| Objective:NAFLD is a clinical syndrome,and has a spectrum ranging from that of fatty liver alone to nonalcoholic steatohepatitis(NASH),which may progress to liver cirrhosis and can progress to liver cell necrosis,fibrosis and cirrhosis of the liver.Several mechanisms like insulin resistance and oxidative stress are likely to contribute to this association,increasing attention has been directed to the role played by adipokines.The carbohydrate response element binding protein(ChREBP),which binds to the carbohydrate response element(ChoRE),regulates the expression of related gene including glycolysis enzyme and lipese.ChREBP has emerged as a central determinant of lipid synthesis in liver through its transcriptional control of key genes of lipogenic pathway(fatty acid synthase and acetyl CoA carboxylase).Recently,the investigation indicates that the expression of ChREBP increases in NAFLD,and has the relate with insulin resistance(IR).In this study,we established rat NAFLD model by high-fat and high-cholesterol diet to try to explore the following vievpoint:(1)To observe the expression of ChREBP mRNA in liver of rats in every stage of NAFLD model and to assess the relationship between intrahepatic ChREBP and NAFLD.(2)To investigate if traditional Chinese medicine----Jiangzhi Yigan Chongji can treat and prevent NAFLD through regulating the expression of ChREBP mRNA.This investigation may provide new clues for the pathogenesis of NAFLD and instruct clinical medication.Methods:48 male Wistar rats were randomly divided into 3 groups after fed with normal diet for 7 days.Normal control group(N group) were fed standard diet.Model control group(M group)were fed with high-fat and high-cholesterol diet which consisting of the standard diet, 13%lard and 2%cholesterol.Administration group(D group) were given Jiangzhi Yigan Chongji by intragastric administration while fed with high fat and high-cholesterol diet.Meanwhile each of the former two groups were divided into 2 subgroups(8 and 12weeks)respectively.Each subgroup has 8 rats.Administration group1,2 were given Jiangzhi Yigan Chongji(1ml/100g rat body weight,its concentration is 0.8g/ ml,twice every day)by intragastric administration separately after the beginning of the study,the end of 8th week,meanwhile,the normal and model group were separately given life drinking water by the same means.All the rats were sacrificed after 8,12 weeks.The following parameters were observed dynamically in each group:Body weight,body lenth,liver and spleen weight were detected,at the same time,liver index,Lee's index and the retios of liver and spleen were calculated;the level of aminotransferase,blood fat and lipid in liver tissue were detected by using automatic biochemistry analyzer;fasting blood glucose was detected by glucose oxidase method;serum and liver free fatty acid were detected by copper staining method;serum insulin and tumor necrosis factorα(TNF-α) level were detected by radio-immunity assay,simultaneously the insulin sensitivity index was calculated;reduced glutathione hormone(GSH),superoxide dismutase(SOD) activity and malondialdehyde(MDA) in serum and liver tissue were measured separately by using colourimetry,xanthinoxidase and thio-barbituric acid;the pathology of liver were observed by hematoxylin and eosin(HE) stain with microscopy;The expression of ChREBP mRNA in rat nonalcoholic fatty liver model induced by high fat diet were assayed with reverse transcriptase-polymerase chain reaction (RT-PCR).Results:In control group,the liver lobules were distinct,the liver cell cords arranged regularly, every biochemical manifestation was all in normal range,the expression of ChREBP mRNA was detectable in liver tissue.At the end of 8th week,the model group shew to be abdomen-form obesity,developed lipid metabolism disorder accompanied with hyperinsulinemia and insulin resistance,increase of aminotransferase,tumor necrosis fatorαin serum,malondialdehyde in serum and liver and decrrease of the vitality of superoxide dismutase,the liver showed typical steatosis accompanied, the expression of ChREBP mRNA in liver was significantly higher than the model group,the treated group shew improvement of every data,the expression of ChREBP mRNA in liver was decreased,and with statistical difference.At the end of 12th week,insulin resistance of the model group shew no progress.Compared with normal group,the level of aminotransferase,TNF-αin serum,blood fat and liver lipid content were increased in model group,at the same time,the content of malondialdehyde was increased,the levels of glutathione,superoxide dismutase,high density lipoprotein cholesterol in serum and liver were obviously reduced,the expression of ChREBP mRNA in liver was more higher than the prophase group(P＜0.01).Hepatocyte steatosis developed and liver presented the pathology of inflammation cell infiltration and sporadic punctiform necrosis,the inflammation score in model group were increased significantly compared with normal group(P＜0.01).In the treated group,every biochemical manifestation shew improvement significantly,the expression of ChREBP mRNA in liver was decreased;steatosis and inflammation of liver show significant improvement,too.Correlation analysis between intrahepatic ChREBP levels in the model groups and the relative parameters:a positive correlation was found between the level of ChREBP and the level of TNF-αin surem at the end of 8th,12thweek(r=0.787,0.888,p＜0.01or＜0.05),the degrees of steatohepatitis at the end of 12th week(r=0.861,0.892,P＜0.01);ChREBP level was negative corelated with ISI(r=-0.834,P＜0.01) at the end of 8th week,but had no corelation at at the end of 12th week.Conclusion:1.The rats NAFLD model could be successfully established with the fed with high-fat and high-cholesterol diet.Insulin resistance,oxidative stress\lipid peroxidation damage and some cytokines such as TNF-αdisorder were well associated with the development of NAFLD.2.The expression of ChREBP mRNA in normal rats liver tissue was detectable.In NAFLD model group,it was significantly increased at the end of 8th week and increased significantly with the time.Correlation analysis revealed that ChREBP might play a key role in the formation of hepatocyte steatosis by triggering insulin resistance,but played capital role in the formation of nonalcoholic steatohepatitis by triggering cytokine production.3.JYC was efective in the treatment and preventment of rats with NAFLD.Its molecular mechanism may be partly through downregulating the expression of ChREBP mRNA of the liver. |
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