木犀草素改善高脂飲食誘導(dǎo)的小鼠非酒精性脂肪肝

王　新,　張　磊,　蔡　皓,　鮑　斌

(合肥工業(yè)大學(xué) 生物與食品工程學(xué)院,安徽 合肥　230009)

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木犀草素改善高脂飲食誘導(dǎo)的小鼠非酒精性脂肪肝

王新,張磊,蔡皓,鮑斌

(合肥工業(yè)大學(xué) 生物與食品工程學(xué)院,安徽 合肥230009)

木犀草素是一種大量存在于蔬菜、水果以及天然藥草中的天然黃酮類化合物,具有抗炎、抗氧化、改善胰島素抵抗和免疫調(diào)節(jié)等作用。肥胖常伴隨非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)的發(fā)生，因此研究木犀草素改善NAFLD具有極其重要的意義。文章用高脂飲食(high fat diet,HFD)喂養(yǎng)C57BL/6小鼠20周來建立非酒精性脂肪肝小鼠模型,同時(shí)設(shè)立低脂飲食(low fat diet,LFD)作為正常對照組，高脂添加0.01%木犀草素(HFD+Lut)作為實(shí)驗(yàn)組。用蘇木精伊紅染色和油紅染色檢測肝臟中脂質(zhì)積累,用實(shí)時(shí)定量熒光聚合鏈?zhǔn)椒磻?yīng)(real-time polymerase chain reaction,Real-Time PCR)檢測脂質(zhì)合成基因及其轉(zhuǎn)錄因子相關(guān)基因的表達(dá)。結(jié)果表明,木犀草素能夠減少小鼠肝臟中脂質(zhì)積累,并且能抑制小鼠肝臟內(nèi)脂質(zhì)合成的基因以及轉(zhuǎn)錄因子的表達(dá),探明了木犀草素能改善HFD導(dǎo)致的NAFLD及其作用機(jī)制。

木犀草素;非酒精性脂肪肝;高脂飲食;小鼠

非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)是指無過量飲酒史,且肝細(xì)胞發(fā)生脂肪病變和脂質(zhì)貯積的一種臨床病理綜合癥,包括單純性脂肪病變、脂肪性肝炎、肝纖維化和肝硬化4個(gè)病理階段[1-2]。隨著肥胖及其相關(guān)代謝綜合癥日益流行,NAFLD現(xiàn)已成為歐美等發(fā)達(dá)國家和我國慢性肝病的重要病因[3]。

在NAFLD患者中,脂質(zhì)代謝紊亂比較常見。肝臟在體內(nèi)脂質(zhì)代謝過程中發(fā)揮著主要的作用,它能夠攝入游離脂肪酸,加工、貯存和輸出脂質(zhì),過程中的任何一環(huán)出現(xiàn)問題都可能導(dǎo)致NAFLD的產(chǎn)生[4]。游離脂肪酸在細(xì)胞中發(fā)揮著重要作用,例如合成細(xì)胞膜、作為能量存儲(chǔ)以及參與細(xì)胞內(nèi)的信號(hào)通路。然而,在很多器官中慢性的游離脂肪酸含量的增加會(huì)破壞代謝途徑,誘導(dǎo)胰島素抵抗(insulin resistance,IR)。肝臟中脂質(zhì)的積累與IR密切相關(guān)[5-6]。脂肪組織胰島素抵抗能增加脂解,并且能增加游離脂肪酸從脂肪組織到肝臟的輸入,減少輸出。除此之外,體內(nèi)活性氧(reactive oxygen species,ROS)含量增加,多種細(xì)胞因子(例如腫瘤壞死因子α(TNF-α)和白細(xì)胞介素-6(IL-6)等)也可能導(dǎo)致肝臟細(xì)胞脂質(zhì)代謝功能異常,從而導(dǎo)致NAFLD的發(fā)生或者加重[7-10]。

木犀草素大量存在于蔬菜、水果以及天然藥草中,屬于天然黃酮類化合物。研究表明，木犀草素具有改善胰島素抵抗、減肥、抗炎、抗氧化、抗腫瘤以及免疫調(diào)節(jié)等作用[11-16]。木犀草素的生理功效與NAFLD的發(fā)病機(jī)制可謂相生相克,鑒于此,本文研究了木犀草素與NAFLD的關(guān)系,以便找到一種能夠預(yù)防和治療NAFLD的天然藥物。

1　材料與方法

1.1材料

1.1.1主要試劑

木犀草素、蘇木精、伊紅和油紅均購自Sigma公司;Trizol RNA提取試劑盒、Oligo d(T)、dNTP均購自Takara公司;逆轉(zhuǎn)錄試劑盒購自Invitrogen公司;實(shí)時(shí)定量熒光聚合鏈?zhǔn)椒磻?yīng)(real-time polymerase chain reaction,Real-Time PCR)試劑SYBR Green Mixture購自Takara公司。

1.1.2實(shí)驗(yàn)小鼠

無特定病原體(specific pathogen free,SPF)級(jí)C57Bl/6小鼠購自北京維通利華實(shí)驗(yàn)室動(dòng)物有限公司,6周齡小鼠隨機(jī)分成3組,每組8只,飼養(yǎng)在SPF級(jí)動(dòng)物房中,環(huán)境溫度為(23±2) ℃;低脂飼料、45%高脂飼料和高脂加0.01%木犀草素飼料(HFD+Lut)均來自Research配方(D12451),Co60輻照殺菌后使用;造模20周以后,稱量小鼠體重,二氧化碳麻醉,心臟取血,解剖收集小鼠肝臟。

1.2方法

1.2.1肝臟組織石蠟包埋以及蘇木精伊紅染色

小鼠新鮮肝臟組織經(jīng)過4%甲醛固定,程序脫水浸蠟,石蠟包埋。石蠟切片,切片厚度5 μm。組織切片經(jīng)過脫蠟,復(fù)水;用蘇木精染色1 h,42 ℃水浴5 min反藍(lán);再用0.2%伊紅染色,95%乙醇分色;經(jīng)過風(fēng)干,中性樹膠封片;光學(xué)顯微鏡200倍視野下觀察拍照。

1.2.2肝臟冰凍切片

將小鼠新鮮肝臟組織包埋在OCT包埋劑(opti-mum cutting temperature compound)中,液氮速凍,-80 ℃冰箱保存。冰凍切片機(jī)切10 μm厚組織切片。將冰凍切片自然風(fēng)干30～60 min,用10%甲醛固定5～10 min。自然風(fēng)干后蒸餾水洗滌。切片自然風(fēng)干,置入丙二醇5 min,轉(zhuǎn)移至已經(jīng)提前預(yù)熱的60 ℃油紅中染色10 min。85%丙二醇分色,蒸餾水中漂洗2次。封片鏡檢拍照。

1.2.3肝臟RNA提取以及Real-Time PCR

稱取20 mg小鼠肝臟組織,按Trizol RNA提取試劑盒提供的方法提取總RNA。Invitrogen 逆轉(zhuǎn)錄試劑盒獲得cDNA后,-20 ℃保存。Real-Time PCR條件為:95 ℃ 3 min完全變性,經(jīng)過95 ℃ 30 s,退火溫度60 ℃,72 ℃延伸20 s 40個(gè)循環(huán)。

1.2.4統(tǒng)計(jì)學(xué)處理

實(shí)驗(yàn)結(jié)果用SPSS統(tǒng)計(jì)學(xué)軟件進(jìn)行方差分析,所有數(shù)據(jù)以(均數(shù)±標(biāo)準(zhǔn)差)表示,*表示與高脂對照組相比,雙側(cè)P<0.05有統(tǒng)計(jì)學(xué)意義上的顯著性差異;**表示與高脂對照組相比,雙側(cè)P<0.01有統(tǒng)計(jì)學(xué)上極顯著性差異;#表示與正常飲食對照組相比,雙側(cè)P<0.05有統(tǒng)計(jì)學(xué)意義上的顯著性差異[17]。

2　結(jié)果與討論

2.1木犀草素對小鼠肝臟質(zhì)量的影響

木犀草素具有抗氧化、抗炎、免疫調(diào)節(jié)、改善胰島素抵抗等作用[11-16],為此推測木犀草素能改善高脂飲食(high fat diet,HFD)相關(guān)的NAFLD的發(fā)生。本文采用含有0.01%木犀草素的高脂飲食誘導(dǎo)小鼠20周,同時(shí)設(shè)置低脂飲食(low fat diet,LFD)作為正常對照組,HFD作為陽性對照組,分析木犀草素對小鼠體重和肝臟質(zhì)量的影響如圖1所示。圖1結(jié)果表明,木犀草素能降低HFD導(dǎo)致的小鼠體重增加以及肝臟質(zhì)量的增加。

圖1　木犀草素對小鼠體重和肝臟的影響

2.2木犀草素對小鼠肝臟脂質(zhì)異常積累的影響

NAFLD顯著的特征是脂類物質(zhì)在肝臟中異常積累,為此使用蘇木精伊紅染色和油紅染色2個(gè)實(shí)驗(yàn)來反映肝臟中脂質(zhì)積累的情況,結(jié)果如圖2、圖3所示。圖2結(jié)果表明,木犀草素能夠減少HFD喂養(yǎng)導(dǎo)致的小鼠肝臟脂質(zhì)積累。與HFD組小鼠相比,給小鼠喂食木犀草素后小鼠肝臟的蘇木精伊紅染色空泡明顯減少,說明肝臟中積累的脂類物質(zhì)明顯減少。肝臟冰凍切片能夠通過油紅將脂類物質(zhì)染成紅色來直觀反映肝臟中脂質(zhì)的積累。

由圖3可看出,在飲食補(bǔ)充木犀草素后肝臟中紅色脂滴明顯減少變小,也證實(shí)木犀草素能明顯減少肝臟中脂類物質(zhì)的積累,減輕NAFLD的發(fā)生。

圖2　石臘切片蘇木精伊紅染色對小鼠肝臟脂質(zhì)積累的影響

圖3　冰凍切片油紅染色對小鼠肝臟脂質(zhì)積累的影響

2.3木犀草素對肝臟脂質(zhì)合成基因表達(dá)的影響

為了研究木犀草素改善小鼠肝臟脂質(zhì)積累的機(jī)制,采用Real-Time PCR檢測了調(diào)控肝臟脂質(zhì)生成基因的表達(dá),結(jié)果如圖4所示。

圖4　木犀草素對小鼠肝臟中脂質(zhì)合成相關(guān)基因表達(dá)的影響

圖4中,相關(guān)基因包括脂肪酸合成酶(FAS)、硬脂酰CoA去飽和酶1(SCD1)和乙酰-CoA羧化酶(ACC)[18]。由圖4可看出,HFD誘導(dǎo)的小鼠經(jīng)木犀草素處理后肝臟脂肪酸合成相關(guān)基因FAS、SCD和ACC的表達(dá)均明顯下降，這說明木犀草素能夠抑制肝臟脂質(zhì)生成,改善肝臟中脂質(zhì)積累。

2.4木犀草素對脂質(zhì)代謝相關(guān)轉(zhuǎn)錄因子的影響

肝臟中膽固醇調(diào)節(jié)元件結(jié)合蛋白1(SREBP1)能夠轉(zhuǎn)移至核內(nèi)激活下游脂質(zhì)合成相關(guān)基因(FAS、SCD1、ACC)的表達(dá)[19]。過氧化物酶體增生物激活受體γ(PPARγ)在體內(nèi)能夠調(diào)節(jié)葡萄糖和脂質(zhì)代謝的穩(wěn)態(tài),通過調(diào)節(jié)SREBP1C的表達(dá)來增強(qiáng)脂質(zhì)的生成。木犀草素對脂質(zhì)代謝轉(zhuǎn)錄因子的影響如圖5所示,圖5結(jié)果表明,木犀草素能夠明顯抑制HFD誘導(dǎo)的SREBP1和PPARγ這2種脂質(zhì)代謝關(guān)鍵轉(zhuǎn)錄因子的表達(dá)。

圖5　木犀草素對脂質(zhì)代謝轉(zhuǎn)錄因子的影響

3　結(jié)　論

隨著當(dāng)前經(jīng)濟(jì)社會(huì)的發(fā)展,生活條件不斷改善,肥胖相關(guān)的NAFLD呈不斷上升的趨勢。肝臟在維持體內(nèi)葡萄糖和脂質(zhì)穩(wěn)態(tài)中發(fā)揮重要作用。肝臟中異常的脂質(zhì)積累會(huì)影響肝臟正常生理功能,嚴(yán)重時(shí)還可能導(dǎo)致肝纖維化、肝硬化、肝臟壞死，甚至患肝癌的風(fēng)險(xiǎn)[3,20]。

本文選用較低劑量(約0.01%)的木犀草素作為食品添加劑,研究了木犀草素與NAFLD關(guān)系。結(jié)果表明,木犀草素具有改善NAFLD的生物活性,HFD小鼠進(jìn)食木犀草素后,肝臟脂質(zhì)沉積明顯減少;通過檢測脂質(zhì)生成關(guān)鍵基因及其轉(zhuǎn)錄因子的表達(dá),發(fā)現(xiàn)在體內(nèi)木犀草素很可能是通過影響調(diào)控脂質(zhì)生成的轉(zhuǎn)錄因子的表達(dá)來抑制下游脂質(zhì)生成的基因表達(dá)。但當(dāng)前研究還處于初始階段,找到木犀草素在體內(nèi)作用的具體方式及其靶標(biāo)將是今后研究的重點(diǎn)。

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(責(zé)任編輯閆杏麗)

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

安徽省自然科學(xué)基金資助項(xiàng)目 (1408085QC48)

王新(1989-),男,山東滕州人,合肥工業(yè)大學(xué)碩士生;

鮑斌(1983-),男,河北石家莊人,博士,合肥工業(yè)大學(xué)講師,碩士生導(dǎo)師.

10.3969/j.issn.1003-5060.2016.07.025

Q786

A

1003-5060(2016)07-0994-05