| Objective:In recent years,alone with the development of social economy and the improvement of people's living standard,there has been an increase in the incidence of nonalcoholic fatty liver disease.NAFLD can even grow into hepatic fibrosis,hepatic cirrhosis and later stage of other hepatic disease.So it has become one of the medical and social problems worldwide.NAFLD is a pathologically clinical syndrome,sharing much similar characteristics with alcoholic fatty liver disease but without alcohol over-ingestion.Many studies have been done to explore its pathogenesis,but there hasn't been any breakthrough yet.Fat metabolism disturbance results in the disequilibrium of internal environment of liver fat,which is the cause of hepatic steatosis.It has been suggested that oxidative stress and resulting lipid peroxidation may play a key role in the development from simple fatty liver to steatohepatitis.Thioredoxin involved in the development of NAFLD as a protein induced by oxidative stress shields the organism in many kinds of diseases.At present,there is no effective preventative and therapeutic ways and means..In this study,we established rat NAFLD model by high-fat and high-cholesterol diet to probe into the following issues:(1)To observe the role of the expression of thioredoxin mRNA in liver of rats in every stage of NAFLD model.(2)To investigate if traditional Chinese medicine——Jiangzhi Yigan Chongji can treat and prevent NAFLD through regulating the expression of thioredoxin mRNA.This investigation can provide new clues for the pathogenesis of NAFLD and instruct clinical medication.Methods:72 male Wistar rats were randomly divided into 3 groups according to the weight level after being 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 consisted of the 85%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 three groups were divided into 3 subgroups(9,13 and 18 weeks)respectively:N1,N2,N3,M1,M2,M3,D1,D2,D3.Each subgroup has 8 rats.The administration group1,2,3 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,7 weeks and 12 weeks,meanwhile,the normal and model group were given drinking water in the same way.The rats were put to death separately after 9,13 or 18 weeks.Blood was taken from the belly aorta and the liver was taken out timely.After that the serum,liver homogenate and liver tissue were prepared according to the routine,and a small amount of liver organization was kept at -70℃.The following parameters were observed dynamically in each group:body weight,body lenth,liver and spleen weight,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 factorαlevel 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 thioredoxin mRNA in rat nonalcoholic fatty liver model induced by high fat diet were assayed with reverse transcriptase-polymerase chain reaction(RT-PCR).Results:After 9 weeks,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 level of glutathione in serum and liver and the expression of thioredoxin mRNA in liver with hepatocyte steatosis shown in hepatic histopathology.Compared with the model group,the treated group shew improvement of every data,and with statistical difference.After 13 weeks,insulin resistance of the model group showed no significant progress.Compared with normal group,the level of aminotransferase,TNF-αin serum,blood fat and liver lipid content were increased greatly in model group,at the same time,the content of malondialdehyde increased,the levels of glutathione,superoxide dismutase,high density lipoprotein cholesterol in serum and liver were obviously reduced,the expression of Trx mRNA in liver was reduced,which is higher than prophase model 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).Lipid metabolism disorder and insulin resistance of the administration group were improved,and the levels of aminotransferase,TNF-αin serum decreased significantly,MDA content decreased,GSH content,SOD activity,HDL-C content in serum and liver increased significantly,the expression of Trx mRNA in liver was increased;steatosis and inflammation of liver show significant improved compared with the model group.After 18 weeks,insulin resistance of the model group remains no progress.The level of aminotransferase,TNF-αin serum,blood fat and liver lipid content increased in model group compared with normal group,the level of aminotransferase,TNF-αin serum,blood fat and liver lipid content increased in model group,meanwhile,MDA content increased,the levels of GSH, SOD,HDL-C in serum and liver became obviously lowered,the expression of Trx mRNA in liver was reduced,which is higher than prophase model group(P＜0.01)Hepatocyte steatosis diffused and the inflammation score increased remarkably.Compared with the model group,the administration group showed improvement on each of data,and with statistical difference.Conclusion:1.Lipid metabolism disorder,oxidative stress,lipid peroxidation damage and some cytokines such as TNF-αdisorder are well associated with the development of NAFLD.2.Insulin resistance plays a key role in the formation of hepatocyte steatosis,but plays no key role in the formation of nonalcoholic steatohepatitis.3.The expression of thioredoxin mRNA decreased in the liver of rats with NAFLD,and might reach the lowest point after developing simple fatty liver,and the down-regulation of Trx mRNA may cause the inflammation injury initially in NAFLD;it is demonstrated for the first time that the expression of Trx mRNA in NAFLD model down-regulates.4.JYC can be used to prevent and treat NAFLD of rats and the possible mechanism of action is as follow:(1)JYC can decrease significantly the content of TC,TG,LDL-C,FFA and increase the HDL-C content,reduce high blood fat and liver lipid,therefore result in the diminution of lipidoses in liver and alleviation of steatosis.(2)JYC can increase insulin sensitivity index to improve insulin resistance.(3)JYC can enhance the activity and content of SOD,GSH,reduce MDA content,lower oxidative stress and lipid peroxidation damage,correct the state of oxidation/anti-oxidation disbalance,obstruct NAFLD development.(4)JYC can reduce the level of ALT,AST in serum,promote the liver function revival.(5)JYC can improve steatosis of liver tissue and have definite anti-inflammatory effect.(6)JYC's molecular mechanism of prevention and treatment may be associated with upregulating the expression of Trx mRNA.(7)JYC can interfere in the genesis and development of NAFLD from multi angles and multi targets,and early and long course of treatment may be better to prevent and cure NAFLD,so this traditional Chinese medicine is deserved to generalization.
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