Effect Of Microbial Pharmaceutics On C-Jun N-terminal Kinase In Nonalcoholic Steatohepatitis

Objective: Nonalcoholic steatohepatitis (NASH) was defined as a syndrome which has a non-alcohol drinking history, or the consumption of alcohol drinking was less than 140g per week in male, 70g in female. It is a common chronic liver disease that characterized by the hepatocytes with diffuse fatty degeneration, ballooning degeneration, lobular inflammation and (or) the collagen deposition in central venous and sinus. NASH is frequently associated with many diseases such as obesity, type 2 diabetes mellitus, hyperlipidemia, and hypertensive disease. c-Jun N-terminal kinase (JNK) is an member of important mitogen-activated protein kinase family. JNK is an important signal transduction system, which is mediated by extracellular stimuli into intracellular responses. In the progression of NASH, the bacterial overgrowth in small intestinal, endotoxin increases in the portal vein, JNK is activated by the Toll-like receptor signaling. We presumed the activated JNK can activate the downstream genes, which correlated with many inflammatory, by phosphorylating c-jun which is one of the transcription factors. These downstream genes releases a great quantity of pro-inflammatory cytokines such as tumor necrosis factorα(TNFα) that effects the hepatocytes by inflammatory reaction.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 correlation of the expression of JNK and c-jun in the liver tissue with the histological manifestations of liver (steatosis, inflammation, fibrosis), TNFα, and the endotoxin in the blood plasma were investigated, to reveal the effect of JNK on the pathogenesis of NASH and to provide theory basis for the prevention and therapy of this kind of disease.Methods: 36 healthy male Wistar rats have been randomly divided into normal control group, high fat diet for 8 weeks, 12 weeks groups, high fat diet for 16 weeks group A, high fat diet for 16 weeks group B, and microbial pharmaceutics treatment group. The rats have been put to death respectively in the 0 week, 8 weeks, 12 weeks and 16 weeks of the model.The serum, plasma and specimens of liver were collected. Cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were measured using an auto-biochemical analyzer. Enzyme-linked immunosorbent assay (ELISA) was used to test serum TNFαin the rat. Tachypleus amebocyte lysate was used to test plasma endotoxin level of the rat. The specimens of liver were divided into four parts. The first part was frozen sections for SudanⅣstaining for hepatocyte steatosis. The second part was fixed in 10% formaldehyde for Hematoxylin-eosin (HE) staining to observe the general pathologic changes of liver and Masson staining for fibrosis. The third part was fixed in 4% paraformaldehyde for immunohistochemical staining to examine the expression of JNK and phosphor-JNK (P-JNK) which was calculated by multifunctional pathological image analyzer. Five areas were chosen randomly from the center and periphery in each section to calculate the average area density under 20 power object lens, and take the average values. The rate of P-JNK/JNK was represented the activity of JNK in the liver. The rest liver were frezzed quickly in liquid nitrogen, then in -80℃frig to detect the expression of JNK mRNA, c-jun mRNA in the liver by reverse transcription polymerase chain reaction (RT-PCR) method. SPSS 13.0 was used to analyse all data.Results:1 The general state of rats: Normal rats were all active and had good appetite with bright hair. The high fat diet rats had bad appetite and the weight increased gradually. The spirit, appetite and activity in the treatment group rats have been ranged between the normal control group and the high fat diet 16 weeks group B.2 Serum biochemistry, circulating TNFαand plasma endotoxin levels in every group: The level of serum TC (2.782±0.312, 3.455±0.324, 3.865±0.183), TG (0.693±0.115, 0.855±0.104, 1.018±0.133), ALT (63.435±15.853, 85.087±14.250, 102.948±12.264), AST (94.553±7.087, 118.442±12.838, 153.435±13.346), TNFα(11.157±2.703, 21.333±4.077, 28.223±2.885) and plasma LPS (0.815±0.107, 1.327±0.209, 2.157±0.238) in the 8wks, 12wks and 16wks A in high fat diet groups respectively higher (P<0.01) than that in normal control (1.843±0.216, 0.430±0.109, 38.400±12.128, 70.447±5.354, 0.478±0.092, 6.127±1.239). All the above indexes of treatment group (3.287±0.360, 0.838±0.136, 81.057±15.508, 104.400±12.268, 1.275±0.341, 19.73±2.885) was significantly lower (P values were <0.05, <0.05, <0.05, <0.01, <0.01, <0.01) compared with high fat diet groups for 16 weeks group B (3.842±0.200, 1.037±0.151, 102.953±12.545, 154.408±14.218, 2.153±0.257, 28.375±2.198).3 Histopathological changes in the liver: The normal rat livers were henna and bright, but the livers in the high fat diet group were greasy and dim. The appearance in the treatment group was ranged between normal control group and high fat diet 16weeks group B. Under the light microscope, the normal hepatocytes arranged in radiation from the central veins with HE staining. In the 8wks, 12wks, 16wks A and B model rats, sections showed a gradual fat and inflammatory cells accumulation in liver cells with the high-fat feeding. The hepatocytes of the treatment group was improved compared with high fat diet for 16 weeks group B in the inflammatory. There was very little collagen in central veins and portal areas in the normal and 8wks, 12wks model rats with Masson staining, but in the 16wks model A and B rats fibrosis presented around the hepatic sinusoids and venofibrosis. Hepatic fibrosis in the treatment group was significantly reduced compared with high fat diet for 16 weeks group B. With SudanⅣstaining, no red granula could be found in hepatocyte cytoplasm in the normal rats, but the number of the red granula increased in the high fat diet groups. The steatosis of hepatocytes in the treatment group was remarkably reduced, compared with high fat diet 16weeks group B.4 The expression of JNK mRNA, c-Jun mRNA in the liver tissue: the expression of JNK mRNA, c-jun mRNA in the liver was detected by RT-PCR. It showed that there was a few of expression of JNK mRNA (0.319±0.027), c-jun mRNA (0.299±0.039) in the liver of normal rats. The expression of JNK mRNA (0.439±0.039, 0.569±0.031, 0.645±0.032), c-jun mRNA (0.385±0.063, 0.525±0.074, 0.611±0.033) in the 8wks, 12wks and 16wks A of high fat diet rats increased with the time (P<0.01). And the expression of JNK mRNA (0.439±0.057), c-jun mRNA (0.367±0.062) in the treatment group was significantly reduced (P<0.01) compared with high fat diet groups for 16 weeks B (0.645±0.027, 0.611±0.029), the difference was significant statistically.5 The expression of JNK, P-JNK in the liver tissue: The rate of P-JNK/JNK was represented the activity of JNK in the liver. Immunohistochemical staining was performed and showed that the activity of JNK in the high fat diet groups (1.698±0.118, 2.215±0.065, 2.376±0.047, 2.617±0.064) increased (P<0.01) gradually with the time. The rate of P-JNK/JNK in the treatment group (2.279±0.067) was significantly reduced (P<0.01) compared with high fat diet groups for 16 weeks B (2.610±0.063), the difference was statistically significant.6 Correlation analysis shows that there is linear correlation between the activity of JNK in the liver tissue and TNFαin the serum, plasma LPS, the r values respectively are 0.776, 0.868, both P values are less than 0.01. Conclusion:1 Rat model of NASH could be developed by feeding with high fat diet gradually. TNFαand LPS play an important role in the development of NASH.2 The activity of JNK and phosphorylated c-jun, they were in the liver tissue of rat with NASH, increased significantly with the time of feeding high-fat diet. It shows that the correlation with serum TNFα, plasma LPS level and the degree of hepatic inflammation was positive, suggesting that JNK plays a key role in the development of NASH.3 Microbial pharmaceutics can add to the normal intestinal flora, reduce the LPS, TNFαin the intestinal, improve inflammation by inhibiting the JNK signaling pathway and the expression of P-JNK to prevent NASH.