values respectively are -0.718, -0.767, -0.795, both P values are less than 0.01.Conclusion:1 Rat model of NASH could be established by feeding with high fat diet gradually; high fat diet can result the decrease and dysfunction of CD1d- dependent NKT cells, promote the release of Th1 cytokines.2 CXCL16 plays an important role in mediating the progress of inflamma- tory lesions in liver tissues with NASH.3 Microbial pharmaceutics can exert its anti-inflammatory influence by increasing hepatic NKT cells, enhancing its immune functions and inhibiting the expression of CXCL16 to suppress the release of Th1 cytokines in NASH.
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