高脂誘導(dǎo)非酒精性脂肪肝對(duì)CYP2A5表達(dá)的影響

崔一喆,王秋菊,李悅,周亞強(qiáng),蘇景(.黑龍江八一農(nóng)墾大學(xué)動(dòng)物科技學(xué)院,黑龍江大慶6339;.黑龍江省動(dòng)物疫病預(yù)防與控制中心,黑龍江哈爾濱50069)

高脂誘導(dǎo)非酒精性脂肪肝對(duì)CYP2A5表達(dá)的影響

崔一喆1,王秋菊1,李悅1,周亞強(qiáng)1,蘇景2
(1.黑龍江八一農(nóng)墾大學(xué)動(dòng)物科技學(xué)院,黑龍江大慶163319;2.黑龍江省動(dòng)物疫病預(yù)防與控制中心,黑龍江哈爾濱150069)

為了研究高脂誘導(dǎo)脂肪肝對(duì)CYP2A5表達(dá)的影響,選取雄性C57BL/6J小鼠18只,隨機(jī)分成對(duì)照組(control)、高脂組(NAFLD)和陽(yáng)性對(duì)照組(PYR),每組6只,分別飼喂基礎(chǔ)飼料和高脂飼料,陽(yáng)性對(duì)照組飼喂基礎(chǔ)飼料在處死前一天給予吡唑誘導(dǎo),連續(xù)8周后小鼠斷頸處死,取肝臟測(cè)定mRNA、CYP2A5的蛋白表達(dá)和酶活性.其結(jié)果陽(yáng)性對(duì)照組CYP2A5的mRNA、蛋白表達(dá)和酶活性與對(duì)照組相比分別增加229%、80%、151%差異極顯著(P<0.01).高脂誘導(dǎo)組與對(duì)照組相比CYP2A5的mRNA、蛋白表達(dá)和酶活性分別增加69%、34%、43%(P<0.01).表明高脂誘導(dǎo)非酒精性脂肪肝可增加CYP2A5的mRNA、蛋白表達(dá)水平和酶活力.

非酒精性脂肪肝;CYP2A5;小鼠

隨著社會(huì)經(jīng)濟(jì)的發(fā)展,非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)的患病率逐年增長(zhǎng),目前已成為危害人類健康的三大肝病之一.同時(shí),NAFLD在雞、狗、豬、牛、羊以及魚(yú)等動(dòng)物也可發(fā)生,是各種動(dòng)物的原發(fā)或者繼發(fā)性疾病.在各種動(dòng)物中,雞、鴨的發(fā)病率較高,給禽類養(yǎng)殖業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失.隨著患病的畜禽進(jìn)入食品消費(fèi)市場(chǎng),又給食品安全帶來(lái)隱患,故而NAFLD不僅是臨床醫(yī)學(xué)、社會(huì)健康問(wèn)題還是食品安全問(wèn)題,研究NAFLD的發(fā)病機(jī)制、疾病的有效預(yù)防和治療均具有重要意義.細(xì)胞色素P450(cytochrome P450,CYP450)是生物體內(nèi)的Ⅰ相代謝酶,在藥物、致癌物、環(huán)境毒物等外源物和內(nèi)源性物質(zhì)的代謝中起重要的作用.CYP2A5在香豆素、尼古丁代謝及外源性藥物和致癌物如黃曲霉毒素、亞硝胺等的激活過(guò)程中發(fā)揮了重要的作用[1-2].由于CYP2A5在肝炎損傷以及不同致癌物的代謝過(guò)程中具有獨(dú)特的上調(diào)特性而被廣泛關(guān)注.許多研究已經(jīng)表明,鼠肝細(xì)胞瘤以及瘤前肝細(xì)胞損傷時(shí)CYP2A5的表達(dá)均顯著升高,因此有學(xué)者提出將CYP2A5作為肝瘤形成的標(biāo)志物[3-6].目前,關(guān)于非酒精性脂肪肝對(duì)(NAFLD)CYP2A5表達(dá)的影響,以及非酒精性脂肪肝對(duì)CYP2A5是否有協(xié)同作用等均未見(jiàn)報(bào)道.因此,本試驗(yàn)旨在研究高脂誘導(dǎo)非酒精性脂肪肝對(duì)CYP2A5表達(dá)的影響.為預(yù)防肝臟相關(guān)性疾病和癌癥,指導(dǎo)臨床合理用藥提供依據(jù).

1　材料與方法

1.1試驗(yàn)動(dòng)物的分組與處理雄性C57BL/6J小鼠18只,由中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所提供,隨機(jī)分為陰性對(duì)照組、高脂組、陽(yáng)性對(duì)照組,適應(yīng)性喂養(yǎng)1周后,進(jìn)入正式試驗(yàn).陰性對(duì)照組飼喂基礎(chǔ)飼料,高脂組飼喂高脂飼料,高脂飼料的配方由前期試驗(yàn)確定(李曉沖等)[7],陽(yáng)性對(duì)照組飼喂基礎(chǔ)飼料在處死前1 d給予吡唑誘導(dǎo)(腹腔注射,150 mg/kg體重),連續(xù)飼喂8周后斷頸處死,取出各試驗(yàn)組小鼠肝臟,立即用生理鹽水沖洗,裝到樣品收集管中暫時(shí)用液氮冷凍-80℃保存.

1.2CYP2A5 mRNA含量的測(cè)定肝臟RNA提取嚴(yán)格按照Gibco TRIZol RNA提取試劑盒說(shuō)明書(shū)操作.CYP2A5引物序列:上游5′-GGACAA-AGAGT TCCTGTCACTGCTTC-3′,下游5′-GTGTTCCACTTT CTTGGTTATGAAGTCC-3′;GADPH引物序列:上游5′-ACAGTCCATGCCATCACTGCC-3′,下游5′-GCCTGCTTCACCACCTTCTTG-3′.引物由北京諾賽生物科技有限公司合成.RT-PCR擴(kuò)增反應(yīng)體系如下:下游引物各0.25 μL,滅菌注射用水7.5 μL,RT-PCR Mix 10 μL,cDNA 2 μL,反應(yīng)總體系20 μL.反應(yīng)條件:94℃預(yù)變性4 min;然后94℃變性30 s,57℃退火30 s,72℃延伸30 s,共30個(gè)循環(huán);72℃終延伸5 min.擴(kuò)增產(chǎn)物用實(shí)時(shí)定量PCR儀Bio-rad IQ5軟件進(jìn)行分析.

1.3CYP2A5蛋白表達(dá)的測(cè)定采用牛血清白蛋白標(biāo)準(zhǔn)品(5 mg/mL BSA)根據(jù)說(shuō)明書(shū)測(cè)定肝微粒體蛋白含量,以緩沖液稀釋,使代測(cè)蛋白樣品終濃度相同,等體積加入2XSDS上樣緩沖液,混合后沸水水浴10 min,冰上冷卻,取樣品10 μg進(jìn)行Western-Blot. CYP2A5首抗用雞抗鼠多克隆抗體(University of Kuopio,Kuopio,Finland),1∶2 000稀釋,二抗用羊抗雞多克隆抗體,1∶2 000稀釋,β-actin作為內(nèi)參.

1.4CYP2A5酶活性的測(cè)定取肝微粒體蛋白0.25 mg,加入50 mol/L的香豆素5 μL,用預(yù)溫(37℃)的50 mmol/L磷酸鉀緩沖液(pH值7.4)調(diào)整懸浮液體積為2.45 mL,裝入37℃預(yù)溫的熒光比色杯中,放入37℃振動(dòng)水浴,用預(yù)溫的輔助液(每毫升含NADP 6.7 mg,葡萄糖-6-磷酸鹽31.0 mg,葡萄糖-6-磷酸脫氫酶24 μL)50 μL啟動(dòng)反應(yīng),10 min后用10%三氯乙酸終止反應(yīng).用960型熒光分光光度計(jì),以355 nm為激發(fā)波長(zhǎng),460 nm為發(fā)射波長(zhǎng)(狹縫5 nm)測(cè)其熒光值,用0.1 mmol/L 7-羥基香豆素做內(nèi)標(biāo),計(jì)算CYP2A5的比活性(nmol/mg),每個(gè)樣品重復(fù)測(cè)定3次.

1.5數(shù)據(jù)分析試驗(yàn)數(shù)據(jù)采用SA9.0軟件中oneway ANOVA及體檢進(jìn)行分析.結(jié)果采用Fig.P曲線擬合軟件繪制柱狀圖和曲線圖.

2　結(jié)果

2.1高脂誘導(dǎo)后CYP2A5 mRNA表達(dá)的變化高脂誘導(dǎo)后小鼠肝臟中CYP2A5基因mRNA表達(dá)結(jié)果如圖1所示.由圖1可見(jiàn),與對(duì)照組相比,高脂組(NAFLD)和陽(yáng)性對(duì)照組(PYR+)分別極顯著提高CYP2A5基因mRNA表達(dá)量69%(P<0.01)和229%(P<0.01),說(shuō)明高脂(NAFLD)具有提高小鼠肝臟中CYP2A5mRNA表達(dá)的能力,但沒(méi)有達(dá)到陽(yáng)性對(duì)照組的程度.

圖1　高脂對(duì)CYP2A5 mRNA表達(dá)的影響

2.2高脂誘導(dǎo)后CYP2A5蛋白表達(dá)的變化高脂誘導(dǎo)后小鼠肝臟中CYP2A5蛋白表達(dá)結(jié)果如圖2所示.由圖2可見(jiàn),與對(duì)照組(Control)相比,高脂組(NAFLD)與陽(yáng)性對(duì)照組(PYR+)小鼠肝臟中CYP2A5蛋白表達(dá)量,分別增加34%(P<0.01)和80%(P<0.01),均極顯著提高.

2.3高脂誘導(dǎo)后CYP2A5酶活性的變化高脂誘導(dǎo)后小鼠肝臟中CYP2A5酶活性變化結(jié)果如圖3所示.由圖3可見(jiàn),與對(duì)照組(Control)相比,高脂組(NAFLD)與陽(yáng)性對(duì)照組(PYR+)小鼠肝臟中CYP2A5酶活性分別增加43%(P<0.01)和151% (P<0.01),均極顯著差異.

3　討論

非酒精性脂肪肝(NAFLD)是一類除外酒精和其他明確的損肝因素所致的,以彌漫性肝細(xì)胞大泡性脂肪變?yōu)橹饕卣鞯呐R床病理綜合征[8].近年來(lái),隨著肥胖和糖尿病的高發(fā),NAFLD的發(fā)病率逐年升高,并被認(rèn)為是隱原性肝硬化的主要原因之一,是僅次于病毒性肝炎的常見(jiàn)肝病[9]. NAFLD目前被認(rèn)為是可以導(dǎo)致終末期肝病的一種疾病狀態(tài),此外,非酒精性脂肪肝還與心血管疾病[10]、梗阻性呼吸暫停[11]具有密切的相關(guān)性.脂肪肝也是動(dòng)物的原發(fā)性或繼發(fā)性疾病,給養(yǎng)殖業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失.

圖2　高脂對(duì)CYP2A5蛋白表達(dá)的影響

圖3　高脂對(duì)CYP2A5酶活性的影響

肝損傷主要發(fā)生在疾病的早期,隨著病程的不斷的發(fā)展成為肝硬化或是肝癌,而CYP2A5的誘導(dǎo)作用發(fā)生在肝細(xì)胞損傷早期,CYP2A5表達(dá)作用增加明顯.本試驗(yàn)采用高脂飼料喂養(yǎng)誘導(dǎo)非酒精性脂肪肝模型小鼠,結(jié)果顯示,CYP2A5 mRNA表達(dá)量增加,Western Blotting分析蛋白表達(dá)大量增加,與對(duì)照組相比明顯增加.CYP2A5活性明顯增高,三者變化相一致.這可能取決于機(jī)體內(nèi)脂質(zhì)內(nèi)環(huán)境穩(wěn)態(tài)或是肝臟脂肪酸組成的特殊變化,高脂飲食可增加線粒體和微粒體對(duì)脂肪酸的氧化而導(dǎo)致氧化應(yīng)激,通過(guò)肝微粒體CYP450酶系表達(dá)及調(diào)控使活性氧增多(ROS),后者與多價(jià)不飽和脂肪酸發(fā)生脂質(zhì)過(guò)氧化反應(yīng)[12],生成過(guò)氧化脂質(zhì)(LPO),LPO不僅使內(nèi)源性ROS增脂肪酸發(fā)生脂質(zhì)過(guò)氧化反應(yīng),LPO不僅使內(nèi)源性ROS增加、毒性增強(qiáng),且可抑制抗氧化系的保護(hù)作用,導(dǎo)致肝損害.CYP2A5是氧化應(yīng)激在損傷肝部的特定區(qū)域催化外源物代謝的主要CYP酶[13].我們推測(cè)非酒精性脂肪肝微粒體CYP2A5表達(dá)增強(qiáng)與脂質(zhì)過(guò)氧化和氧自由基的增加有關(guān),體內(nèi)脂質(zhì)的不斷積累將損害肝細(xì)胞結(jié)構(gòu)和功能,從而引起CYP2A5的改變.本研究說(shuō)明,非酒精性脂肪肝小鼠肝臟CYP2A5表達(dá)的增強(qiáng)與非酒精性脂肪肝引起的肝臟損害密切相關(guān),CYP2A5參與了非酒精性脂肪肝的發(fā)生發(fā)展.

目前,肝損傷尤其是非酒精性脂肪肝已成為全球共同關(guān)注的社會(huì)健康問(wèn)題,食譜、環(huán)境或疾病等均會(huì)影響CYP2A5的表達(dá),對(duì)該酶的調(diào)節(jié)將會(huì)直接影響煙癮、吸煙行為、外源物的致癌作用及藥物的療效.關(guān)于非酒精性脂肪肝損傷時(shí)CYP2A5的誘導(dǎo)與表達(dá)的機(jī)制尚不清楚.

4　結(jié)論

本試驗(yàn)發(fā)現(xiàn),高脂飼料誘導(dǎo)下,小鼠非酒精性脂肪肝從mRNA、蛋白質(zhì)及酶活性水平3個(gè)方面均能顯著增強(qiáng)CYP2A5的表達(dá).

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The expression of CYP2A5 in nonalcoholic fatty liver disease induced by high-fat diet

CUI Yi-zhe1,WANG Qiu-ju1,LI Yue1,ZHOU Ya-qiang1,SU Jing2
(1.College of Animal science and Veterinary,Heilongjiang Bayi Agricultural University,Daqing 163319,China; 2.Animal Diseases Prevention and Control center in Heilongjiang province,Harbin 150069,China)

This study was conducted to examine the effect of an experimental mouse model of NAFLD induced by high-fat feed on hepatic Cyp2A5 expression in vivo.Eighteen male C57BL/6J mice were randomly selected and divided into control group, high fat group and positive control group,and each group had 6 rats.Rats were fed with standard diet or high-fat diet.The positive control was fed with standard feed on the day before the rats were killed to pyrazole.At 8 weeks later,mice were executed,and CYP2A5 expression and enzyme activity were analyzed.The results showed that Pyrazole treatment resulted in statistically signifi?cant increases in levels of CYP2A5 mRNA,protein and catalytic activity by 229,80 and 151%,respectively(P<0.01).In high fattreated livers,CYP2A5 expression was significantly increased compared to that of controls at the mRNA(69%),protein(34%), and activity(43%)levels(P<0.01).These findings show that high fat diet induced non-alcoholic fatty liver disease may increase the expression of CYP2A5 mRNA,protein level and enzyme activity.

non-alcoholic fatty liver disease;cytochrome P450 2A5;mouse

WANG Qiu-ju

R 575.5

A

0529-6005(2016)02-0096-03

2014-10-20

黑龍江八一農(nóng)墾大學(xué)校培育科研項(xiàng)目(X2R2014-05)

崔一喆(1979-),男,講師,博士,從事獸醫(yī)藥理學(xué)與毒理學(xué)研究,E-mail:cyz_79@yeah.net

王秋菊,E-mail:wqj_9@126.com