| Objective: Under the guidance of TCM theory, on the basis of previous clinical and experimental studies, by feeding with high lipid food and celio-injecting tetracycline to make rat non-alcoholic fatty liver disease(NAFLD) model, discuss the effect of XiaoZhiHuGan(XZHG) capsule on hepatocyte cytochrome P450 2E1 (CYP2E1) and peroxisome proliferator activated receptor-alpha(PPARα) mRNA expression. Through comparing with the liver weight, liver index, serum ALT, AST, TC, TG, liver homogenate free fatty acid and liver pathologic changes in different groups, profoundly clarify the molecular mechanism and target of XZHG capsule in treating NAFLD, reveal the function of CYP2E1 and PPARαin the NAFLD model and their inner relation , provide theoretical basis for treatment of NAFLD by traditional Chinese medicine(TCM) on celled and molecular levels..Methods: By feeding with high lipid food and celio-injecting tetracycline, the rats were made into NAFLD model.72 SD male rats were subdivided randomly into control group(KB), model group(MX), DongBaoGanTai(DBGT) group(DB), XZHG capsule high dose group (XG), middle dose group(XZ), low dose group(XD). Every group were 12 rats. All rats were free to drink ,eat and feed in coops in animal experimental lab , where temperature was 20～25℃,light and dark were equally 12 hours. After the rats were fed one week normally, excepting the control group fed with normal food , the other groups were fed with fat food (84% basic food +10% pig fat+5% yolk+1% cholesterol) everyday and were celio-injected 2% tetracycline (15mg/100g weight) in the first time ,which have to do every six days by the dose. 10mg/100g weight. From the second week, model group were fed physiological saline and the other groups were fed corresponding medicines once a day except the control group,: the dose of XZHG capsule for XG, XZ, XD group are respectively 0.15g/100g, 0.075g/100g, 0.0375g/100g, which was 20, 10, 5 times of the adult's normal dose one day; the dose of DBGT for DB group is 0.06g/100g, which was 10 times of the adult's. The rats were weighed every week and the dose adjusted according to the weight. 7 weeks later, all the groups were killed. Methods: fasting during the previous night, the rats were anesthetized by cello-injecting 10% cholral hydrate solution (0.3ml/100g weight). The blood were got from the aorta ventralar, the liver were picked off and weighed, 500mg liver tissue got from the liver were conserved in the degree of-70℃liquid-nitrogen in order to extract the tissue total RNA. Another 0.5cm×lcm×0.3cm liver tissue were got from the left liver, and were fixed by 10% formalin solution for the purpose of pathologic slices for light microscope. Another 500 mg tissue were made of 10% liver homogenate. After 30min steady placement under room temperature, the serum was separated from blood. The parameters include: liver wet weight, liver index, pathologic changes, serum ALT, AST, TC, TG level; liver homogenate FFA content, the level of liver CYP2E1 and PPARa mRNA expression.Results:1. Compared with KB group: in MX group, the liver wet weight, liver index, serum ALT, AST, TC, TG, CYP2E1 mRNA expression level and the liver homogenate FFA content increased obviously(P＜0.01); the level of PPARa mRNA expression decreased obviously(P＜0.01); liver cells appear moderate or severe steatosis.2. Compared with MX group: the liver index, serum ALT, AST, TC, TG and liver homogenate FFA of the four treated groups were all decreased in some degree, the curative effects of XG and XZ groups are better(P＜0.01 or P＜0.05). CYP2E1 mRNA expressions level in XG, XZ, XD and DB groups decreased obviously (P＜0.01). PPARa mRNA expressions of XG and XZ groups were increased obviously (P＜0.01).3. Compared with DB group: in XG and XZ groups, pathological histology were improved obviously and the liver wet weight were decreased; serum ALT, AST, TC, TG and liver homogenate FFA level were decreased obviously (P＜0.01 or p＜0.05), CYP2E1 mRNA expressions were inhibited more than DB group (P＜0.01 or P＜0.05), PPARa mRNA expressions were increased (P＜0.05).4. Compared with XD group: XG and XZ groups have remarkably superior on decreasing liver homogenate FFA, inhibiting CYP2E1 mRNA expressions and activating PPARa mRNA expressions (P＜0.01 or P＜0.05); there was no difference between XG and XZ groups (P＞0.05).Conclusions:1. By feeding with high lipid food and celio-injecting tetracycline, NAFLD model were made in rats successfully. The method of making model take less time and easily succeed, so it is an ideal method.2. XZHG capsule can decrease obviously liver wet weight, liver index, serum ALT, AST, TC, TG, liver homogenate FFA level, and improve pathologic changes. So it has remarkable effect in treating NAFLD.3. The lever of liver CYP2E1 and PPARa mRNA expression were associated with NAFLD. Their tendence are opposite.4. XZHG capsule can correct the disorder of fat lipid in liver through activating PPARa and hold back lipid peroxisome reaction through inhibiting CYP2E1, which may be important molecular mechanism to treat NAFLD of XZHG capsules.5. XZHG capsule activates PPARa and inhibit CYP2E1 at the same time, so it could not aggravate liver injury from lipid peroxisome reaction. It plays the important role of decreasing fat lipid, protecting liver and preventing NAFLD.6. No matter what from the serum parameters, pathologic changes or CYP2E1 and PPARa gene expressions, XZHG capsule has so much superiorities more than DB group.7. With the dose increasing, the serum parameters and pathologic changes of XZHG capsule groups get better, which indicate the curative effect relate to the dose in choosen scope. But most parameters are not different between XG and XZ groups; XZ group is better than XG group in interfering CYP2E1, PPARa gene expressions, which is in line with the clinical dose. |
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