| Objective: Non-alcohol fatty liver disease(NAFLD) is one of the most common complications in diabetic patients, which main pathological characteristic is liver steatosis, hepatitis, liver cirrhosis, and liver cancer. The exact pathogenesis of this disease is yet to be clarified. Insulin resistance and two-hit hypothesis were reported to be relative to NAFLD. Recent reports supported that activation of C-Jun N-terminal protein kinase(JNK) may participate the pathogenesis of NAFLD, which included liver insulin resistance and oxidative stress. As a pro-inflammatory cytokine, tumor necrosis factor-α(TNF-α) induced the activation of JNK signaling pathway, promoted the expression of nuclear factor kappa B(NF-κB), caused liver damage. Liraglutide, a Glucagon-like peptide-1(GLP-1) analogue, not only increases insulin secretion, improves insulin sensitivity and protects pancreatic beta cells, but also decrease liver weight, reduce fat accumulation in liver. However, the mechanism of Liraglutide on NAFLD remains poorly understood. In this study, we developed a rat model of type 2 diabetes to explore the relationship between JNK phosphorylation and NAFLD in type 2 diabetes rats and the intervention of liraglutide.Methods: 24 healthy male wistar rats, adaptive fed for one week, were randomly assigned to normal control group(NC, n = 6) and experiment group EX, n = 18). NC group was fed with standard rodent chow diet, while EX group was fed with high-fat, high-sugar diet, which composed of(by mg) 20% sugar, 10% lard, 2.5% cholesterol, 1% cholic and 66.5% standard chow diet. 4 weeks later, EX rats were intraperitoneally injected with 1% streptozotocin(STZ, 30 mg/kg), NC group was simultaneously injected with equal volume of citric acid buffer. Two weeks later, tail vein blood glucose was measured after 8 hours fasting, and those with blood glucose≥7.8 mmol/L were considered DM rats(among 18 EX rats, 14 developed to diabetes). Diabetic rats were randomly divided into 2 subgroups: diabetic blank control group(DM, n=7), and T2 DM + liraglutide intervented diabetic group(LIR, n=7). The LIR group was given liraglutide 100μg·kg-1 subcutaneously twice one dayily for 8 weeks, the NC group and DM group were given the same amount of sterile distilled water. Body weight was measured weekly(The intervention last for eight weeks). At the end of the experiment, the rats were executed to collect blood from cardiac apex, biochemical indicators, such as total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), TNF-α, and NF-κB were detected. Hematoxylin and eosin(HE) staining were performed to observe the histomorphology of general condition of rats liver tissue, Oil Red O staining to observe the extent of fatty degeneration in liver. The protein expression of JNK and p-JNK in the liver was measured by immunohistochemistry. The p-JNK positive cells were stained brown-yellow in the nuclear,while the JNK positive cells were stained brown-yellow material in the cytoplasm. All the results were observed in microscope and semi-quantitatively analyzed by manual counting method. Numerical variables with a normal distribution were presented as the mean ± standard deviation. Comparisons among groups were performed with ANOVA, followed by a SNK-q test to compare two individual groups. Data with non-normal or homogeneous were presented as the median(minimum and maximum), and comparisons among groups were performed with non-parametric test. Correlation analysis was performed with the linear correlation analysis. Statistical analyses were performed using SPSS statistical software(version 13.0; SPSS Inc.) A two-tailed P < 0.05 was considered to be statistically significant.Results:1 The general situation of ratsThe rats in NC group were in good condition, whose body weight showed a trend of increase, whereas there were no obvious change on diet and urine volme in NC group.There were an increase trend of diet and urine volme in DM and LIR group, some of them had yellow, dark, messy, wet hair. There was no statistically differences on weight among 3 groups(NC group 320.7±12.0g;DM group 326.3±69.6g;LIRgroup 316.3±34.2g;χ2=0.309, P=0.857)2 At the end of six weekIn NC group, The FBG was 6.50±0.52mmol/L, and in EX group it was 15.90±4.85mmol/L, FBG was significantly higher in EX group than NC group(P<0.01).3 Biochemical indexes at the end of fourteen weekNC group: FBG 6.33±0.69 mmol/L, TC 2.18±0.39 mmol/L, TG 0.80±0.26 mmol/L, LDL-C 1.24±0.47 mmol/L, HDL-C 1.55±0.33 mmol/L, ALT 19.67±2.16 U/L, AST 104.50±11.48 U/L, TNF-α 0.83±0.14 ug/ml, NF-κB 78.68±12.15 μmol/L.DM group: FBG 12.99±2.07 mmol/L, TC 4.15±0.41 mmol/L, TG 1.83±0.40 mmol/L, LDL-C 2.53±0.44 mmol/L, HDL-C 0.68±0.23 mmol/L, ALT 139.0±20.74 U/L, AST 150.00±24.75 U/L, TNF-α 1.19±0.16 ug/ml, NF-κB 170.22±21.53 μmol/L.LIR group: FBG 8.87±1.82 mmol/L, TC 3.42±0.82 mmol/L, TG 1.20±0.30 mmol/L, LDL-C 1.86±0.28 mmol/L, HDL-C 0.92±0.11 mmol/L, ALT 67.14±8.11 U/L, AST 164.70±25.49 U/L, TNF-α 0.95±0.06 ug/ml, NF-κB 116.14±32.63μmol/L.The level of FBG, TC, TG, LDL-C, ALT, AST, TNF-α and NF-κB was significantly higher in DM group than those of NC group(P < 0.05), whereas HDL was significantly decreased in DM group than NC group(P < 0.05). After 8 weeks of liraglutide intervation, the level of FBG, TC, TG, LDL-C, ALT, TNF-α, and NF-κB was significantly decreased compared with DM group(P < 0.05). There was an increased trend of HDL and decreased trend of AST in LIR group compared with DM group, but no statistically significant difference was found(PHDL-C=0.070;PAST=0.228).4 Pathological changes of liverUnder HE stainingNC group: under microscopy, liver cells neatly and radially around liver lobules central vein, the structure of hepatic cords was also intact.DM group: The structure of hepatic cords was disorder, and swelling and ballooning degeneration were found in hepatocytes. There was some spotted and focal necrosis in liver lobules.LIR group: pathological alterations such as ballooning degeneration and inflammation can be improved by liraglutide treatment when compared with DM group.Oil Red O stainingNC group: The cytoplasm of the liver cell was stained bule colour, there were barely lipid droplets in liver cells.DM group: Liver cells which locatd aroud the central vein and portal area were found full of lipid droplets. Diffuse fatty degeneration was observed in hepatic tissue.LIR group: Red lipid drops were significantly reduced in liver cells after liraglutide intervention when compared with those of DM group.5 Immunohistochemical resultsThere was significantly difference in the expression of p-JNK/JNK among three groups(χ2=12.606,P=0.002. The expression of p-JNK/JNK was significantly higher in DM group than NC group(Z=-2.935,P=0.003, liraglutide treatment significantly downregulated the expression of p-JNK/JNK(Z=-2.859,P=0.004). The level of TNF-α(r=0.518,P=0.019) and NF-κB(r=0.529,P=0.012) was positive correlated with p-JNK/JNK.Conclution:1 Activation of JNK pathway plays an important role in the development of diabetic non-alcohol fatty liver disease.2 Liraglutide can ameliorate diabetic non-alcohol fatty liver disease through decreasing JNK phosphorylation and improving liver inflammation reaction. |
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