| Objective:To explore the possible mechnisms of JNK signal pathway on hepatocyte apoptosis,we intervened NAFLD rats with recombinant adenovirus expressing dominant-negative type c-junN-terminal kinasel (Ad-DN-JNK1) and its effect on amelioration of hepatocyte apoptosis were examed.Methods:Fourty-eight male SD rats were randomly divided into normal diet group(NG, n=16), which were fed on normal diet, and high-fat diet group(HG, n=32),which were fed on high-fat diet(normal diet adding 2% cholesterol and 14% lard). At the end of the 8th week,8 rats were randomly drawn from each group, blood were obtained from portal vein. After sacrificed, rat liver was then removed for the identification of NAFLD model. The rest 8 rats in NG were continue fed with normal chow until the end of 12w to serve as control group,while the rest 24 rats in HG were divided randomly into three groups, HG12w,adenovirus vector carrying green fluorescence protein (Ad-GFP) group and Ad-DN-JNK1 group respectively,and continue fed with high fat diet until the end of 12w. At the end of 8w,both NG12w group and HG12w group rats were tail vein injected 1ml normal saline as placebo control, Ad-GFP rats were tail vein injected lml 2.0×1010pfu of Ad-GFP as a adenovirus control group, Ad-DN-JNK1 group 1ml 2.0×1010pfu pfu of Ad-DN-JNK1 as a treatment group.Fasted overnight, rats were killed and weighed; blood were drawn from rat portal vein blood, centrifugal separation of serum for the determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), fasting blood sugar (FBS), serum insulin (FIns), free fatty acids (FFAs), tumor necrosis factor-α(TNF-α); Fasting insulin resistance index(FIRI) was calculated; the liver was then removed and weighed, liver index was calculated; a small part of liver tissue were cut into pieces, homogenized in a glass homogenizer. The liver homogenate was then used for determination of FFAs, SOD, MDA content; The right lobe rat liver were isolated for tissue biopsy, HE stained to evaluate the degree of liver steatosis Hepatic steatosis was assessed by using H-E stain sections, the expression of JNK1 in liver tissue was detected by using immunohistochemistry method. Hepatocyte apoptosis was detected by using TUNEL method.Results:(1)The weight,liver index,levels of serum ALT,AST,TG,TC,FIns,FFAs,TNF-α,FIRI and levels of liver homogenate FFAs in HG were significantly increased compared to the corresponding period of NG(P<0.05); liver homogenate SOD were significantly decreased (p<0.05). However, there was no significant changes in FBS in each group (p>0.05); also, these parameters differences are not reach significant level between Ad-GFP group and HG12w. (2)Pairwise comparison among Ad-DN-JNK1 group,HG12w group and Ad-GFP group show that body weight, liver index, levels of serum ALT,AST,TG,TC,FIns,FFAs,TNF-α,FIRI and levels of liver homogenate FFAs are decreased,except for homogenate SOD, which shows increase trend,while the levels of JNK1 protein expression,the number of apoptosis cell are decreased, which shows significant different (p<0.05). Comparison beteen Ad-DN-JNK1 group and NG12w revealed that the body weight, liver index, serum AST, FIns, FIRI, were not significantly different (p>0.05). The remaining indicators of serum and liver homogenate, the levels of JNK1 protein expression,the number of apoptotic cells did not recover to normal levels, the differences were significant (p<0.05). (3)Vacuolar hepatic steatosis was observed in HG8w rats under the light microscope; rat liver in HG12w group showed diffuse Vacuolar fatty degeneration, accompanied by inflammatory cell infiltration; All rats from Ad-GFP and HG12w progressed to non-alcoholic steatohepatitis. However, hepatic steatosis has been improved and no inflammation was observed in Ad-DN-JNK1 group. (4)A positive correlation was found between the expression intensity of JNK1 and hepatic steatosis(Pearson correlation:0.822,p<0.01).Conclusion:1.The models of the simple hepatic steatosis could be established in SD rats fed with high fat diet continuously for 8 weeks,NASH can be formed at 12 weeks2. High-fat diet can induce hepatic JNK1 protein expression, increase the number of apoptotic cells. as the time prolonged, apoptosis is aggravated in a time-dependence manner. A positive correlation was found between the expression intensity of JNK1 and hepatic steatosis3. Hepatocyte apoptosis could be improved by using Ad-DN-JNK1 in rat with NAFLD. Targeting JNK pathways may become a new strategy for the trentment of NAFLD;...
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