Pathogenesis Of Nonalcoholic Fatty Liver Disease And Effects Of Adenovirus Mediated WT-JNK1 On It

Chapter 1 Model Establishment of Insulin Resistance Nonalcoholic Fatty Liver Disease SD RatObjective:To create a SD rat model of insulin resistance nonalcoholic fatty liver disease.Methods:Thirty-two male SD rat were randomly divided into a normal diet group(NG)and a high-fat diet group(HG).NG was fed on standard diet(19%energy from fat),HG was fed on high-fat diet(45% energy from fat),both groups had been fed lasting for 8weeks.At the end of 8 week,insulin sensitivity was measured with glucose infusion rate(GIR)by the euglycermic hyperinsulinemia clamp technique; Detected the fasting blood sugar(FBS),fast insulin(FINS),triglyceride (TG),total cholesterol(TC),alanine aminotransferase(ALT),aspartate amino transferase(AST),by drawing blood from portal vein;To measure liver wet weight(LWW)by freeing liver,and calculated liver index(LWW(g)/weight(g)×100%);To assessment of hepatic steatosis in all rats were observed under light microscope by HE stain and Sudanâ…¢staining.Results:Liver tissue pathology showed that the hepatic cells of HG rats presented diffused macrovesicular steatosis,and developed fatty liver disease;NG rats showed no abnormal.Compared with NG,the liver index,GIR,serum ALT,AST,insulin,BS,TG,TC,FFA of HG rats increased obviously,there were significant differences between two groups(P<0.05).Conclusion:NAFLD IR SD rat model was successfully established by feeding on high-fat diet lasted for 8 weeks.The metabolic characteristic of this model were IR,hyperinsulinemia,hperlipoidemia,serum transaminase increasing. Chapter 2 Initial Investigation of IR NAFLD Rat Model Induced by High-fat DietObjective:To investigate the molecular mechanism of IR NAFLD induced by high-fat diet:the changes of liver JNK1 protein and insulin signaling molecular IRS-1 Ser³⁰⁷phosphorylation level,to explain the molecular mechanism of JNK1 protein's effects on IR during NAFLD forming;Effects of oxidative stress and tumor necrosis factor-α(TNF-α) on IR NAFLD forming.Methods:SD rats were randomly divided into a NG and a HG.After being fed for 8 weeks,all rats were put to death and the specimens were saved.Detected liver homogenate superoxide dismutase(SOD),malondialdehyde(MDA),free fatty acids(FFA)and TNF-α.The expression of JNK1 protein in liver was measured by immunohistochemistry.JNK1 protein content and IRS-1 Ser³⁰⁷ phosphorylation level were measured by western-blot.Results:Compared with NG,the liver homogenate MDA,TNF-αof HG notablely increased,and SOD notablely decreased,there were significant differences between two groups(P<0.05).JNK1 protein immunohistochemistry showed:compared with NG,JNK1 antibody positive cells' cytoplasm stain of HG rats presented brown.JNK1 antibody positive cells of NG rats had not been observed,JNK1 antibody positive cells' rate of riG rats was about 10%.The level of JNK1 protein express and IRS1 Ser³⁰⁷phosphorylation of HG marked higher than NG, there were significant differences between two groups(P<0.05).Conclusion:The molecular mechanism of IR NAFLD induced by high-fat diet might be:the express of JNK1 in liver increased,and interfered the signal transmission of insulin and insulin receptor by up-regulating IRS-1 Ser³⁰⁷phosphorylation level,finally,physiology effects of insulin were weakened and lead to IR;TNF-αand Oxidative stress-lipid peroxidation injury were one of the mechanism of IR NAFLD induced by high-fat diet. Chapter3 Construction of WT-JNK1 recombinant adenovirus by using AdEasy adenovirus vector systemObjective:To construct replication deficient recombinant adenovirus expressing wild-type JNK1(WT-JNK1)by homologous recombination.Methods:The linearized recombinant shuttle vector pAdTrack-CMV-WT-JNK1 was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells to construct replication deficient recombinant adenovirus,then,the recombinant adenovirus was identified by using PCR and DNA sequencing,finally,amplified and assembled recombinant adenovirus, detected virus titer.Results:JNK1 recombinant adenoviral vector could be effectively transfected HEK 293 cells and successfully packed intracellulare enzyme.The assembled virus obtained definite gene fragment by using PCR amplification,and was identified successfully by sequencing. Meanwhile,2.5×10¹⁰pfu/ml high titer recombinant adenovirus was also amplified.Conclusion:The research successfully constructed recombinant adenovirus,and the achievement laid a foundation for further investigation of the effects of JNK1,and laid a foundation for prevention and treatment of the diseases which related to JNK1. Chapter 4 To investigate the effects and pathogen of WT-JNK1 over-expression by using adenovirus vector on NAFLDObjective:to study the effects of over-expression WT-JNK1 on NAFLD's forming and further investigate the effects of JNK1 on IR NAFLD by using adenovirus vector.Methods:Rats were randomly divided into high fat control group,Ad-GFP group,Ad-WT-JNK1 group,all groups were fed on high fat diet. At the 1^stweek of experiment,high fat control group rats were injected Sodium Chloride through caudal vein,Ad-GFP group rats were injected 2×10¹⁰pfu Ad-GFP through caudal vein,Ad-WT-JNK1 group rats were injected 2×10¹⁰pfu Ad-WT-JNK1 through caudal vein.At the end of 8 week,measured GIR by the euglycermic hyperinsulinemia clamp technique;Drew blood from portal vein and saved specimen,detected FBS,FINS,TG,TC,ALT,AST,FFAs,SOD,MDA and TNF-α; Observed steatosis of liver tissue;Measured expression of JNK1 protein in liver by immunohistochemistry,Measured JNK1 protein content and IRS-1 Ser³⁰⁷phosphorylation level by western-blot.Results:Compared with high fat control group and Ad-GFP group, the liver tissue steatosis of Ad-WT-JNK1 group aggravated notablely, appeared inflammatory changing,TG,TC,ALT,AST increased notablely, hyperinsulinemia and IR level aggravated notablely;MDA,TNF-αincreased notablely,SOD level decreased notablely,express of JNK1 protein and IRS-1 Ser³⁰⁷phosphorylation level increased notablely,there were significant differences among three groups(P<0.05).Compared with high fat control group,there were no differences in weight,liver index,biochemistry index,GIR,liver tissue steatosis,JNK1 protein expression and IRS-1 Ser³⁰⁷phosphorylation level of Ad-GFP group。After being injected adenovirus,skin hypersensitiveness,response abnormal and death of animals had not been observed。Conclusion:After being transfect WT-JNK1 recombinant adenovirus gene of SD rats,JNK1 protein was up-regulated obviously, and IR was induced by affecting IRS-1 Ser³⁰⁷phosphorylation, meanwhile,the form and development of NAFLD aggravate.Adenovirus could be applied safely for pathogensis study of SD rats with IR NAFLD.