Investigation On Recombinant Adenovirus Mediated With DN-JNK1 Transgenic Prevention Of Non Alcoholic Fatty Liver Disease In SD Rats And Its Mechanism

Chapter 1 Investigation on the construction and mechanisms of non alcoholic fatty liver disease model in SD ratObjective:To construct a SD rat non alcoholic fatty liver disease (NAFLD) model with insulin resistance(IR) and to investigate the molecular mechanisms involved in the formation of NAFLD in high-fat induced-rat.Methods:Male SD rats(n=64) were divided randomly into two groups:normal diet group(NG,n=32) and high-fat diet group(HG,n=32), both groups had been fed for eight weeks.At the end of the first and the fourth weekend,eight rats were randomly chosen from two groups respectively.After performing hyperinsulinemic-euglycemic clamp to detect the insulin sensitivity,rats were sacrificed and liver samples were taken,the expression of c-Jun N-terminal kinasel(JNK1) protein and phosporylation of insulin receptor substrate-1 Serine 307(IRS-1^Ser307) of the rat liver tissue were detected by using western blot technology.At the end of the eighth week,eight rats that been randomly selected from both groups respectively,were conducted clamp test.Meanwhile,among the rests,blood was draw from portal vein and assayed for Triglyceride(TG), total cholesterol(TC),alanine aminotransferase(ALT),aspartate amino transferase(AST),fast blood sugar(FBS) and fast insulin(FINS).The rat liver samples were harvested after killing for identifying model,then tumor necrosis factor-a(TNF-α),free fatty acids(FFAs),malonaldehyde (MAD) and superoxide dismutase(SOD) of liver homogenates were detected,the expression of JNK1 protein and phospalation of IRS-1^Ser307 of the liver tissue were detected by using western blot technology.Results:In HG,the glucose infusion rate(GIR) level decreased,and the expression of JNK1 protein and phosphorylation of IRS-1^Ser307 in rat liver tissue increased,these index presented time-dependent manner,and significant differences were found in multiple comparison of the same group and same index at different periods(p＜0.05).However,the GIR, JNK1 protein expression and IRS-1^Ser307 phosphorylation of NG in different periods showed no significant differences(p＞0.05).In contrast to NG,at the end of the eighth week,IR appeared in HG,the liver tissue of HG presented significant steatosis,the body weight,liver index,TG, TC,ALT,AST,MDA,TNF-α,FFAs and FINS of HG rats increased obviously,and the level of SOD decreased.There were significant differences between two groups(p＜0.05).There was no significant difference between the two groups in serum FBS(p＞0.05).Conclusion:NAFLD SD rat model was successfully established by feeding on high-fat diet lasted for 8 weeks.The metabolic characteristic of this model were IR,hyperlipidemia,hyperinsulinemia and serum transaminase increasing.The high-fat diet could induce the level of TNF-αand FFAs increased,lead to oxidative stress and abnormal lipid metabolism,these factors play an interaction on the JNK signal pathway, activate the expression of JNK1 protein,up-regulate the IRS-1^Ser307 phosphorylation level,induce and aggravate IR,finally,NAFLD ensued.Chapter 2 Construction and function identification of recombinant adenovirus expressing dominant-negative type c-Jun N-terminal kinase1 by using AdEasy adenovirus vector systemObjective:To construct and identify recombinant adenovirus expressing dominant-negative type c-Jun N-terminal kinase 1 (Ad-DN-JNK1) and to identify its function through animal experiment.Methods:The linearized recombinant shuttle vector pAdTrack-CMV-DN-JNK1 was co-transformed with backbone vector pAdEasy-1 into Bacillus coli BJ5138 for recombinant adenoviral vector. The recombinant adenoviral vector was transfected into HEK293 packing cells to construct recombinant adenovirus Ad-DN-JNK1,which then, identified by PCR analysis and DNA sequence analysis.The JNK1 protein expression and the level of insulin receptor substrate-1 Serine 307 (IRS-1^Ser307) phosphorylation in SD rat were detected by western blot.Results:JNK1 recombinant adenoviral vector could be effectively transfected HEK293 and successfully packed intracellularly,the significant expression of green fluorescent protein(GFP) could be observed after 5 days.The virus obtained specific JNK1 gene fragment was assembled by using PCR amplification and was identified successfully by sequencing.The titer of the prepared Ad-DN-JNK1 is 2.5×10¹⁰ pfu/ml.Animal experiment revealed that the rat liver tissue which had been infected by Ad-DN-JNK1 presented increased expression of JNK1 protein and decreased level of IRS-1^Ser307 phosphorylation. There was significant difference in the comparison between two groups (p＜0.05).Conclusion:Our study successfully constructed JNK1 recombinant adenovirus and related particles.Animal studies reveal that Ad-DN-JNK1 can effectively mediate DN-JNK1 gene protein expression,and down-regulate IRS-1^Ser307 phosphorylation level.The achievement laid a foundation for further research on the effects of JNK1 and transgenic therapy of Ad-DN-JNK1 for related diseases. Chapter 3 Investigation on recombinant adenovirus mediated with DN-JNK Transgenic prevention of non alcoholic fatty liver disease in SD rats and Its MechanismObjective:To study the preventive effect and mechanism of recombinant adenovirus expressing dominant-negative type c-Jun N-terminal kinasel in the development of non alcoholic fatty liver disease(NAFLD) in SD rats.Methods:Male SD rats(n=128) were divided randomly into four groups with 32 rats in each group:normal diet control group(NC), high-fat diet control group(HC),adenovirus vector carrying green fluorescence protein(Ad-GFP) group and Ad-DN-JNK1 group.The NC group was fed with regular diet,while the other three groups were fed with high-fat diet.All groups were fed for eight weeks.At the first week, rats from both NC and HC groups were injected with 1ml normal saline as control,rats from Ad-GFP group were injected with 1ml 2.5×10¹⁰pfu Ad-GFP as adenovirus control,and rats from Ad-DN-JNK1 group were injected with 1ml 2.5×10¹⁰pfu.All rats were injected from caudal vein.At the first and the fourth weekend,eight rats were selected randomly from the four groups respectively.After performing hyperinsulinemic-euglycemic clamp to detect the insulin sensitivity,rats were sacrificed and liver samples were taken,the expression of JNK1 protein and phosporylation of insulin receptor substrate-1 Serine 307 (IRS-1^Ser307) in the liver tissue were detected by western blot.At the end of the eighth week,eight rats were selected randomly from the four groups respectively,and were conducted clamp test.While among the rest, bloods were draw from portal veins and assayed for Triglyceride(TG), total cholesterol(TC),alanine aminotransferase(ALT),aspartate amino transferase(AST),fast blood sugar(FBS),and fast insulin(FINS).The rat liver samples were harvest after killing,tumor necrosis factor-a (TNF-α),free fatty acids(FFAs),malonaldehyde(MAD) and superoxide dismutase(SOD) of liver homogenates were detected,the expression of JNK1 protein and phosporylation of IRS-1^Ser307 in the rat livers were detected by using western blot.Results:Rat livers from Ad-DN-JNK1,HC,and Ad-GFP group showed a increased expression of JNK1 protein in an obvious time-dependent manner,significant differences were found in multiple comparison of the same group at different periods(p＜0.05).The decreased level of GIR and increased level of IRS-1^Ser307 phosphorylation in both HC and Ad-GFP groups were time-dependent,and significant differences were found in multiple comparison of the same group and same index at different periods(p＜0.05).The levels of GIR and IRS-1^Ser307 phosphorylation at different stages in Ad-DN-JNK1 and NC group showed no significant difference(p＞0.05).Compared with HC and Ad-GFP group at the end of the eighth week,the rat livers of Ad-DN-JNK1 and NC group presented no significant steatosis and insulin resistance(IR),and lever of TG,ALT,AST,FINS,TNF-α,MDA, FFAs and IRS-1^Ser307 phosphorylation decreased,the level of SOD increased,there were significant differences in multiple comparison(p＜0.05).In contrast to NC group,the expression of JNK1 protein of liver tissue in Ad-DN-JNK1,HC and Ad-GFP group increased, which showed a significant differences(p＜0.05).The differences were not observed between Ad-DN-JNK1 and NC group,HC and Ad-GFP group.Meanwhile,there was no allergy or death etc.after adenovirus injection.Conclusion:Ad-DN-JNK1 is a deactivated JNK1 protein,because of blockade of JNK pathway,the phosporylation of IRS-1Ser307 was inhibited,so the IR delayed,and finally the occurrence and development of NAFLD was prevented.Adenovirus vector was a safe and effective gene vector,the recombinant adenovirus DN-JNK1 could be applied safely to investigate the pathogenesis of IR NAFLD in SD rats.