【作者】 谭莺；

【导师】 刘慧霞；

【作者基本信息】 中南大学 ， 老年医学， 2010， 博士

【摘要】 目的：高脂饲料喂养SD大鼠构建非酒精性脂肪肝病(nonalcoholic fatty liver disease, NAFLD)模型,动态观察NAFLD大鼠血清学和肝匀浆指标、IR及肝组织病理改变等指标的变化。方法：雄性SD大鼠192只,随机分为对照组(NG)和高脂组(HG)各96只,对照组喂饲普通饲料,高脂组喂饲高脂饲料。于第1周(w)开始、第2、4、8、12、16w末随机从各组抽取16只大鼠,其中8只行正糖高胰岛素钳夹实验技术检测葡萄糖输注率(GIR),另8只测量体重、肝指数,门静脉采血予以生物化学法检测空腹血糖(FBS)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)；放射免疫法检测空腹胰岛素(FIns)、肿瘤坏死因子a (TNF-a);分光光度计检测游离脂肪酸(FFAs);制备肝匀浆分光光度计测定超氧化物歧化酶(SOD)、丙二醛(MDA)；大鼠肝组织行HE染色和苏丹Ⅳ染色,光镜下对肝脏脂肪变性情况进行评定。结果：与同期NG比较：HG大鼠体重、肝指数、血清ALT、AST、TG、TC、FIns、FFAs、TNF-a及肝匀浆MDA在第8、12、16w末均明显升高,肝匀浆SOD水平则降低,差异均具有统计学意义(p值均<0.05)HG各组间两两比较：在第8、12、16w末随着高脂饲料喂养时间的延长,除肝匀浆SOD水平逐渐降低外,肝指数、血清各项指标及肝匀浆MDA水平均逐渐升高,差异均具有统计学意义(p值均<0.05)。与同期NG比较,在第4、8、12、16w末,HG大鼠的GIR水平降低；HG各组间两两比较,差异均具有统计学意义(p值均<0.05)。肝组织病理学检查显示HG大鼠第8w末形成单纯性脂肪肝,随着喂养时间的延长,HG大鼠肝细胞脂肪变性逐渐加重,第12w末形成了非酒精性脂肪性肝炎(non alcoholic steatohepatitis, NASH)。结论：高脂饮食喂饲SD大鼠8w成功构建单纯性脂肪肝模型,12w可形成NASH模型；肝脏脂肪沉积,TNF-α、FFAs.氧化应激反应都参与了NAFLD的发生发展；IR出现在肝脏脂肪变性之前,IR可能是NAFLD发病的始动因素。第二部分动态研究非酒精性脂肪肝病中JNK1蛋白表达及其对胰岛素信号通路的影响目的：动态观察非酒精性脂肪肝病(non alcoholic fatty liver disease, NAFLD)中c-jun氨基末端激酶1(c-Jun N-terminal kinase-1,JNK1)蛋白表达及其对胰岛素(Insulin, INS)信号通路的影响,探讨JNK1、INS信号通路蛋白变化及其与IR和NAFLD的关系。方法：雄性SD大鼠192只,随机分为对照组(NG)和高脂组(HG)每组各96只,对照组喂饲普通饲料,高脂组喂饲高脂饲料。于第1周(w)开始、第2、4、8、12、16w末随机从各组抽取8只大鼠,处死后取肝组织行Western blot检测JNKl、胰岛素受体底物1(insulin receptor substrate-1,IRS-1)、胰岛素受体底物1丝氨酸307磷酸化(phospho-IRS-1 Ser307,p-IRS-1Ser307)、蛋白激酶B(Protein kinase B,PKB)、蛋白激酶B丝氨酸473磷酸化(phospho-PKBSer473,p-PKBSer473)水平。结果：第2、4、8、12、16w末,HG与同期NG比较：肝组织JNK1蛋白表达、p-IRS1Ser307水平增高,p-PKBSer473水平降低；HG各组间上述各项指标比较,从第2w末开始,随着时间的延长,除p-PKBSer473水平逐渐降低外,JNK1蛋白表达及p-IRS1Ser307水平均逐渐升高,差异均具有统计学意义(p值均<0.05)。NG各组间比较JNK1蛋白表达、p-IRS1Ser307和p-PKBSer473水平,差异均无统计学意义(p值均>0.05)。各组间两两比较IRS-1和PKB蛋白表达,差异均无统计学意义(p值均>0.05)。JNK1的蛋白表达强度与IR呈正相关(Pearson相关系数为0.931,p值<0.01)。结论：高脂饮食可诱导肝组织JNK1蛋白表达增高,增加的JNK1结合IRS-1后可以上调p-IRS-1Ser307水平,下调p-PKBSer473水平,从而干扰INS信号传导,导致IR,引起NAFLD; TNF-α、FFAs、氧化应激反应通过促进JNK1活性增强,进一步加重IR从而参与NAFLD的发生发展过程；肝组织的R出现在全身IR和肝脏脂肪变性之前,IR可能是NAFLD发病的启动因素。第三部分应用表达DN-JNK1的重组腺病毒治疗SD大鼠非酒精性脂肪肝病及其机制研究目的：应用表达失活型c-jun氨基末端激酶1的重组腺病毒(recombinant adenovirus expressing adenovirus expressing dominant negative type c-Jun N-terminal kinase1, Ad-DN-JNK1)治疗SD大鼠非酒精性脂肪肝病(non alcoholic fatty liver disease, NAFLD),探讨其对NAFLD的治疗作用及作用机制。方法：雄性SD大鼠96只,随机分为对照组(NG)32只、高脂组(HG)64只,对照组喂饲普通饲料,高脂组喂饲高脂饲料。于第8周(w)末随机从各组抽取16只大鼠,其中8只行正糖高胰岛素钳夹实验技术(钳夹技术)检测葡萄糖输注率(GIR),另8只门静脉抽血和肝组织病理切片鉴定成模后,将余下的16只对照组大鼠继续喂饲普通饲料至12w末称为12w对照组(NG12w)；余下的高脂组大鼠随机分为12w高脂组(HG12w),携带绿色荧光蛋白的腺病毒(Ad-GFP)组和Ad-DN-JNK1组,各组均16只,此3组均喂饲高脂饲料至12w末。第8w末,NG12w和HG12w大鼠均从尾静脉注入1ml生理盐水作为安慰剂对照,Ad-GFP组大鼠尾静脉注入1ml2.5×1010pfu的Ad-GFP作为腺病毒对照,Ad-DN-JNK1组大鼠尾静脉注入1ml 2.5×1010pfu的Ad-DN-JNK1作为治疗组。12w末随机从各组抽取8只大鼠行钳夹技术检测胰岛素抵抗(IR)情况,各组余下8只大鼠门静脉采血生物化学法检测空腹血糖(FBS)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)；放射免疫法检测空腹胰岛素(FIns)、肿瘤坏死因子-a (TNF-a),分光光度计测定游离脂肪酸(FFAs);制备肝匀浆测定TG、TC、FFAs(检测方法同上),分光光度计法检测超氧化物歧化酶(SOD)、丙二醛(MDA)；另取部分肝组织行Western Blot技术检测肝组织c-jun氨基末端激酶1(c-Jun N-terminal kinase-1, JNK1)、胰岛素受体底物1(insulin receptor substrate-1,IRS-1)、蛋白激酶B(Protein kinase B,PKB)蛋白表达和胰岛素受体底物1丝氨酸307磷酸化(phospho-IRS-1 Ser307,p-IRS-1 Ser307)、蛋白激酶B丝氨酸473磷酸化(phospho-PKBSer473,p-PKBSer473)水平。部分肝组织行HE染色、苏丹Ⅳ染色,光镜下评定肝脏脂肪变性情况。结果：第8w末高脂组大鼠已构建成单纯性脂肪肝IR模型。与NG12w大鼠比较,HG12w和Ad-GFP组的体重、肝指数、血清ALT、AST、TG、TC、FFAs、FIns、TNF-a及肝匀浆TG、TC、FFAs、MDA均明显升高,肝匀浆SOD降低；葡萄糖输注率(GIR)降低；肝组织JNK1蛋白表达、p-IRS-1Ser307水平增高,p-PKBSer473水平降低,差异均具有统计学意义(p值均<0.05)。Ad-DN-JNK1组与HG12w及Ad-GFP组大鼠分别比较上述各项指标,Ad-DN-JNK1组的体重、肝指数、血清ALT、AST、TG、TC、FFAs、FIns、TNF-a及肝匀浆TG、TC、FFAs、MDA均明显降低,肝匀浆SOD升高；GIR升高；肝组织JNK1蛋白表达、p-IRSlSer307水平降低,p-PKBSer473水平升高,差异均具有统计学意义(p值均<0.05)。Ad-DN-JNK1组与NG12w组比较,体重、肝指数、AST、FIns、GIR水平、JNK1蛋白表达、p-IRS1Ser307、p-PKBser473水平差异均无统计学意义(p值均>0.05),其余血清学及肝匀浆指标差异均具有统计学意义(p值均<0.05)。各组间两两比较IRS-1和PKB蛋白表达,差异均无统计学意义；HG12w与Ad-GFP组比较血清和肝匀浆各项指标以及JNK1、INS信号通路蛋白表达及磷酸化水平,差异均无统计学意义(p值均>0.05)。HG12w及Ad-GFP组大鼠均进展为非酒精性脂肪性肝炎(non alcoholic steatohepatitis, NASH);而Ad-DN-JNK1组大鼠肝脏脂肪变性明显改善,未出现炎症反应。结论：腺病毒载体是安全有效的基因治疗载体,应用Ad-DN-JNK1治疗SD大鼠NAFLD,可明显改善NAFLD,其作用机制可能为：Ad-DN-JNK1抑制JNK1活性后,下调p-IRS-1Ser307水平,升高其下游p-PKBSer473水平,进而促进INS信号传导,减弱IR,并降低TNF-α、FFAs水平,减少氧化应激反应,减轻肥胖和肝脏脂肪变性,最终改善NAFLD。更多还原

【Abstract】 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.更多还原