木犀草素改善高脂饮食诱导的小鼠非酒精性脂肪肝

王　新,　张　磊,　蔡　皓,　鲍　斌

(合肥工业大学 生物与食品工程学院,安徽 合肥　230009)



木犀草素改善高脂饮食诱导的小鼠非酒精性脂肪肝

王新,张磊,蔡皓,鲍斌

(合肥工业大学 生物与食品工程学院,安徽 合肥230009)

木犀草素是一种大量存在于蔬菜、水果以及天然药草中的天然黄酮类化合物,具有抗炎、抗氧化、改善胰岛素抵抗和免疫调节等作用。肥胖常伴随非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)的发生，因此研究木犀草素改善NAFLD具有极其重要的意义。文章用高脂饮食(high fat diet,HFD)喂养C57BL/6小鼠20周来建立非酒精性脂肪肝小鼠模型,同时设立低脂饮食(low fat diet,LFD)作为正常对照组，高脂添加0.01%木犀草素(HFD+Lut)作为实验组。用苏木精伊红染色和油红染色检测肝脏中脂质积累,用实时定量荧光聚合链式反应(real-time polymerase chain reaction,Real-Time PCR)检测脂质合成基因及其转录因子相关基因的表达。结果表明,木犀草素能够减少小鼠肝脏中脂质积累,并且能抑制小鼠肝脏内脂质合成的基因以及转录因子的表达,探明了木犀草素能改善HFD导致的NAFLD及其作用机制。

木犀草素;非酒精性脂肪肝;高脂饮食;小鼠

非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)是指无过量饮酒史,且肝细胞发生脂肪病变和脂质贮积的一种临床病理综合症,包括单纯性脂肪病变、脂肪性肝炎、肝纤维化和肝硬化4个病理阶段[1-2]。随着肥胖及其相关代谢综合症日益流行,NAFLD现已成为欧美等发达国家和我国慢性肝病的重要病因[3]。

在NAFLD患者中,脂质代谢紊乱比较常见。肝脏在体内脂质代谢过程中发挥着主要的作用,它能够摄入游离脂肪酸,加工、贮存和输出脂质,过程中的任何一环出现问题都可能导致NAFLD的产生[4]。游离脂肪酸在细胞中发挥着重要作用,例如合成细胞膜、作为能量存储以及参与细胞内的信号通路。然而,在很多器官中慢性的游离脂肪酸含量的增加会破坏代谢途径,诱导胰岛素抵抗(insulin resistance,IR)。肝脏中脂质的积累与IR密切相关[5-6]。脂肪组织胰岛素抵抗能增加脂解,并且能增加游离脂肪酸从脂肪组织到肝脏的输入,减少输出。除此之外,体内活性氧(reactive oxygen species,ROS)含量增加,多种细胞因子(例如肿瘤坏死因子α(TNF-α)和白细胞介素-6(IL-6)等)也可能导致肝脏细胞脂质代谢功能异常,从而导致NAFLD的发生或者加重[7-10]。

木犀草素大量存在于蔬菜、水果以及天然药草中,属于天然黄酮类化合物。研究表明，木犀草素具有改善胰岛素抵抗、减肥、抗炎、抗氧化、抗肿瘤以及免疫调节等作用[11-16]。木犀草素的生理功效与NAFLD的发病机制可谓相生相克,鉴于此,本文研究了木犀草素与NAFLD的关系,以便找到一种能够预防和治疗NAFLD的天然药物。

1　材料与方法

1.1材料

1.1.1主要试剂

木犀草素、苏木精、伊红和油红均购自Sigma公司;Trizol RNA提取试剂盒、Oligo d(T)、dNTP均购自Takara公司;逆转录试剂盒购自Invitrogen公司;实时定量荧光聚合链式反应(real-time polymerase chain reaction,Real-Time PCR)试剂SYBR Green Mixture购自Takara公司。

1.1.2实验小鼠

无特定病原体(specific pathogen free,SPF)级C57Bl/6小鼠购自北京维通利华实验室动物有限公司,6周龄小鼠随机分成3组,每组8只,饲养在SPF级动物房中,环境温度为(23±2) ℃;低脂饲料、45%高脂饲料和高脂加0.01%木犀草素饲料(HFD+Lut)均来自Research配方(D12451),Co60辐照杀菌后使用;造模20周以后,称量小鼠体重,二氧化碳麻醉,心脏取血,解剖收集小鼠肝脏。

1.2方法

1.2.1肝脏组织石蜡包埋以及苏木精伊红染色

小鼠新鲜肝脏组织经过4%甲醛固定,程序脱水浸蜡,石蜡包埋。石蜡切片,切片厚度5 μm。组织切片经过脱蜡,复水;用苏木精染色1 h,42 ℃水浴5 min反蓝;再用0.2%伊红染色,95%乙醇分色;经过风干,中性树胶封片;光学显微镜200倍视野下观察拍照。

1.2.2肝脏冰冻切片

将小鼠新鲜肝脏组织包埋在OCT包埋剂(opti-mum cutting temperature compound)中,液氮速冻,-80 ℃冰箱保存。冰冻切片机切10 μm厚组织切片。将冰冻切片自然风干30～60 min,用10%甲醛固定5～10 min。自然风干后蒸馏水洗涤。切片自然风干,置入丙二醇5 min,转移至已经提前预热的60 ℃油红中染色10 min。85%丙二醇分色,蒸馏水中漂洗2次。封片镜检拍照。

1.2.3肝脏RNA提取以及Real-Time PCR

称取20 mg小鼠肝脏组织,按Trizol RNA提取试剂盒提供的方法提取总RNA。Invitrogen 逆转录试剂盒获得cDNA后,-20 ℃保存。Real-Time PCR条件为:95 ℃ 3 min完全变性,经过95 ℃ 30 s,退火温度60 ℃,72 ℃延伸20 s 40个循环。

1.2.4统计学处理

实验结果用SPSS统计学软件进行方差分析,所有数据以(均数±标准差)表示,*表示与高脂对照组相比,双侧P<0.05有统计学意义上的显著性差异;**表示与高脂对照组相比,双侧P<0.01有统计学上极显著性差异;#表示与正常饮食对照组相比,双侧P<0.05有统计学意义上的显著性差异[17]。

2　结果与讨论

2.1木犀草素对小鼠肝脏质量的影响

木犀草素具有抗氧化、抗炎、免疫调节、改善胰岛素抵抗等作用[11-16],为此推测木犀草素能改善高脂饮食(high fat diet,HFD)相关的NAFLD的发生。本文采用含有0.01%木犀草素的高脂饮食诱导小鼠20周,同时设置低脂饮食(low fat diet,LFD)作为正常对照组,HFD作为阳性对照组,分析木犀草素对小鼠体重和肝脏质量的影响如图1所示。图1结果表明,木犀草素能降低HFD导致的小鼠体重增加以及肝脏质量的增加。

图1　木犀草素对小鼠体重和肝脏的影响

2.2木犀草素对小鼠肝脏脂质异常积累的影响

NAFLD显著的特征是脂类物质在肝脏中异常积累,为此使用苏木精伊红染色和油红染色2个实验来反映肝脏中脂质积累的情况,结果如图2、图3所示。图2结果表明,木犀草素能够减少HFD喂养导致的小鼠肝脏脂质积累。与HFD组小鼠相比,给小鼠喂食木犀草素后小鼠肝脏的苏木精伊红染色空泡明显减少,说明肝脏中积累的脂类物质明显减少。肝脏冰冻切片能够通过油红将脂类物质染成红色来直观反映肝脏中脂质的积累。

由图3可看出,在饮食补充木犀草素后肝脏中红色脂滴明显减少变小,也证实木犀草素能明显减少肝脏中脂类物质的积累,减轻NAFLD的发生。

图2　石腊切片苏木精伊红染色对小鼠肝脏脂质积累的影响

图3　冰冻切片油红染色对小鼠肝脏脂质积累的影响

2.3木犀草素对肝脏脂质合成基因表达的影响

为了研究木犀草素改善小鼠肝脏脂质积累的机制,采用Real-Time PCR检测了调控肝脏脂质生成基因的表达,结果如图4所示。

图4　木犀草素对小鼠肝脏中脂质合成相关基因表达的影响

图4中,相关基因包括脂肪酸合成酶(FAS)、硬脂酰CoA去饱和酶1(SCD1)和乙酰-CoA羧化酶(ACC)[18]。由图4可看出,HFD诱导的小鼠经木犀草素处理后肝脏脂肪酸合成相关基因FAS、SCD和ACC的表达均明显下降，这说明木犀草素能够抑制肝脏脂质生成,改善肝脏中脂质积累。

2.4木犀草素对脂质代谢相关转录因子的影响

肝脏中胆固醇调节元件结合蛋白1(SREBP1)能够转移至核内激活下游脂质合成相关基因(FAS、SCD1、ACC)的表达[19]。过氧化物酶体增生物激活受体γ(PPARγ)在体内能够调节葡萄糖和脂质代谢的稳态,通过调节SREBP1C的表达来增强脂质的生成。木犀草素对脂质代谢转录因子的影响如图5所示,图5结果表明,木犀草素能够明显抑制HFD诱导的SREBP1和PPARγ这2种脂质代谢关键转录因子的表达。

图5　木犀草素对脂质代谢转录因子的影响

3　结　论

随着当前经济社会的发展,生活条件不断改善,肥胖相关的NAFLD呈不断上升的趋势。肝脏在维持体内葡萄糖和脂质稳态中发挥重要作用。肝脏中异常的脂质积累会影响肝脏正常生理功能,严重时还可能导致肝纤维化、肝硬化、肝脏坏死，甚至患肝癌的风险[3,20]。

本文选用较低剂量(约0.01%)的木犀草素作为食品添加剂,研究了木犀草素与NAFLD关系。结果表明,木犀草素具有改善NAFLD的生物活性,HFD小鼠进食木犀草素后,肝脏脂质沉积明显减少;通过检测脂质生成关键基因及其转录因子的表达,发现在体内木犀草素很可能是通过影响调控脂质生成的转录因子的表达来抑制下游脂质生成的基因表达。但当前研究还处于初始阶段,找到木犀草素在体内作用的具体方式及其靶标将是今后研究的重点。

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(责任编辑闫杏丽)

Effect of luteolin on the alleviation of high fat diet induced non-alcoholic fatty liver disease in mice

WANG Xin,ZHANG Lei,CAI Hao,BAO Bin

(School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China)

Luteolin is a natural flavonoid compound which exists in fruits, vegetables and natural herbs. It has been reported that luteolin has the properties of anti-inflammation, anti-oxidation, improving insulin sensitivity and immune regulation. Obesity is often accompanied with non-alcoholic fatty liver disease(NAFLD), so it is of importance to study the effect of luteolin on NAFLD. In this paper, the NAFLD model of C57BL/6 mice induced by high fat diet(HFD) for 20 weeks was established. The mice fed with low fat diet(LFD) were set as normal control group, and the mice fed with HFD adding 0.01% luteolin were set as the test group. The liver lipid accumulation was detected by using the hematoxylin and eosin(HE) staining and Oil Red O staining procedures. And the lipid metabolism-related gene expression was quantified by using real-time polymerase chain reaction(PCR). The results showed that luteolin decreased the lipid accumulation in liver of mice, and improved the expression of genes related to lipid synthesis and transcription factors. The presented study verified the effect of luteolin on the improvement of NAFLD caused by HFD and the possible mechanism of action.

luteolin; non-alcoholic fatty liver disease(NAFLD); high fat diet(HFD); mice

2015-03-23；

2015-05-13

安徽省自然科学基金资助项目 (1408085QC48)

王新(1989-),男,山东滕州人,合肥工业大学硕士生;

鲍斌(1983-),男,河北石家庄人,博士,合肥工业大学讲师,硕士生导师.

10.3969/j.issn.1003-5060.2016.07.025

Q786

A

1003-5060(2016)07-0994-05