| Objectives: With the improvement of the standard of living, more and more people are suffering nonalcoholic steatohepatitis (NASH). Its pathomechanism is not yet elucidate at present. The abnormality of liver lipid metabolism is known as one of the most important and elementary component element, the activation of kupffer cell which induce the releasing of the inflammatory transmitter is a important stage of fatty liver to NASH. Our topic had lucubrate the two aspects. The treatment of NASH has still no satisfactory medicine. Chinese traditional medicine possesses some superiority on it. My tutor, professor Zhang Chi-zhi, realized in his clinical work that the principle of its treatment should be to invigoration liver and kidney, to clear away heat and promote diuresis. He chose Qi Ji Preparation which made up of medlar fruit and silybin to treat NASH and gained good effects. Using the animal model of NASH recognized by the world and linking to the evolution of modern research of NASH, we observed the liver lipid metabolism and inflammatory reaction in the state of NASH and threw light on the mechanism of Qi Ji Preparation to prevent and cure it by the technique of pathology, biochemistry and molecular biology in the research, to elucidate mechanism of Qi Ji Preparation preventing and curing NASH. The topic had provided scientific evidence for the clinical application of Qi Ji Preparation, and had set up foundation for exploitation of new drug preventing and curing NASH.Methods: the research was divided into three partsThe primary part: the establishment of rat models of NASH and the effect of Qi Ji Preparation on NASH of ratsForty clearing graded rats, weighing 190±20g, were randomly divided into 4 groups, namely normal control group, blank model group, Qi Ji Preparation groups and Compound Methionine and Choline Tablet control group. To adopt the high fat diet confected 88% ordinary diet and 10% lard and 2% cholesterin to replicate NASH model of rat. Then common state of rats were observed when the model was being replicated, the content of serum triglyceride(TG), total cholesterol (TCH), low density lipoprotein(LDL), aminotransferase (ALT) and aspartate aminotransferase (AST) were detected with Olympus Au560 entire automated biochemical analysis instrument. Hepatic tissues were stained with HE dyeing, the degree of hepatic steatosis and inflammation activity were evaluated under the optical microscope.The second part: The effect of Qi Ji Preparation on the liver lipid metabolism of NASH of rat (experiment one to four)The NASH model of rat was duplicate adopted high fat diet. The rats were randomly divided into normal control group, blank model group, Qi Ji Preparation groups and Compound Methionine and Choline Tablet control group. To detect content of free fatty acids (FFA), superoxide dismutase (SOD), malondialdehyde (MDA) of every rat's hepatic tissue according to the instructions of the kits. The mRNA expression of peroxisome proliferator activated receptor a (PPARa) and liver fatty acid binding protein(L-FABP) of hepatic tissue was detected useing in situ hybridization, and the protein expression of L-FABP of hepatic tissue was detected by western blot.The third part: The effect of Qi Ji Preparation on inflammatory reaction of NASH of rat (experiment one to two)The NASH model of rat was duplicated adopted high fat diet. The rats were randomly divided into normal control group, blank model group, Qi Ji Preparation groups and Compound Methionine and Choline Tablet control group. The change of kupffer cell was investigated through detecting CD14 and lysozyme protein expression of every hepatic tissue by immunohistochemistry. To observe the protein expression of one of inflammatory mediators such as intercellular adhesion molecule-1(ICAM-1) of every hepatic tissue by immunohistochemistry.Results:The primary part: the rats' models establishment of NASH and the effect of Qi Ji Preparation on NASH of ratThe NASH model of rat was duplicated successfully. Compared with that of the blank model group, the content of serum TG,TCH,LDL of Choline Tablet control group decreased significantly (P＜0.01 or P＜0.05). The difference of the degree of hepatic steatosis and inflammation activity between the blank model group and Compound Methionine and Choline Tablet control group wasn't significant in statistics(P＞0.05). Compared with that of the blank model group, the content of serum TG,TCH,LDL,ALT,AST of Qi Ji Preparation groups decreased significantly (P＜0.01 or P＜0.05). the effects have an advantage to that of Compound Methionine and Choline Tablet control group (P＜0.05), the degree of hepatic steatosis and inflammation activity of Qi Ji Preparation groups obviously improve(P＜0.01), and the degree of the inflammation activity was lessen than Compound Methionine and Choline Tablet control group (p＜0.05).The second part: The effect of Qi Ji Preparation on the liver lipid metabolism of NASH of rat(experiment one to four)1. Compared with that of the normal control group, the content of hepatic FFA of the blank model group stepped up significantly (P＜0.01). Compared with that of the blank model group, the content of hepatic FFA of Qi Ji Preparation groups decreased significantly (P＜0.01), the effect surpass to that of Compound Methionine and Choline Tablet control group (P＜0.05).2. Compared with that of the normal control group, the SOD activity of hepatic tissue of the blank model group descended obviously and the MDA content of hepatic tissue of the blank model group stepped up significantly (P＜0.01). Compared with that of the blank model group, Qi Ji Preparation and Compound Methionine and Choline Tablet could raise the SOD activity and degrade the MDA content (P＜0.01 or P＜0.05), the effect of Qi Ji Preparation surpass to that of Compound Methionine and Choline Tablet(P＜0.01 or P＜0.05).3. Compared with that of the normal control group, the PPARa mRNA expression of hepatic tissue of the blank model group was degraded obviously(P＜0.01). Compared with that of the blank model group, Qi Ji Preparation and Compound Methionine and Choline Tablet could reinforce the PPARa mRNA expression of hepatic tissue(P＜0.05), the difference of the effect between Qi Ji Preparation group and Compound Methionine and Choline Tablet control group wasn't significant in statistics(P＞0.05).4. Compared with that of the normal control group, the L-FABP mRNA and protein expression of hepatic tissue of the blank model group was reinforced obviously(P＜0.01). Compared with that of the blank model group, Qi Ji Preparation and Compound Methionine and Choline Tablet could weaken the L-FABP mRNA and protein expression of hepatic tissue(P＜0.01), the effect of Qi Ji Preparation surpass to that of Compound Methionine and Choline Tablet(P＜0.01 or P＜0.05).The third part: The effect of Qi Ji Preparation on inflammatory reaction of NASH of rat(experiment one to two)1. Compared with that of the normal control group, the CD14 and lysozyme protein expression of hepatic tissue of the blank model group was reinforced obviously(P＜0.01). Compared with that of the blank model group, Qi Ji Preparation and Compound Methionine and Choline Tablet could weaken the CD14 and lysozyme protein expression of hepatic tissue(P＜0.01), the effect of Qi Ji Preparation surpass to that of Compound Methionine and Choline Tablet(P＜0.01).2. Compared with that of the normal control group, the ICAM-1 protein expression of hepatic tissue of the blank model group was reinforced obviously(P＜0.01). Compared with that of the blank model group, Qi Ji Preparation and Compound Methionine and Choline Tablet could repress the ICAM-1 protein expression of hepatic tissue(P＜0.01), the effect of Qi Ji Preparation surpass to that of Compound Methionine and Choline Tablet(P＜0.01).Conclusion:1. The animal model establishmentTo adopt the high fat diet to replicate NASH models of rats, the blood fat was rised and the liver function was damaged and the changes of hepatic tissue accorded with pathological changes of NASH in rats of the blank model group. The rate of the animals models establishment was 100%. 2. The research on pathomechanism of NASH of rat2.1 The disord of liver lipid metabolism in NASH of rat: in NASH of rat, the content of hepatic FFA stepped up significantly, the liver lipid peroxidation enhanced, the PPARa mRNA expression of hepatic tissue degraded, the L-FABP mRNA and protein expression of hepatic tissue reinforced obviously. All of that are nosogenesis of NASH.2.2 Liver inflammatory reaction in NASH of rat: in NASH of rat, the endotoxin binding receptor as CD14 protein expression of hepatic tissue reinforced obviously, kupffer cell was activated leaded to the ICAM-1 protein expression of hepatic tissue reinforced obviously. Their synthetic actions are reasons of NASH development.3. The preventive and therapeutic effect and mechanism of Qi Ji Preparation on NASH of ratQi Ji Preparation could cut down serum lipid, ameliorate liver function and the degree of hepatic steatosis and inflammation activity. Qi Ji Preparation has the multitarget site effects of preventing and curing NASH, and the effects surpass to that of Compound Methionine and Choline Tablet control group. The concrete mechanism of action is as follows:3.1 The regulation of liver lipid metabolism of Qi Ji Preparation in NASH of rat: the content of hepatic FFA of Qi Ji Preparation groups decreased significantly. Accordingly, Qi Ji Preparation decreased the liver intake of FFA and TG accumulation. Qi Ji Preparation can raise liver competence of anti- oxidation and relieve oxidative stress and decrease lipid peroxidation. Qi Ji Preparation could reinforce the PPARa mRNA expression of hepatic tissues and weaken the L-FABP mRNA and protein expression of hepatic tissue. The mechanism of action of Qi Ji Preparation on NASH is related to the rectification of liver lipid metabolic disturbance.3.2 The influence of Qi Ji Preparation on hepatic inflammatory reaction of rats with NASH: Qi Ji Preparation has the effects of preventing and curing NASH through restraining the endotoxin binding receptor as CD14 and kupffer cell expression and ICAM-1 protein expression of hepatic tissue.
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