摘要：目的：采用高脂饮食复制非酒精性脂肪肝病(Non-alcoholic fatty liver disease，NAFLD)大鼠模型，观察决明子蒽醌苷治疗前后大鼠肝功能、血脂、炎性因子，以及肝脏组织中TLR4和NF-ΚB的表达特点。方法：72只雄性SD大鼠在适应性喂养10天后随机分为正常组、模型组、阳性药物多烯磷脂酰胆碱干预组、决明子蒽醌苷低、中和高剂量治疗组，每组12只，除正常组外，其余五组给予高脂饲料8周构建NAFLD模型，模型构建成功后，正常组和模型组予以生理盐水灌胃，决明子蒽醌苷低、中和高剂量组分别予决明子蒽醌苷5、10和20mg/kg/d，阳性对照组予以多烯磷脂酰胆碱23mg/kg/d治疗，6周后隔夜禁食12h称重后处死，计算肝指数，收集血液检测肝功能（AST和ALT）、血脂（TG和TC）炎性因子（IL-1a、IL-1b、IL-6和TNF-a），收集肝脏检测肝脏病理学以及肝脏组织中TLR4和NF-ΚB蛋白和mRNA表达特点。结果：与正常组相比，模型组肝重、肝指数、AST、ALT、TG、TC、IL-1α、IL-1β、IL-6和TNF-α均明显升高(P<0.05)，与模型组相比，阳性药物组和决明子蒽醌苷三个剂量组上述指标均明显降低(P<0.05)，决明子蒽醌苷高剂量组肝重、肝指数、AST、ALT、TG、TC、IL-1β、IL-6和TNF-α均明显低于阳性药物组(P<0.05)，而大鼠体重各组间并没有明显差异，此外决明子蒽醌苷三个剂量组IL-1α明显低于多烯磷脂酰胆碱干预组；HE染色正常组大鼠肝小叶结构完整，细胞核分布均匀，模型组大鼠有明显弥漫性肝细胞脂肪变性，多烯磷脂酰胆碱干预组和决明子蒽醌苷三个剂量治疗组脂肪变性程度和脂肪空泡数量均呈不同程度减少。Western Blotting和RT-PCR可见，与正常组相比，模型组TLR4和NF-ΚB 蛋白和mRNA表达均明显升高(P<0.05)，多烯磷脂酰胆碱和决明子蒽醌苷治疗后，二者均明显下降 (P<0.05)，此外决明子蒽醌苷中剂量和高剂量治疗组明显低于多烯磷脂酰胆碱干预组(P<0.05)。结论：决明子蒽醌苷可明显改善NAFLD大鼠肝功能、降低血脂，这与其可以降低肝脏中TLR4和NF-ΚB表达，进而改善机体炎症刺激有关。
关键词：决明子蒽醌苷，非酒精性脂肪性肝；Toll样受体4；核因子-kB
中图分类号： R285.5 文献标识码： A
The Affection of cassia glycosides on SREBP-1c and PPARα in liver of Nonalcoholic Fatty Liver Disease Rats
Abstract : Objective: Using a high-fat diet replicate non-alcoholic fatty liver disease (NAFLD) rat model, observe the liver function, blood lipids, inflammatory factors and TLR4 and NF-ΚB expression in liver tissue in rats before and after treatment with cassia glycosides. Methods: 72 male SD rats were randomly divided into the normal group, model group, positive drug polyene phosphatidylcholine group, cassia glycoside low, medium and high dose treatment groups after 10 days of adaptive feeding, n=12. Except the normal group, the others five groups were given a high-fat diet for 8 weeks to build the NAFLD model, After the model was successfully built, the normal group and model group were given normal saline gavage, and the cassia glycoside low, medium and high dose groups were given 5, 10 and 20 mg/kg/d cassia glycoside treatment, positive control group were given23 mg/kg/d the polyene phosphatidylcholine treated. After 6 weeks of treatment, the rats were fasted overnight and weighed, then the liver was weighed and the liver index was calculated. Blood was collected to detect liver function (AST and ALT), lipids (TG and TC) and inflammatory factors (IL-1a, IL-1b, IL-6, and TNF-a), the liver was collected for detected the liver pathology and the TLR4 and NF-ΚB protein and mRNA expression in liver. Results: Compared with the normal group, the liver weight, liver index, AST, ALT, TG, TC, IL-1α, IL-1β, IL-6 and TNF-α in the model group were significantly higher (P<0.05). Compared with the model group, the above-mentioned indexes in the positive drug group and the three doses of cassia glycoside were significantly lower (P<0.05). In addition, the liver weight, liver index, AST, ALT, TG, TC, liver weight, IL-1β, IL-6 and TNF-α in the high-dose cassia glycoside group were significantly lower than those indexes in the positive drug group (P<0.05), but there was no significant difference between the weights of the rats. In addition, IL-1α in the three dose groups of cassia glycoside was significantly lower it in the polyene phosphatidylcholine intervention group; The hepatic cell was lobule in normal groups, and the distribution of nucleus was uniform, The result of HE in the model group had obvious hepatic steatosis, in addition polyene phosphatidylcholine and cassia glycoside group have an varying degrees reduced. Western Blotting and RT-PCR showed that compared with the normal group, the expression of TLR4 and NF-ΚB protein and mRNA in the model group were significantly increased (P<0.05), after treatment with polyene phosphatidylcholine and cassia glycoside, they were decreased significantly (P<0.05). In addition, the medium and high-dose cassia glycoside group was significantly lower than the polyene phosphatidylcholine group (P<0.05). Conclusion: Cassia glycoside could obviously improve the liver function and reduce blood lipids of NAFLD rats, which is related with Cassia glycoside can reduce the expression of TLR4 and NF-ΚB in liver, further improve the inflammatory.
Keywords：cassia glycosides；nonalcoholic fatty liver disease；Toll-like receptors（TLR4）；nuclear factor-kappa B（NF-ΚB）
前言
非酒精性脂肪性肝病(Non-alcoholic fatty liver disease，NAFLD)是临床中常见的一种肝脏脂肪浸润、但无过量饮酒史的一种临床病例综合征 [1]。根据其在病理发展过程，可分为单纯性非酒精脂肪肝(non-alcoholic fatty liver，NAFL)、非酒精脂肪性肝炎(non-alcoholic steatohepatitis，NASH)和以及由其发展而来的非酒精脂肪性肝硬化等 [2]。近年来NAFLD在世界范围内广泛流行，且呈逐步年轻化趋势。以发达国家为例，包括日本、美国、以及欧洲各国等，本病发病率在正常人群中约为17%-33%，而在肥胖人群中高达80% [3]，此外美国对12岁以下的儿童流行病学统计 [4]发现，本病发生率约为1/10。在我国虽然没有对NAFLD进行过大样本统计学研究，但众多的文献 [5-7]均显示随着人们生活水平的提高，我国NAFLD发生率也逐年升高，其约占总人群的12-24%。NAFLD是一种进展性病变，控制不佳可在短期内给肝脏带来不可逆转的损害，因此围绕NAFLD的治疗是当前的研究热点。本研究成功复制了NAFLD大鼠模型，从炎症因子TLR4和NF-ΚB探讨决明子蒽醌苷对其的作用，以为NAFLD的治疗提供参考。

1、材料和方法
1.1实验材料
决明子蒽醌苷自制，采用含有5%的决明子蒽醌苷（购于陕西元朗生物工程有限公司）按照文献提取 [8]（蒽醌苷总含量为93.6%）；
多烯磷脂酰胆碱胶囊（polyene phosphatidylcholine）由赛诺菲(北京)制药有限公司提供；谷草转氨酶（Aspartate aminotransferase，AST）、谷丙转氨酶（Alanine aminotransferase，ALT）、白细胞介素1α（Interleukin-1α，IL-1α）、白细胞介素1β（Interleukin-1β，IL-1β）、白细胞介素6（Interleukin-6，IL-6）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α)试剂盒购于武汉博士德生物有限公司提供；总胆固醇（total cholesterol，TG）和甘油三脂（Triglyceride，TC）试剂盒购于南京建成生物工程研究所；Toll样受体4（Toll-like receptors，TLR4）、核因子-kB（nuclear factor-kappa B，NF-ΚB）和甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH）抗体购于美国Santa公司提供；二抗购于艾默生生物有限公司提供；PVDF膜购于赛诺菲(北京)制药有限公司；无水乙醇、多聚甲醛、磷酸二氢钠等均为国产分析纯，由实验室提供。
1.2实验仪器与设备
电热恒温鼓风干燥箱（上海喆图仪器有限公司）；-86摄氏度冰箱（美的公司）；电子天平（常州万泰天平仪器有限公司）；普通冰箱（海尔公司）；酶标仪和高速冷冻离心机（赛伯乐仪器有限公司）；分光光度计（上海美析仪器公司）；蛋白电泳仪（美国伯乐公司）； RT-qRCR仪（罗氏诊断产品有限公司）。
1.3实验动物
清洁级雄性SD大鼠72只，大鼠体重160~180 g，动物合格证号：SCXK(滇)2015.0002，由昆明医科大学实验动物中心提供。大鼠在购买后均饲养于标准饲养间，由专业人员饲养，室温18℃～22℃、湿度45～55％，日常明暗各12小时；高脂和普通饲料、饲养用垫料也均有由昆明医科大学实验动物中心提供。
作者：赵梓铭¹ 武俊紫¹ 姚政¹ 李玉晶^1，2  侯伟¹   陈文慧 ^* 石安华