TLR介導(dǎo)的Nrf2抗氧化通路在對(duì)乙酰氨基酚藥物性肝損傷中的保護(hù)作用

顧佳毅　郁豐榮

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·論著·

TLR介導(dǎo)的Nrf2抗氧化通路在對(duì)乙酰氨基酚藥物性肝損傷中的保護(hù)作用

顧佳毅郁豐榮

目的研究?jī)?nèi)毒素(LPS)及Toll 樣受體(TLR)在對(duì)乙酰氨基酚(APAP)藥物性肝損傷中的保護(hù)作用及其相關(guān)機(jī)制。方法雄性 C57BL/6 小鼠40只，分為 4 組，每組 10 只?？瞻讓?duì)照組腹腔注射0.9%氯化鈉溶液，LPS組腹腔注射LPS 10 μg/kg，APAP組腹腔注射APAP 300 mg/kg，LPS+APAP組在APAP造模前16 h給予LPS 10 μg/kg預(yù)處理。通過比較各組血清 ALT 和 AST水平，并通過HE染色評(píng)價(jià)肝組織損傷程度，觀察LPS對(duì)小鼠肝損傷的保護(hù)作用。測(cè)定相應(yīng)時(shí)間點(diǎn)的肝臟組織丙二醛(MDA)、還原型谷胱甘肽(GSH)的變化以及肝組織DHE染色，評(píng)價(jià)小鼠氧化應(yīng)激水平。應(yīng)用Western印跡及RT-PCR檢測(cè)肝臟Nrf2，Gclc及HO-1的表達(dá)水平。結(jié)果LPS預(yù)處理可明顯減輕APAP所致的肝臟氧化應(yīng)激反應(yīng)及肝損傷程度。LPS預(yù)處理組的小鼠血清ALT(518.3±142.3對(duì)4542±498.4 U/L)、AST(643.3±105.6對(duì)5432.1±569.2 U/L)水平及肝組織MDA(78.0±14.5對(duì)141.7±26.4 mmoL/mg)水平與模型組相比明顯降低,而GSH (6.2±1.7對(duì)3.5±1.0 μmol/g)水平明顯升高(P<0.05),肝組織病理損傷明顯減輕。同時(shí)，LPS預(yù)處理可明顯促進(jìn)Nrf2及其下游抗氧化基因的表達(dá)。結(jié)論LPS在小鼠APAP肝損傷中起到保護(hù)作用，作用機(jī)制與Nrf2抗氧化通路的激活相關(guān)，可能成為藥物性肝損傷的新的治療策略。

對(duì)乙酰氨基酚；藥物性肝損傷；氧化應(yīng)激；Nrf2；HO-1

對(duì)乙酰氨基酚(acetaminophen，APAP)是一種解熱鎮(zhèn)痛藥，在治療劑量下，APAP具有較好的療效及安全性[1]。然而，APAP會(huì)導(dǎo)致部分患者肝損傷甚至急性肝功能衰竭。目前APAP肝損傷已取代病毒性肝炎成為歐美等發(fā)達(dá)國(guó)家急性肝衰竭的首要病因[1-3]。很多嚴(yán)重的APAP肝衰竭患者需要肝移植術(shù)才能得到救治[4-6]。研究認(rèn)為，APAP本身并不具有直接殺傷肝細(xì)胞的作用，APAP的肝細(xì)胞毒性主要是由其活性代謝產(chǎn)物導(dǎo)致的[7]。在常規(guī)劑量下，90%的APAP可以通過醛糖酸化和硫酸化作用后經(jīng)尿液排除[8]，10%的APAP經(jīng)過CYP450代謝轉(zhuǎn)化而產(chǎn)生高活性代謝產(chǎn)物NAPQI[9-13]。在正常情況下，肝細(xì)胞通過還原型谷胱甘肽(GSH)結(jié)合NAPQI轉(zhuǎn)化為無毒物質(zhì)[14]。但在GSH耗竭的情況下，如過量飲酒，饑餓，APAP過量或營(yíng)養(yǎng)不良等，NAPQI無法被有效中和而產(chǎn)生肝毒性[15,16]。NAPQI可造成細(xì)胞膜的脂質(zhì)過氧化, 并影響線粒體、內(nèi)質(zhì)網(wǎng)等細(xì)胞器的功能, 導(dǎo)致肝細(xì)胞氧化應(yīng)激和線粒體功能失調(diào)，從而致肝細(xì)胞損傷及壞死[17]?；钚匝踝杂苫? reactive oxygen species，ROS)是介導(dǎo)氧化應(yīng)激的主要物質(zhì)。研究證明，減少ROS產(chǎn)生是防治APAP肝損傷的關(guān)鍵，增強(qiáng)肝細(xì)胞抗氧化應(yīng)激的能力可有效避免APAP肝損傷[18-22]。

轉(zhuǎn)錄因子Nrf2是體內(nèi)抗氧化相關(guān)基因的直接調(diào)控因子，主要包括Gclc、HO-1、G6pdh等，而這些抗氧化基因則是體內(nèi)清除ROS的重要因子[23]。已報(bào)道有多種藥物可以通過激活Nrf2抗氧化通路對(duì)APAP所致肝損傷起保護(hù)作用[24,25]。

研究證明，LPS激活TLR后可促進(jìn)肝組織內(nèi)Nrf2及其下游基因的表達(dá)[26]。因此，推測(cè)LPS對(duì)APAP肝損傷的保護(hù)作用是通過激活Nrf2抗氧化通路實(shí)現(xiàn)的。

資料和方法

一、化學(xué)試劑

APAP 與 LPS 購自美國(guó) Sigma 公司;DHE購自上海碧云天公司；MDA和GSH檢測(cè)試劑盒購自南京建成生物工程研究所;Western 抗體購于美國(guó)abcam公司。動(dòng)物飼料購于上海斯萊克實(shí)驗(yàn)動(dòng)物有限公司。APAP溶于PBS中, 濃度為20 mg/mL, 55 ℃水浴鍋加熱至完全溶解, 現(xiàn)用現(xiàn)配;LPS溶于0.9%氯化鈉溶液，濃度為1 μg/mL。

二、實(shí)驗(yàn)動(dòng)物及分組

清潔級(jí)C57BL/6 雄性小鼠40只，8～10周齡, 體質(zhì)量20～25 g,購自上海斯萊克實(shí)驗(yàn)動(dòng)物有限公司。小鼠均于SPF環(huán)境下晝夜規(guī)律喂養(yǎng)，自由進(jìn)食、飲水。所有小鼠在APAP造模前進(jìn)食過夜，APAP造模均在早上9點(diǎn)進(jìn)行，LPS預(yù)處理在造模前16 h進(jìn)行。采用隨機(jī)數(shù)表法將小鼠分為4組，每組10只。①空白對(duì)照組：腹腔注射0.9%氯化鈉溶液(劑量同LPS組)，預(yù)處理；②APAP組：給予APAP腹腔注射，劑量為300 mg/kg；③LPS組：給予LPS腹腔注射(10 μg/kg)預(yù)處理；④LPS+APAP組：LPS處理后禁食16 h，給予APAP造模。所有小鼠在APAP造模后恢復(fù)禁食。

三、標(biāo)本收集

APAP造模后1 h，收集各組5只小鼠標(biāo)本用于檢測(cè)GSH和MDA。造模后6 h，收集各組5只小鼠的血清和肝組織標(biāo)本。用于檢測(cè)血清ALT, AST，組織病理損傷以及肝組織內(nèi)Nrf2的表達(dá)情況。

四、肝功能及肝組織檢測(cè)

血清ALT、AST 檢測(cè)采用全自動(dòng)生化分析儀(美國(guó)德靈公司)。

小鼠肝組織用體積分?jǐn)?shù)為0.04的甲醛固定, 脫水, 石蠟包埋, 切片(厚5 μm), HE染色, 于顯微鏡下觀察肝組織病理改變。

新鮮組織制作冰凍切片后，將二氫乙啶(DHE)終濃度稀釋至10 μmoL，將其滴在冰凍組織切片上，37 ℃避光孵育30 min。PBS清洗3遍，用熒光顯微鏡觀察DHE染色情況。紅色熒光的強(qiáng)度代表ROS量的多少。

五、RT-PCR

按照Trizol試劑盒操作提取組織總mRNA，并測(cè)定濃度。用TAKARA反轉(zhuǎn)錄試劑盒進(jìn)行反轉(zhuǎn)錄，20 μL體系。10 μL擴(kuò)增體系，LightCycler○R480 即時(shí)PCR系統(tǒng)進(jìn)行擴(kuò)展并測(cè)定熒光強(qiáng)度。

六、Western印跡

取肝臟組織適量，加入蛋白裂解液RIPA進(jìn)行蛋白提取，用BSA法定量，調(diào)整蛋白濃度后加入加樣緩沖液100 ℃加熱變性，進(jìn)行聚丙烯酰胺凝膠電泳分離及轉(zhuǎn)膜，5% BSA封閉液常溫封閉1 h，一抗4 ℃孵育過夜，用TBST液洗膜3次，每次5 min，熒光二抗37 ℃孵育1 h后洗膜10 min×4次，Odyssey 掃膜儀進(jìn)行圖像掃描分析。

七、統(tǒng)計(jì)學(xué)處理

結(jié)　　果

一、LPS預(yù)處理對(duì)APAP肝損傷的影響

與對(duì)照組相比，單純給予LPS處理對(duì)WT小鼠的轉(zhuǎn)氨酶水平無明顯影響(P>0.05)。而APAP組小鼠造模后 6 h 血清 ALT和AST較對(duì)照組明顯升高，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；與APAP組相比，LPS+APAP組的血清 ALT明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，見表1。HE染色顯示APAP可以引起小鼠肝組織大片的小葉中央型壞死，而LPS預(yù)處理可明顯減輕APAP的損傷程度，見圖1。

圖1　各組小鼠肝組織鏡下表現(xiàn)(HE，×200)

組別鼠數(shù)ALT(U/L)ALT(U/L) 對(duì)照545.0±11.945.1±11.8 APAP54542.0±498.45432.1±569.2 LPS546.7±13.347.8±14.2 LPS+APAP5518.3±142.3643.3±105.6*

注：*與APAP組相比，P<0.05

注：與空白對(duì)照組相比，*P<0.05；與APAP組相比，#P<0.05圖3　LPS預(yù)處理可促進(jìn)Nrf2抗氧化通路的激活

二、LPS對(duì)APAP肝毒性過程中氧化應(yīng)激反應(yīng)的影響

與對(duì)照組相比，APAP處理可引起小鼠肝組織內(nèi)MDA水平的明顯升高(P<0.05)，同時(shí)造成GSH水平的明顯降低(P<0.05)。而LPS預(yù)處理可明顯減少APAP損傷后的MDA水平(P<0.05)，同時(shí)減輕APAP對(duì)GSH的消耗(P<0.05)，見表2。DHE染色也是反映氧化應(yīng)激的重要指標(biāo)，如圖2所示，APAP組的熒光強(qiáng)度明顯高于對(duì)照組，而LPS預(yù)處理可明顯減輕這一趨勢(shì)。

圖2　各組小鼠肝組織免疫熒光染色結(jié)果

組別鼠數(shù)MDA(mmoL/mg)GSH(μmoL/g) 對(duì)照551.7±12.910.8±2.6 APAP5141.7±26.43.5±1.0 LPS553.3±14.011.7±3.0 LPS+APAP578.0±14.56.2±1.7*

注：*與APAP組相比，P<0.05

三、LPS對(duì)Nrf2抗氧化通路的影響

與對(duì)照組相比, APAP處理組小鼠肝組織內(nèi)Nrf2的蛋白表達(dá)量無明顯變化，而給予LPS預(yù)處理后，無論是否給予APAP造模，肝組織內(nèi)Nrf2的水平均明顯升高(P<0.05)。RT-PCR結(jié)果顯示，Nrf2的重要的下游抗氧化基因Gclc和HO-1均可被LPS明顯激活，見圖3。

討　　論

APAP是臨床常用藥, 過量使用易導(dǎo)致急性藥物性肝損傷, 患者預(yù)后較差。因此探究APAP的肝毒性機(jī)制從而研究防治策略是非常有必要的。已有大量研究證實(shí)，氧化應(yīng)激反應(yīng)是APAP肝毒性過程中的最重要機(jī)制，大量的ROS生成，可損傷線粒體、內(nèi)質(zhì)網(wǎng)等重要細(xì)胞器的功能，進(jìn)而導(dǎo)致細(xì)胞壞死。本研究也發(fā)現(xiàn)，APAP處理的小鼠肝組織內(nèi)氧化應(yīng)激反應(yīng)及ROS水平明顯增加。因此，抗氧化應(yīng)激是防治APAP肝損傷的關(guān)鍵之一。而Nrf2作為一種重要的核轉(zhuǎn)錄因子，可以調(diào)控一系列抗氧化基因的表達(dá)。Nrf2抗氧化通路已成為APAP肝損傷領(lǐng)域的研究熱點(diǎn)和重點(diǎn)[27,28]。

TLR是一種病原體識(shí)別受體，可以識(shí)別病原相關(guān)分子模式，通過刺激細(xì)胞內(nèi)炎性信號(hào)通路刺激炎性因子的合成和釋放，繼而促進(jìn)炎性反應(yīng)的發(fā)生[29]。LPS是革蘭陰性桿菌細(xì)胞壁的主要組分之一[30]。LPS是TLR的自然配體，該受體的激活可對(duì)多種信號(hào)通路產(chǎn)生重要影響。TLR在多種肝臟疾病中均扮演重要角色，包括缺血再灌注損傷、非酒精性脂肪性肝病、酒精性肝病、自免肝損傷等[31-34]。研究發(fā)現(xiàn)，苯甲醇可以通過激活TLR4對(duì)APAP肝損傷起保護(hù)作用[35]。盡管LPS預(yù)處理對(duì)APAP肝損傷的保護(hù)作用已有相關(guān)報(bào)道，但具體機(jī)制尚不明確。本研究發(fā)現(xiàn)，LPS激活TLRs/Nrf2抗氧化通路，具有減輕氧化應(yīng)激損傷的作用。

低劑量LPS預(yù)處理可明顯減輕APAP引起的肝組織壞死。進(jìn)一步研究發(fā)現(xiàn)，LPS可明顯增強(qiáng)Nrf2在小鼠肝組織內(nèi)的表達(dá)，同時(shí)其代表性的抗氧化基因Gclc及HO-1的mRNA表達(dá)水平也明顯升高，提示低劑量LPS可以誘導(dǎo)Nrf2抗氧化通路的表達(dá)。同時(shí)驗(yàn)證了LPS對(duì)APAP肝毒性過程中的氧化應(yīng)激反應(yīng)的影響。MDA是檢測(cè)動(dòng)物體內(nèi)氧化應(yīng)激水平和脂質(zhì)過氧化反應(yīng)的良好指標(biāo)，在多種氧化應(yīng)激相關(guān)的肝損傷過程中MDA均會(huì)明顯升高，而GSH是體內(nèi)最重要的抗氧化物質(zhì)，也是中和APAP毒性的主要解毒物質(zhì)。本研究顯示，LPS+APAP組的小鼠肝組織內(nèi)MDA含量較APAP組減少，而GSH水平明顯增多。DHE染色是反映組織內(nèi)ROS含量的重要指標(biāo)，DHE結(jié)果與MDA結(jié)果趨勢(shì)一致。

綜上所述，LPS通過TLR預(yù)激活Nrf2抗氧化通路，減輕APAP引起的氧化應(yīng)激反應(yīng)，從而明顯緩解肝臟損傷。Nrf2抗氧化通路的深入研究可能為藥物性肝損傷的臨床防治提供一種新的策略。

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(本文編輯：錢燕)

TLR protects liver against acetaminophen-induced Hepatotoxicitin via activating the Nrf2 antioxidant signaling pathway

GUJia-yi，YUFeng-rong.

DepartmentofGastroenterologicalSurgery，RenJiHospital，SchoolofMedcine，ShanghaiJiaotongUniversity，Shanghai200127，ChinaCorrespondingauthor:YUFengrong,Email：rry@126.com

ObjectiveTo investigate the protective effect of Lipopolysaccharide (LPS) and toll-like receptors (TLRs) on acetaminophen (APAP)-induced liver injury and its potential mechanism. MethodsForty male mice were randomly divided into 4 groups. Mice in control group were intraperitneally (i.p.) injected with saline, in LPS group were i.p. given with 10 μg/kg LPS, and in APAP group were i.p. administrated with APAP (300 mg/kg). LPS+APAP group were i.p. pretreated with LPS (10 μg/kg) 16 h before APAP (300 mg/kg) injection. Serum and liver tissue among 4 groups were collected for further analysis. Liver injury was assessed by detection of serum ALT and AST levels and HE staining of liver tissue. The oxidative stress was evaluated by measuring hepatic glutathione (GSH) and malondialdehyde (MDA) levels, and Dihydroethidium (DHE) staining. Expression of Nrf2, Gclc and HO-1 were measured by western blot and real time-polymerase chain reaction. ResultsCompared with APAP groups, LPS+APAP group showed lower serum levels of ALT (518.3±142.3 vs. 4542±498.4 U/L,P<0.05) and AST (643.3±105.6 vs. 5432.1±569.2 U/L,P<0.05), milder liver tissue damage, lower MDA levels (78.0±14.5 vs.141.7±26.4 mmol/mg tissue,P<0.05) and higher GSH levels (6.2±1.7 vs. 3.5±1.0 μmol/g tissue,P<0.05), which revealed that APAP-induced liver injury and oxidative stress response were significantly attenuated by LPS pretreatment. Moreover, LPS pretreatment could enhance the expression of Nrf2 and other antioxidant genes significantly. ConclusionLPS plays an important role in protection against APAP-induced hepatotoxicity in mice via activating antioxidant signaling pathway of Nrf2, which might become a new strategy for APAP-induced liver injury therapy.

Acetaminophen；Drug induced liver injury; Oxidative stress；Nrf2；HO-1

200127上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院胃腸外科

郁豐榮，Email：rry@126.com

2016-07-26)