| Objective:To establish the model of nonalcoholic fatty liver disease(NAFLD) by feeding Sprague-Dawley (SD) rats with high-fat diet. To make dynamic observation on the change of indicators of serum, liver homogenates,IR and histopathology on the whole process of constructing the model of NAFLD in SD rats.Methods:Male SD rats(n=192) were randomly divided into two groups;control group(NG,n=96) fed on normal diet, and high-fat diet group(HG,n=96) fed on high-fat diet. At the beginning of the 1st week(w) and at the end of the 2end,4th,8th,12th,16 th w,8 rats in each group were randomly chosen to test glucose infusion rate(GIR) by euglycemic hyperinsulinemic clamp.Meanwhile,among the rests,8 rats that been randomly selected from both groups respectively,which blood was obtained from portal vein before liver removal.The blood of rats were assayed for alanine aminotransferase(ALT),aspartate aminotransfer-ase(AST), triglyceride (TG),total cholesterol(TC) and fasting blood sugar (FBS) by biochemistry automatic analyzer.The fast insulin(FIns) and tumor necrosis factor-α(TNF-α) were determined with radioimmunoassay and Free fatty acids(FFAs) were analyzed with spectrophotometer in blood.Superoxide dismutase(SOD) and malondialdehyde(MDA) of the liver homogenates were tested by spectrophotometer.Liver tissue of all rats were stained with hematoxylin-eosin and SudanⅣ.Hepatic stetosis was observed by microscope. Results:Compared with NQthe weight,liver index, ALT, AST,TG, TC,FIns,FFAs,TNF-a and MDA levels increased,while the SOD level decreased in HG at the end of the 8th,12th,16th(p<0.05). From the 8th to 16thw, liver index and the levels of indicators of serum and liver homogenates progressively increased,while SOD level gradually reduced in HG (p<0.05). Compared with NQGIR in HG were reduced at the end of the 4th,8th,12th,16th w.GIR gradually reduced in HG from 4th to 16thw, and it presented time-dependant manner(p<0.05).Hepatocytes fatty degeneration in HG aggravated progressively.Hepatic steatosis and nonalcoholic steatohepatitis(NASH) were obtained respectively by feeding on high-fat diet for 8 weeks and 12 weeks.Conclusion:The models of the hepatic steatosis and NASH could be established respectively by feeding on high-fat diet for 8 weeks and 12 weeks in SD rats.Hepatic lipid accumulation,TNF-a, FFAs and oxidative stress reaction have involved in the progression of NAFLD. IR occurred before hepatic steatosis.And IR could be the initiating factor of NAFLD. Objective:To make dynamic observation on the expression of JNK1(C-Jun N-terminal kinase-1) and its effect on insulin signal transduction pathway in nonalcoholic fatty liver disease(NAFLD).Methods:Male SD rats(n=192) were randomly divided into two groups:control group(NG,n=96) fed on normal diet,high-fat diet group(HG,n=96) fed on high-fat diet. At the beginning of the 1st week(w) and at the end of the 2nd,4th,8th,12th,16th w,8 rats taken randomly from each group were sacrificed and liver samples were taken.The expression of JNK1 protein,insulin receptor substrate-1 (IRS-1) protein,Protein kinase B(PKB),phospho-IRS-1 Ser307(p-IRS-1Ser307)and phospho-PKBSer473 (p-PKBSer473) in liver tissue were detected by Western blot.Results:The expression of JNK1 protein and p-IRS-1Ser307 in liver tissue increased,while expression of p-PKBSer473 decreased at the end of the 2nd,4th,8th,12th,16thw in HG compared with NG.The expression of JNK1 protein and p-IRS-1 Ser307 in liver tissue progressively increased while expression of p-PKBSer473 gradually reduced in HG from the 2nd to the 16thw,and the levels presented time-dependant manner(p<0.05). No differences were observed in IRS-1Ser307 and PKBSer473 phosphorylation in NG at different periods.(p>0.05).No differences were observed concerning the expression of IRS-1 and PKB in multiple comparison in each group(p>0.05). A positive correlation was found between the expression intensity of JNK1 and Insulin resistance(IR) (Pearson correlation:0.931,p<0.01).Conclusion:The high-fat diet could increase the expression of JNK1. Increased JNK1 promoted p-IRS-1 Ser307 up-regulated and p-PKBSer473 down-regulated,and these changes disturbed the insulin signal transduction.Finally,it led to IR,so NAFLD ensued.Furthermore, tumor necrosis factor-a,free fatty acids and oxidative stress reaction mediated IR through JNK1 activation.IR of liver occurred before systemic IR and liver steatosis.And IR could be the initiating factor of NAFLD. Objective:To investigate the therapeutic effect and mechanism of recombinant adenovirus expressing dominant-negative type c-Jun N-terminal kinase1(Ad-DN-JNK1) on NAFLD in SD rats.Methods:Male SD rats (n=96) were randomly divided into two groups:control group(NG,n=32) fed on normal diet,and high-fat diet group(HG,n=64) fed on high-fat diet.After 8 weeks treatment,8 Rats that been chosen randomly from each group were tested glucose infusion rate(GIR) by euglycemic hyperinsulinemic clamp.Meanwhile,among the rests,8 rats that been randomly selected from both groups respectively, which blood was obtained from portal vein before liver removal.The rats liver samples were harvested for identifying model.The rest 16 rats in NG were named control group 12w(NG12w).And the rest 48 rats in HG were divided randomly into three groups with 16 rats in each group:HG12w,adenovirus vector carrying green fluorescence protein (Ad-GFP) group and Ad-DN-JNK1 group.The NG12w fed on normal diet, while the other three groups fed on high-fat diet.Rats from NG12w and HG12w groups were injected with 1ml normal saline as control;rats from Ad-GFP group were injected with lml 2.5×1010pfu Ad-GFP as adenovirus control, and rats from Ad-DN-JNK1 group were injected with 1ml 2.5×1010pfu Ad-DN-JNK1 as treatment group at the end of the 8thw.All rats were injected from caudal vein. At the end of the 12thw,8 rats that been randomly selected from each group were tested GIR by euglycemic hyperinsulinemic clamp.And blood samples were obtained from portal vein before liver removal of the rest rats in each group.Rats blood were tested for alanine aminotransferase (ALT),aspartate aminotr-ansferase(AST),triglyceride(TG),total cholesterol(TC) and fasting blood sugar(FBS) by biochemistry automatic analyzer.The fast insulin(FIns) and tumor necrosis factor-a(TNF-a) were determined with radioimmunoassay and Free fatty acids(FFAs) were analyzed with spectrophotometer in blood. Superoxide dismutase(SOD) and malondialdehyde(MDA) of liver homogenates were tested by spectrophotometer.TG,TC and FFAs of liver homogenates were analyzed with the technique as previously described.The expression of c-Jun N-terminal kinase-1(JNK1)protein, insulin receptor substrate-1 (IRS-1) protein,Protein kinase B(PKB),phospho-IRS-1Ser307(p-IRS-1Ser307)and phospho-PKBSer473(p-PKBSer473) in the liver tissue were detected by Western blot.The liver tissue of all rats were stained with hematoxylin-eosin and Sudan IV.The hepatic stetosis was observed by microscope.Results:The model of hepatic stetosis with insulin resistance(IR) in SD rats were established in HG at the end of the 8thw.Compared with NG12w,the weight,liver index, ALT,AST,TG,TC,FIns,FFAs and TNF-a levels in blood and the levels of TG,TC,FFAs and MDA of the liver homogenates increased while SOD level of liver homogenates and GIR decreased in HG12w and Ad-GFP group(p<0.05).Compared with NG12w,increases in JNK1 and p-IRS1Ser307 and decrease in p-PKBSer473 were observed in HG12w and Ad-GFP group (p<0.05). Compared with HG12w and Ad-GFP group,all the index in serum and liver homogenates decreased except SOD in liver homogenates and GIR increased,and the expression of JNK1 and p-IRS1Ser307 decreased while p-PKBSer473 increased in Ad-DN-JNK1 group (p<0.05).No significant differences were observed in the weight,liver index, and levels of AST,FIns,GIR and the expression of JNK1,p-PKBSer473 and p-IRSlSer307 (p>0.05) except the rest indicators of serum and liver homogenates in Ad-DN-JNK1 group compared with NG12w (p<0.05).There were no obvious difference on the expression of IRS-1 and PKB in multiple comparison in each group(p>0.05).No difference were observed about all indicators in HG12w compared with Ad-GFP group.The model of NASH was established in HG12w and Ad-GFP group.Hepatic steatosis has been improved and no inflammation was observed in Ad-DN-JNK1 group.Conclusion:Adenovirus vector was a safe and effective gene vector.NAFLD could be improved by using Ad-DN-JNK1.The mechanism is possibly that Ad-DN-JNK1 inhibit the activation of JNK1,thereby reducing the phosphorylation of IRS-1Ser307 and enhancing the phosphorylation of PKBSer473,and then accelerating the insulin signal transduction pathway,so IR decreased.Furthermore,TNF-a,FFAs and oxidative stress reaction reduced; Obesity and hepatic steatosis alleviated.Finally NAFLD has been improved.
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