對(duì)乙酰氨基酚誘導(dǎo)的急性肝損傷和肝衰竭模型中線粒體基因組轉(zhuǎn)錄改變

明雅南　李春敏　張靜怡　劉曉琳　茅益民

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·論著·

對(duì)乙酰氨基酚誘導(dǎo)的急性肝損傷和肝衰竭模型中線粒體基因組轉(zhuǎn)錄改變

明雅南李春敏張靜怡劉曉琳茅益民

200001上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院消化內(nèi)科，上海市消化疾病研究所

【摘要】目的通過(guò)探索對(duì)乙酰氨基酚(APAP)誘導(dǎo)的急性肝損傷和急性肝衰竭模型中線粒體基因組轉(zhuǎn)錄水平的改變與疾病進(jìn)展之間的關(guān)聯(lián)，為AILI預(yù)后的預(yù)測(cè)提供新的生物標(biāo)記。方法將90只小鼠隨機(jī)分為3組：對(duì)照組、APAP導(dǎo)致的DILI組(AILI，300 mg/kg)和APAP導(dǎo)致的急性肝衰竭組(AILF，750 mg/kg)。禁食16 h后，腹腔注射體積相近的0.9%氯化鈉溶液和不同劑量APAP，在0、1、3、6、12 h等不同時(shí)間點(diǎn)時(shí)，每組隨機(jī)選取6只小鼠處死，留取小鼠的血漿和肝臟組織。檢測(cè)每只小鼠ALT、AST、ROS變化水平; 提取肝臟總RNA，采用RT-PCR技術(shù)檢測(cè)線粒體基因組基因的轉(zhuǎn)錄水平。結(jié)果與對(duì)照組相比，腹腔注射APAP后，兩組均可見(jiàn)轉(zhuǎn)氨酶明顯升高，AILI組在6-12 h時(shí)ALT達(dá)到峰值(5 000～10 000 U/L)；AILF組12 h時(shí)ALT水平超過(guò)10 000 U/L，明顯高于AILI組(P<0.05)。APAP處理后，兩組小鼠均可見(jiàn)典型的3區(qū)微泡性脂肪變，AILF組可見(jiàn)典型的急性肝衰竭的大塊性壞死。與對(duì)照組相比，AILI和AILF組肝臟中ROS從1 h開(kāi)始，各時(shí)間點(diǎn)均可見(jiàn)顯著升高，且1 h時(shí)，AILF組ROS的生成量約為AILI組的2.5倍(P<0.05)。AILI組COX1在6 h時(shí)顯著升高(P<0.05)，而對(duì)照組和AILF組均未見(jiàn)顯著升高。與對(duì)照組相比，3 h時(shí)，AILI組和AILF組可見(jiàn)CYTB、COX2和ATP8顯著降低(P<0.05)；6 h時(shí)，AILI組和AILF組COX1、ND1、ND5和ATP8轉(zhuǎn)錄水平顯著降低(P<0.05)；12 h時(shí)，AILI組和AILF組中NADH各亞基均可見(jiàn)顯著降低(P<0.05)。AILI組與AILF組相比， 6 h時(shí)，AILF組中 ATP6的轉(zhuǎn)錄水平顯著低于AILI組和對(duì)照組(P<0.05)；12 h時(shí)，AILF組的肝臟中CYTB、COX2、ATP8以及NADH的2、3、5和6亞基的轉(zhuǎn)錄水平顯著低于AILI組(P<0.05)。結(jié)論線粒體基因組除COX1在AILI組明顯上調(diào)，其他存在明顯改變的基因均出現(xiàn)降低趨勢(shì),且AILF組變化更明顯。線粒體基因組轉(zhuǎn)錄水平改變對(duì)于預(yù)測(cè)AILI預(yù)后有潛在價(jià)值。

【關(guān)鍵詞】對(duì)乙酰氨基酚；肝損傷；肝衰竭；線粒體；轉(zhuǎn)錄

藥物的肝臟毒性是新藥研發(fā)失敗及上市后被撤回最主要的原因之一，盡管藥物導(dǎo)致肝損傷的機(jī)制復(fù)雜，但線粒體功能損傷是其的重要環(huán)節(jié)[1，2]。線粒體基因組(mtDNA)位于線粒體內(nèi)膜上的環(huán)狀DNA，是可編碼13種合成線粒體呼吸鏈和氧化磷酸化功能蛋白必備的RNA、rRNA和tRNA，但由于缺少組蛋白保護(hù)，而且相對(duì)于細(xì)胞核DNA對(duì)復(fù)制和轉(zhuǎn)錄過(guò)程中出現(xiàn)各種錯(cuò)誤后完善的修復(fù)機(jī)制，線粒體DNA的修復(fù)機(jī)制并不完善，導(dǎo)致其更易受到氧化應(yīng)激產(chǎn)物(ROS)的損傷[3-5]，影響線粒體氧化磷酸化功能，進(jìn)而影響能量生成。有研究提示，血漿中線粒體生物標(biāo)記物明顯升高與預(yù)后不良明顯相關(guān)，線粒體結(jié)構(gòu)和功能?chē)?yán)重受損者，存活率更低[6]。

對(duì)乙酰氨基酚(APAP)是最常見(jiàn)的固有型藥物性肝損傷(DILI)的代表藥物，APAP過(guò)量應(yīng)用已成為西方國(guó)家急性肝衰的重要原因之一[7]。P450酶代謝產(chǎn)生的毒性產(chǎn)物N-乙酰對(duì)苯醌亞胺(NAPQI)造成線粒體結(jié)構(gòu)和功能的損傷，是其主要的發(fā)病機(jī)制。肝內(nèi)過(guò)多的NAPQI能夠耗竭肝內(nèi)解毒NAPQI的谷胱甘肽(GSH)，并損害線粒體復(fù)合體Ⅱ和Ⅲ的酶活性[8-9]，最終導(dǎo)致線粒體功能受損，產(chǎn)生大量的ROS。本研究通過(guò)構(gòu)建APAP導(dǎo)致的DILI動(dòng)物模型(AILI)和急性肝衰竭動(dòng)物模型(AILF)，采用實(shí)時(shí)PCR技術(shù)對(duì)兩種具有不同預(yù)后的動(dòng)物模型在不同時(shí)間點(diǎn)mtDNA的轉(zhuǎn)錄水平進(jìn)行分析，探索mtDNA在DILI的發(fā)生發(fā)展過(guò)程中的變化特點(diǎn)，以發(fā)現(xiàn)可以預(yù)測(cè)疾病預(yù)后的潛在生物標(biāo)記。

資料和方法

一、實(shí)驗(yàn)動(dòng)物與試劑儀器

90只C57BL/6小鼠購(gòu)于上海斯萊克動(dòng)物實(shí)驗(yàn)中心；APAP、戊巴比妥鈉購(gòu)于sigma公司(美國(guó))；全自動(dòng)生化分析儀(SIEMEN SADVIA 1800，美國(guó))；全自動(dòng)脫水機(jī)(ASP300)、石蠟包埋機(jī)(EG1150C)、脫蠟機(jī)(Auto tainer XL)等均來(lái)自于德國(guó)Leica公司；Bio-Rad CFX96熒光定量PCR儀(美國(guó))；Trizol、Prime Script TM反轉(zhuǎn)錄試劑盒、SYBR@Premix Ex TaqTM II購(gòu)于 Takara公司(日本)；PCR引物購(gòu)于上海生工有限公司。

90只小鼠，隨機(jī)分為對(duì)照組、AILI組和AILF組，每組30只；腹腔分別注射體積相近的0.9%氯化鈉溶液、APAP-300 mg/kg、APAP-750 mg/kg。處理后的動(dòng)物在0、1、3、6、12 h，每組隨機(jī)抽出6只小鼠，麻醉，取血，留取肝臟組織。提取肝臟總RNA，檢測(cè)AILI組和AILF組小鼠肝臟組織ROS 的生成量和mtDNA轉(zhuǎn)錄水平，觀察其隨時(shí)間變化的特點(diǎn)。

二、血漿轉(zhuǎn)氨酶檢測(cè)

戊巴比妥鈉麻醉小鼠，抗凝管眼球取血，取血后，3000×g，4℃離心，取上清液，稀釋10倍，檢測(cè)小鼠的ALT和AST。

三、ROS檢測(cè)

新鮮的肝臟100 mg，用組織清洗液清洗后，用勻漿器制成肝臟勻漿，按照試劑盒說(shuō)明檢測(cè)肝臟勻漿中ROS的產(chǎn)生量，并采用BCA的方法檢測(cè)肝臟勻漿的蛋白濃度，校正肝臟內(nèi)ROS的產(chǎn)量。

四、實(shí)時(shí)PCR檢測(cè)線粒體RNA

TRIZOL試劑盒提取組織總RNA。NanoDrop ND-1000測(cè)定RNA的濃度、質(zhì)量和純度，A260/A280>1.8作為RNA合格的標(biāo)準(zhǔn)，根據(jù) PrimeScript反轉(zhuǎn)錄試劑盒合成cDNA，然后按照SYBR@Premix Ex Taq II說(shuō)明擴(kuò)增線粒體基因，mRNA水平以18S作為內(nèi)參對(duì)照，結(jié)果以相對(duì)含量表示不同基因的轉(zhuǎn)錄水平。

五、統(tǒng)計(jì)學(xué)分析

統(tǒng)計(jì)分析應(yīng)用SPSS 19.0軟件，作圖應(yīng)用GraphPad Prime 6.01軟件。計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差表示，組間比較采用t檢驗(yàn)，P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié)果

一、動(dòng)物模型建立

與對(duì)照組相比，腹腔注射APAP后，AILI組和AILF組均可見(jiàn)轉(zhuǎn)氨酶明顯升高，AILI組在6～12 h時(shí)ALT達(dá)到高峰(5 000～10 000 U/L)；AILF組12 h時(shí)ALT水平超過(guò)10 000 U/L，明顯高于AILI組(P<0.05)。見(jiàn)圖1。12 h時(shí)，AILF組肝臟組織學(xué)可見(jiàn)典型的急性肝衰竭表現(xiàn)-大塊性肝壞死，病變周?chē)?xì)胞也表現(xiàn)為明顯的彌漫性的肝細(xì)胞變性；AILI組損傷范圍局限，損傷周?chē)母渭?xì)胞形態(tài)結(jié)構(gòu)正常。上述組織學(xué)特點(diǎn)提示，APAP誘導(dǎo)的AILI和AILF造模成功。

二、ROS的表達(dá)變化

與對(duì)照組相比，APAP處理后1 h，AILI組和AILF組可見(jiàn)肝內(nèi)ROS生成均明顯增多，且AILF組約為AILI組2.5倍(P<0.05)，兩組肝內(nèi)ROS均隨時(shí)間延長(zhǎng)，含量越高(P<0.05)。見(jiàn)表1。

三、線粒體基因組轉(zhuǎn)錄水平變化

各組mtDNA檢測(cè)結(jié)果顯示AILI組COX1在6 h時(shí)顯著升高(P<0.05)，而對(duì)照組和AILF組均未見(jiàn)顯著升高。與對(duì)照組相比，3 h時(shí)，AILI組和AILF組可見(jiàn)CYTB、COX2和ATP8顯著降低(P<0.05)；6 h時(shí)，AILI組和AILF組COX1、ND1、ND5和ATP8轉(zhuǎn)錄水平顯著降低(P<0.05)；12 h時(shí)，AILI組和AILF組中NADH各亞基均可見(jiàn)顯著降低(P<0.05)。AILI組與AILF組相比， 6 h時(shí)，AILI組COX1顯著高于AILF組(P<0.05)，AILF組中ATP6的轉(zhuǎn)錄水平顯著低于AILI組和對(duì)照組(P<0.05)；12 h時(shí)，AILF組的肝臟中CYTB、COX2、ATP8以及NADH的2、3、5和6亞基的轉(zhuǎn)錄水平顯著低于AILI組(P<0.05)。見(jiàn)表2，表3。

注：AILI組和AILF組與對(duì)照組比較*P<0.05；AILI組和AILF組比較§P<0.05。

組別0h1h3h6h12h對(duì)照組927.02±183.471013.68±23.37993.68±141.48936.68±128.03977.35±42.11AILI組1060.35±162.721739.15±655.62*3519.14±806.51*4069.00±1046.64*6371.32±1910.92*AILF組993.68±262.374518.59±913.71*§4061.37±422.22*5015.99±654.29*5555.80±977.36*

討論

AILI發(fā)生過(guò)程中，線粒體氧化應(yīng)激并生成ROS[10]，損傷線粒體蛋白和DNA，并導(dǎo)致線粒體膜通透性轉(zhuǎn)變孔(mitochondrial membrane permeability transition，MPT)開(kāi)放，MPT的發(fā)生最終導(dǎo)致線粒體膜電位改變、ATP生成中斷，胞內(nèi)離子穩(wěn)態(tài)平衡被打破，肝細(xì)胞腫脹壞死。因此，線粒體結(jié)構(gòu)和功能損傷是DILI早期重要的發(fā)病機(jī)制[11,12]。mtDNA是線粒體內(nèi)重要的組成成分，能夠編碼表達(dá)13種呼吸鏈復(fù)合體的必需蛋白，其數(shù)量減少和功能改變可能直接影響肝細(xì)胞代謝狀態(tài)及存活[13]。

表2　不同組別各時(shí)間點(diǎn)NADH亞基中線粒體編碼基因的轉(zhuǎn)錄水平

注：*：與對(duì)照組比較，P<0.05；§：與AILI組比較，P<0.05

表3　CYTB、COX以及ATP等線粒體編碼基因的轉(zhuǎn)錄水平

注：*：與對(duì)照組比較，P<0.05；§：與AILI組比較，P<0.05

本研究結(jié)果顯示，腹腔注射不同劑量APAP后，早期即可見(jiàn)ROS明顯升高，且衰竭組明顯高于損傷組。線粒體是ROS產(chǎn)生的主要細(xì)胞器，mtDNA由于無(wú)組蛋白保護(hù)，而且相對(duì)于細(xì)胞核DNA對(duì)復(fù)制和轉(zhuǎn)錄過(guò)程中出現(xiàn)各種錯(cuò)誤后完善的修復(fù)機(jī)制，線粒體DNA的修復(fù)機(jī)制并不完善，過(guò)量ROS使其更易受損傷。Suliman等[14]的研究發(fā)現(xiàn)，氧化應(yīng)激可能是mtDNA損傷的原因，嚴(yán)重的mtDNA損傷，會(huì)進(jìn)一步減少ATP的生成，并增加ROS在肝內(nèi)的累積，因此，本研究中觀察到的AILI和AILF組中ROS明顯增加，與既往的研究報(bào)道一致。

從mtDNA的轉(zhuǎn)錄水平變化來(lái)看，除AILI組COX1的轉(zhuǎn)錄升高，其他差異表達(dá)的mtDNA的轉(zhuǎn)錄水平均表現(xiàn)為降低趨勢(shì)，并隨時(shí)間延長(zhǎng)更為明顯。本研究中發(fā)現(xiàn)肝臟組織中mtDNA轉(zhuǎn)錄水平明顯降低可能與預(yù)后不良密切相關(guān)，推測(cè)急性肝衰竭時(shí)肝臟組織中mtDNA大量釋放入血后導(dǎo)致的血漿mtDNA明顯升高，可以作為預(yù)測(cè)預(yù)后的潛在生物標(biāo)記，循環(huán)中高水平的mtDNA與AILI患者預(yù)后不良密切相關(guān)。

有研究發(fā)現(xiàn)，mtDNA降低20%～40%就會(huì)直接影響線粒體功能，持續(xù)mtDNA減少能夠降低線粒體呼吸鏈NAD+ FAD的再生功能，最終影響脂質(zhì)和丙酮酸鹽氧化并產(chǎn)生大量的ROS[15,16]。

長(zhǎng)期高脂飲食以及急性酒精性肝損傷模型均出現(xiàn)mtDNA減少和損傷[17,18]。ROS對(duì)mtDNA的直接損傷導(dǎo)致正常mtDNA發(fā)生突變，以及細(xì)胞膜裂解后將mtDNA釋放到血液循環(huán)可能是導(dǎo)致AILI動(dòng)物模型中mtDNA減少的重要因素。釋放的mtDNA可作為配體活化TLR9，引起NASH肝臟的炎性反應(yīng)[19]。另外有研究認(rèn)為，mtDNA作為損傷相關(guān)模式分子激活DILI的無(wú)菌性炎性反應(yīng)[20]。從上述針對(duì)其他肝病的證據(jù)來(lái)看，mtDNA的減少不僅是疾病發(fā)生早期重要的機(jī)制，也可能是導(dǎo)致疾病進(jìn)展的關(guān)鍵因素之一。相反，通過(guò)藥物改善肝臟內(nèi)mtDNA的水平可能阻斷毒物引起的肝衰竭[21]。

本研究?jī)H對(duì)AILI和AILF發(fā)生過(guò)程中mtDNA的轉(zhuǎn)錄水平改變進(jìn)行相應(yīng)研究，并未對(duì)其原因進(jìn)行深入的探討，而mtDNA轉(zhuǎn)錄降低是由于數(shù)量的減少還是刺激導(dǎo)致其異常突變，還需要通過(guò)更多的實(shí)驗(yàn)提供證據(jù)。此外，肝臟組織中mtDNA轉(zhuǎn)錄水平降低的程度對(duì)于預(yù)測(cè)疾病預(yù)后具有潛在的價(jià)值，但尚需要更多研究加以驗(yàn)證。COX1在AILI組的變化趨勢(shì)與其他mtDNA明顯不一致，可能與線粒體及肝細(xì)胞的適應(yīng)性反應(yīng)有關(guān)，對(duì)于COX1在不同疾病和生理過(guò)程中的作用和意義還需進(jìn)一步的實(shí)驗(yàn)證實(shí)。

本研究描述了AILI和AILF發(fā)生和發(fā)展過(guò)程中mtDNA轉(zhuǎn)錄水平隨時(shí)間變化的特點(diǎn)，結(jié)果提示，mtDNA轉(zhuǎn)錄水平變化差異可能具有預(yù)測(cè)預(yù)后的價(jià)值，但其轉(zhuǎn)錄改變機(jī)制還需要更多的基礎(chǔ)和臨床研究加以證實(shí)。

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(本文編輯：錢(qián)燕)

基金項(xiàng)目：“十二五”科技重大專(zhuān)項(xiàng)(2012ZX09303-001，2012ZX09401004)

通信作者：茅益民，Email：maoym11968@163.com

Corresponding author:MAO Yi-min, Email: maoym11968@163.com

(收稿日期：2016-04-13)

The transcriptional change of mitochondrial genome in mice models for acetaminophen-induced acute liver injury and failure

MINGYa-nan，LIChun-min，ZHANGJing-yi，LIUXiao-lin，MAOYi-Min.DivisionofGastroenterologyandHepatology,RenjiHospital,SchoolofMedicine,ShanghaiJiaoTongUniversity,ShanghaiInstituteofDigestiveDisease,Shanghai200001,China

【Abstract】ObjectiveTo explore the relationship between transcription level of mitochondrial DNA (mtDNA) and disease progression in the mice models of acetaminophen (APAP)- induced acute liver injury (AILI) and acute liver failure (ALF), and to find the relative new biomarkers for outcome. MethodsNinety mice were randomly divided into three groups, including control group, AILI group (300 mg/kg) and ALF group (750 mg/kg). After fasting 16 h, all mice were intraperitoneally injected with same volume of saline or different doses of APAP. At different time points of 0, 1, 3, 6 and 12 h, 6 mice randomly selected from each group were sacrificed for blood and liver, respectively. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and reactive oxygen (ROS) levels were detected, and liver total RNA was extracted for RT-PCR to detect changes in transcription of mitochondrial genome. ResultsCompared with the control group, ALT and AST levels in AILI and ALF group were significantly increased. In AILI group, ALT peaked at 6-12 h (5000-10000 IU/L), and ALT exceeded 10000 IU/L at 12 h in ALF group, which showed significantly difference (P<0.05). Microbubbles steatosis in three zones was observed in both AILI and ALF groups, and massive hepatic necrosis (MHN) were found merely in ALF group. In 1 h after APAP injection, ROS in ALF group was about 2.5 times as much as that in AILI group, which significantly increased at all time points in both group. Contrasting with control and ALIF groups, COX1 transcriptional level in AILI group increased significantly at 6 h. In AILI and ALF group, CYTB, COX2 and ATP8 reduced significantly at 3 h (P<0.05), COX1,ND1,ND5 and ATP8 significantly decreased at 6 h (P<0.05), and transcription level of other subunits of NADH significantly decreased at 12 h (P<0.05) comparing with those in control group. Furthermore, ATP6 at 6h in ALF group was obviously lower than that in AILI and control group (P<0.05). At 12 h, the majority of mtDNA (CYTB, COX2, ATP8, ND2, ND3, ND5 and ND6 ) had significant differences between AILI and ALF group. ConclusionThere are significant difference in mtDNA transcription between AILI and ALF groups. Besides COX1, other mtDNA showed significant decreases in AILI and ALF group compared with those in control group, especially in ALF group. mtDNA transcriptional changes may have great potential as biological makers to predict outcomes of AILI.

【Key words】APAP; Liver injury; Liver failure; Mitochondrial; Transcription