摘要
目的：本课题以中医理论为指导，在前期实验研究和临床观察基础上，以高脂饲料联合四环素腹腔注射制备大鼠非酒精性脂肪性肝病(non alcoholic fatty liver disease，NAFLD)模型，探讨消脂护肝胶囊对模型鼠肝组织细胞色素P450 2E1(cytochrome P450 2E1，CYP2E1)和过氧化物酶体增殖物激活受体a(peroxisome proliferator-activated receptor-a，PPARa)基因表达的影响，并通过对各组肝湿重、肝指数、血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)、肝匀浆游离脂肪酸(FFA)和肝脏病理组织学变化等指标的比较，深入阐释消脂护肝胶囊防治NAFLD的分子机制，揭示CYP2E1和PPARa在NAFLD发生发展中的作用及二者之间的相关性，从细胞和分子水平为NAFLD的中医药防治提供理论依据。
方法：SD雄性大鼠72只，随机分为6组，分别为空白组(KB组)、模型组(MX组)、东宝肝泰组(DB组)和消脂护肝胶囊高(XG组)、中(XZ组)、低(XD组)剂量组，每组12只。各组动物均自由饮水和进食，分笼喂养于20～25℃明暗各12h的动物实验室内。常规饲养1周后，除KB组予基础饲料外，其余5组均饲以高脂饲料(84％基础饲料+10％猪油+5％蛋黄粉+1％胆固醇)，并于造模第1天腹腔注射2％四环素溶液0.75ml／100g，1周后每6天一次腹腔注射四环素0.5ml／100g，共计6次。自造模的第2周起，除KB组外，各组均按1ml／(100g·d)体积分别灌服生理盐水及相应药物，每日1次。消脂护肝胶囊高、中、低剂量组分别按0.15g／100g、0.075g／100g、0.0375g／100g剂量灌胃，用量相当于成人用药量的20倍、10倍、5倍；东宝肝泰灌胃剂量为0.06g／100g，用量相当于成人用药量的10倍。每周称鼠重一次，并相应调整用药剂量。第7周(末次腹腔注射后1周)实验结束时处死全部实验动物。方法：隔夜禁食，次日以10％水合氯醛溶液0.3ml／100g腹腔注射麻醉后，自腹主动脉采血；之后迅速取出肝脏称肝湿重；取肝组织500mg放入-70℃液氮保存待提取肝组织总RNA；取肝左叶1块0.5cm×1cm×0.3cm组织置于10％甲醛溶液固定，备作光镜病理切片；再取肝左叶1块500mg制成10％肝匀浆；所采血液室温静置30min后分离血清备用。检测指标：肝湿重、肝指数、病理组织学变化、血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇(TC)、肝匀浆FFA含量、肝组织CYP2E1、PPARa mRNA相对表达量等。
结果：1．与KB组比较：MX组肝湿重、肝指数、血清ALT、AST、TC、TG水平、肝匀浆FFA含量、肝组织CYP2E1基因表达水平均有显著升高(P＜0.01)，PPARa基因表达水平显著降低(P＜0.01)；病理组织学呈中～重度脂肪变。2．与MX组比较：各用药组肝湿重、血清ALT、AST、TC、TG和肝匀浆FFA含量均有不同程度下降，以XG、XZ组疗效最优(P＜0.01或P＜0.05)。XG、XZ、XD组及DB组肝组织CYP2E1基因表达水平均有显著降低(P＜0.01)。XG、XZ组PPARa基因表达水平显著升高(P＜0.01)。3．与DB组比较：XZ组肝湿重改善明显；血清ALT、AST、TG、肝匀浆FFA含量明显降低(P＜0.01或P＜0.05)；XZ组抑制CYP2E1基因表达效果明显优于DB组(P＜0.01或P＜0.05)；XZ组的PPARa基因表达水平较DB组明显升高(P＜0.05)。4．与XD组比较：XG、XZ组在改善病理学变化、降低肝匀浆FFA含量和血清TG、ALT水平、抑制肝组织CYP2E1基因表达、升高PPARa基因表达水平方面有明显优势(P＜0.01或P＜0.05)：XG、XZ组相比，除AST、FFA含量XG组优于XZ组(P＜0.05)之外，其余指标二者之间比较无显著性差异(P＞0.05)。
结论：1．采用高脂饲料联合四环素腹腔注射可以成功复制大鼠NAFLD模型，造模时间短，成功率高，是一种较为理想的造模方法。2．消脂护肝胶囊可有效降低NAFLD模型大鼠的肝湿重、肝指数、血清ALT、AST、TC、TG及肝匀浆FFA水平，改善病理组织学变化，具有显著的抗脂肪肝作用；3．肝组织CYP2E1和PPARa基因表达水平与NAFLD的形成和发展密切相关，二者之间呈负相关关系；4．消脂护肝胶囊可激活PPARa mRNA表达，纠正肝脏脂质代谢紊乱；同时可抑制CYP2E1 mRNA表达，阻止脂质过氧化反应；这可能是其防治NAFLD的重要分子机制。5．消脂护肝胶囊增强大鼠PPAR a的表达的同时又可抑制CYP2E1表达，故不会加重脂质过氧化作用对肝脏的损伤，而是最终发挥了降低血脂、保肝降酶、阻止或减轻脂肪肝形成的作用。6．无论从生化指标、病理学改变还是从CYP2E1和PPARa基因表达情况来看，消脂护肝胶囊防治NAFLD的效果均优于东宝肝泰对照组；7．随着剂量的增加，消脂护肝胶囊生化指标(TC、TG、ALT、AST)及病理学获得更明显的改善，说明该药在所选范围内存在一定量效关系。而从高、中剂量组来看，多数指标无显著差异；从对CYP2E1和PPARa基因表达的影响来看，XZ组更显优势，说明三个剂量组中以XZ组剂量最优，这与临床用药剂量范围基本一致。
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.
引文
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