4-HNE通過抑制TNF-α介導(dǎo)的NF-κB活化誘導(dǎo)酒精性肝損傷*

于晨輝， 杜仲燕， 高 佳， 王偉茜， 竇曉兵

(浙江中醫(yī)藥大學(xué)生命科學(xué)學(xué)院，浙江 杭州 310053)

4-HNE通過抑制TNF-α介導(dǎo)的NF-κB活化誘導(dǎo)酒精性肝損傷*

于晨輝， 杜仲燕， 高 佳， 王偉茜， 竇曉兵△

(浙江中醫(yī)藥大學(xué)生命科學(xué)學(xué)院，浙江 杭州 310053)

目的通過體外細(xì)胞及體內(nèi)動(dòng)物實(shí)驗(yàn)，研究腫瘤壞死因子α(TNF-α)誘導(dǎo)的4-羥基壬烯酸(4-HNE)致敏肝細(xì)胞發(fā)生死亡的作用及機(jī)制。方法以人肝細(xì)胞株HepG2及小鼠原代肝細(xì)胞為細(xì)胞模型，通過乳酸脫氫酶(LDH)釋放及MTT比色法檢測4-HNE對TNF-α誘導(dǎo)的肝細(xì)胞死亡的作用，利用Western blotting技術(shù)檢測細(xì)胞內(nèi)4-HNE與蛋白質(zhì)形成的加合物水平，通過Western blotting和ELISA技術(shù)檢測細(xì)胞核內(nèi)NF-κB(p65)的表達(dá)及其與DNA結(jié)合活性。以C57BL/6小鼠為動(dòng)物模型，利用HE染色、ELISA、Western blotting及TUNEL等技術(shù)，檢測長期攝入酒精前后動(dòng)物肝組織形態(tài)、甘油三酯(TG)水平、4-HNE水平、TNF-α水平及血漿丙氨酸氨基轉(zhuǎn)移酶(ALT)活性的變化。結(jié)果(1) 4-HNE可以顯著增加HepG2細(xì)胞及小鼠原代肝細(xì)胞對TNF-α殺傷作用的敏感性，從而使TNF-α誘導(dǎo)4-HNE致敏的肝細(xì)胞死亡。(2) 4-HNE可顯著提高HepG2細(xì)胞內(nèi)4-HNE-蛋白質(zhì)加合物的水平。(3) 4-HNE抑制HepG2細(xì)胞內(nèi)TNF-α介導(dǎo)的NF-κB活化。(4) 長期攝入酒精導(dǎo)致小鼠肝細(xì)胞內(nèi)4-HNE和TNF-α水平升高，引起肝細(xì)胞內(nèi)TG水平升高，血漿ALT活性升高，肝細(xì)胞死亡增多。結(jié)論長期攝入酒精使肝細(xì)胞發(fā)生氧化應(yīng)激，其產(chǎn)物4-HNE可作為一種肝細(xì)胞致敏因子，通過抑制肝細(xì)胞內(nèi)TNF-α介導(dǎo)的NF-κB抗細(xì)胞凋亡信號(hào)通路，誘導(dǎo)酒精性肝損傷。這可能是一種新的酒精性肝病發(fā)病機(jī)制。

酒精性肝病； 4-羥基壬烯酸； 腫瘤壞死因子α； 核因子κB； 肝細(xì)胞

酒精性肝病(alcoholic liver disease, ALD)是由于長期過度飲酒導(dǎo)致的肝臟疾病[1]。近年來我國ALD的發(fā)病率及死亡率也呈逐年增長趨勢，已成為僅次于病毒性肝炎的第二大肝病。有研究顯示，酒精性肝硬化占肝硬化發(fā)病總數(shù)的比例已從1999年的10.8%上升為2003年的24.0%[2-3]。雖然許多研究已證實(shí)，氧化應(yīng)激在ALD的發(fā)生發(fā)展過程中起著十分關(guān)鍵的作用，抗氧化劑對ALD具有一定的預(yù)防作用[4]，但是ALD的發(fā)病機(jī)制復(fù)雜，氧化應(yīng)激可以影響細(xì)胞內(nèi)許多信號(hào)調(diào)控通路[5]，其具體機(jī)制尚未完全明確，很大程度上限制了抗氧化劑在ALD臨床治療上的應(yīng)用。目前為止，沒有任何能夠安全有效治療ALD的臨床用藥。長期攝入酒精導(dǎo)致肝細(xì)胞發(fā)生氧化應(yīng)激和脂質(zhì)過氧化反應(yīng)，4-羥基壬烯酸(4-hydroxynonenal, 4-HNE)是氧化應(yīng)激和脂質(zhì)過氧化反應(yīng)的主要產(chǎn)物之一，在ALD的發(fā)生發(fā)展過程中起著至關(guān)重要的作用[6]。許多臨床研究、流行病學(xué)調(diào)查及動(dòng)物實(shí)驗(yàn)研究均表明，ALD患者血漿中4-HNE水平顯著增加。此外，1989年，McClain等最初發(fā)現(xiàn)酒精性肝炎患者體內(nèi)的腫瘤壞死因子α(tumor necrosis factor α, TNF-α)明顯高于正常人[7]。TNF-α具有多種生物學(xué)功能，在生理狀態(tài)下，TNF-α介導(dǎo)的抗凋亡通路的活化使肝細(xì)胞對TNF-α處于耐受狀態(tài)。然而在酒精等某些病理因素作用下，肝細(xì)胞可以變得對TNF-α敏感，導(dǎo)致細(xì)胞的死亡[8]。以上研究提示，長期攝入酒精可產(chǎn)生氧化應(yīng)激，而氧化應(yīng)激是誘發(fā)肝細(xì)胞死亡的重要觸發(fā)機(jī)制,4-HNE作為其主要產(chǎn)物之一，是否也可以通過增加肝細(xì)胞對TNF-α介導(dǎo)的細(xì)胞毒作用的敏感性，誘導(dǎo)肝細(xì)胞死亡呢？在本研究中我們采用人肝細(xì)胞株HepG2和小鼠原代肝細(xì)胞作為細(xì)胞模型、長期攝入乙醇的C57BL/6小鼠作為動(dòng)物模型，檢測4-HNE對TNF-α誘導(dǎo)的肝細(xì)胞死亡的作用，從細(xì)胞信號(hào)調(diào)控及蛋白質(zhì)翻譯后修飾水平進(jìn)一步揭示ALD的發(fā)病機(jī)制，為ALD的臨床治療提供新的策略，也為以4-HNE為靶點(diǎn)的新型藥物研發(fā)提供重要的理論指導(dǎo)和依據(jù)。

材 料 和 方 法

1材料

1.1動(dòng)物 8周齡雄性C57BL/6小鼠，體重(25.0±0.5)g，由Jackson Laboratory提供。

1.2細(xì)胞 人肝細(xì)胞株HepG2購自中科院上海細(xì)胞庫；小鼠原代肝細(xì)胞購自Celsis IVT。

1.3試劑 4-HNE購自Cayman Chemical。TNF-α購自Perro Tech。LDH測試盒購自Key Gen。β-actin 抗體購自Santa Cruz。4-HNE抗體購自R&D。NF-κB(p65)抗體購自CST。ELISA試劑盒購自Pierce。其它實(shí)驗(yàn)試劑均購自Sigma Aldrich。

2方法

2.1LDH測定 按2×108/L細(xì)胞密度制備單細(xì)胞懸液，每孔1 mL接種于24孔培養(yǎng)板中，每組重復(fù)3孔進(jìn)行細(xì)胞培養(yǎng)及藥物孵育。根據(jù)LDH檢測試劑盒說明測定細(xì)胞培養(yǎng)液中LDH的活性，分析各組間的細(xì)胞毒性作用。

2.2MTT比色法 制備單細(xì)胞懸液，細(xì)胞按2×108/L密度接種于96孔培養(yǎng)板內(nèi)，每孔100 μL，每組重復(fù)5孔。置37 ℃、5%CO2培養(yǎng)箱中培養(yǎng)24 h，待細(xì)胞貼壁后，棄掉原培養(yǎng)基，向每孔加入含4-HNE的培養(yǎng)基100 μL，預(yù)處理2 h， 再加入TNF-α孵育16 h。加入20 μL MTT在培養(yǎng)箱內(nèi)培養(yǎng)4 h后，棄去孔中所有液體，各孔加入150 μL DMSO，振蕩10 min后在酶標(biāo)儀570 nm波長處測定吸光度。

2.3Western blotting 細(xì)胞培養(yǎng)及藥物孵育后，PBS洗滌細(xì)胞2次，加入蛋白裂解液，置冰浴裂解30 min。以12 000 ×g、4 ℃離心10 min。取上清，用BCA試劑盒測定提取液中的蛋白質(zhì)濃度。將各組調(diào)整至相同蛋白濃度后，取20 μg樣本經(jīng)SDS-PAGE電泳、轉(zhuǎn)移電泳、抗體免疫、顯影、曝光等步驟檢測蛋白表達(dá)情況(β-actin作為內(nèi)參照)。

2.4ELISA檢測 細(xì)胞培養(yǎng)藥物孵育后按試劑盒(碧云天)說明書操作抽提核蛋白，根據(jù)ELISA試劑盒(Pierce)說明書操作，經(jīng)包被，樣本孵育，封閉，Ⅰ抗、Ⅱ抗孵育，顯色，終止等步驟檢測細(xì)胞核內(nèi)NF-κB(p65)與96孔板中特異性DNA序列的結(jié)合活性。

2.5動(dòng)物模型制備 采用含乙醇的液體飼料(alcohol fed，AF)及不含乙醇但含有與AF組相同熱卡的對照組飼料(pair fed，PF)喂飼小鼠5周(試喂1周后，從第2周開始乙醇液體飼料中乙醇含量從占總熱卡的30%遞增到36%，每周增加2%)，以建立正常對照組(PF)和ALD模型組(AF)。小鼠的進(jìn)食量和體重每天、每周分別記錄，5周后處死小鼠，提取并保存肝組織和血漿。

3統(tǒng)計(jì)學(xué)處理

利用SPSS 10.0統(tǒng)計(jì)軟件，數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，組間均數(shù)比較采用單因素方差分析。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

14-HNE顯著增加肝細(xì)胞對TNF-α殺傷作用的敏感性

用4-HNE(0～60 μmol/L)預(yù)處理肝細(xì)胞2 h，再加入40 μg/L TNF-α孵育16 h，LDH檢測結(jié)果顯示，40 μg/L TNF-α或20 μmol/L 4-HNE單獨(dú)作用HepG2肝細(xì)胞，細(xì)胞活性與對照組相比無明顯差異(P>0.05)，若先用20 μmol/L 4-HNE預(yù)處理HepG2肝細(xì)胞2 h，再加入40 μg/L TNF-α孵育16 h，可檢測到細(xì)胞活性顯著降低(P<0.05)，且呈現(xiàn)明顯的劑量效應(yīng)，見圖1A。MTT比色法檢測4-HNE對TNF-α介導(dǎo)的肝細(xì)胞增殖的作用，得到了與LDH檢測相一致的結(jié)果，見圖1B。先用20 μmol/L 4-HNE預(yù)處理HepG2肝細(xì)胞2 h，再加入40 μg/L TNF-α孵育16 h，熒光顯微鏡下被Hoechst 33342染色的細(xì)胞數(shù)量明顯減少，見圖1C。MTT檢測4-HNE對TNF-α介導(dǎo)的小鼠原代肝細(xì)胞的增殖作用，得到了與HepG2細(xì)胞相一致的結(jié)果，見圖1D。

Figure 1. 4-HNE significantly increased the sensitivity of hepatocytes to the killing effect of TNF-α. A: HepG2 cells were pretreated with 4-HNE (20, 40 or 60 μmol/L) for 2 h, followed by 40 μg/L TNF-α incubation for 16 h, and then the activity of LDH in cell medium was determined (n=3); B: HepG2 cells were pretreated with 20 μmol/L 4-HNE for 2 h, followed by TNF-α (10 or 40 μg/L) incubation for 16 h, and then the proliferation of the cells was tested by MTT assay(n=6); C: HepG2 cells were pretreated with 20 μmol/L 4-HNE for 2 h, followed by 40 μg/L TNF-α incubation for 16 h, and then Hoechst 33342 staining was performed (×40); D: primary mouse hepatocytes were pretreated with 20 μmol/L 4-HNE for 2 h, followed by 40 μg/L TNF-α incubation for 24 h, and then MTT assay was conducted(n=6). Mean±SD.*P<0.05,**P<0.05vscontrol (0 μmol/L 4-HNE + 0 μg/L TNF-α);△P<0.05vs20 μmol/L 4-HNE + 40 μg/L TNF-α or 40 μmol/L 4-HNE alone;#P<0.05vs40 μmol/L 4-HNE + 40 μg/L TNF-α or 60 μmol/L 4-HNE alone.

圖14-HNE顯著增加肝細(xì)胞對TNF-α殺傷作用的敏感性

24-HNE對HepG2細(xì)胞內(nèi)4-HNE-蛋白質(zhì)加合物形成的影響

不同濃度的4-HNE作用于HepG2細(xì)胞，隨著4-HNE濃度的增高，細(xì)胞內(nèi)4-HNE-蛋白質(zhì)加合物明顯增多,當(dāng)4-HNE為80 mol/L時(shí)，4-HNE與蛋白質(zhì)形成加合物的量是對照組的4.83倍(P<0.01)，見圖2A。用20 μmol/L 4-HNE作用HepG2細(xì)胞2 h，細(xì)胞內(nèi)4-HNE-蛋白質(zhì)加合物明顯增多(P<0.05)，作用4 h表達(dá)量進(jìn)一步增加，約為對照組的10倍(P<0.01)，作用8 h與4 h比無明顯變化,見圖2B。

Figure 2. Effect of 4-HNE on 4-HNE-protein adduct formation in HepG2 cells detected by Western blotting. A: HepG2 cells were incubated with exogenous 4-HNE at the indicated concentrations for 4 h; B: HepG2 cells were incubated with exogenous 4-HNE (20 μmol/L) for the indicated time periods. Mean±SD.n=3.*P<0.05,**P<0.01vscontrol (0 μmol/L or 0 h).

圖24-HNE對HepG2細(xì)胞4-HNE-蛋白質(zhì)加合物形成的影響

34-HNE抑制HepG2細(xì)胞內(nèi)TNF-α介導(dǎo)的NF-κB活化

用20 μmol/L 4-HNE預(yù)處理細(xì)胞2 h，再用100 μg/L TNF-α孵育4 h，細(xì)胞核內(nèi)NF-κB(p65)與DNA的結(jié)合活性比單獨(dú)用100 μg/L TNF-α孵育4 h明顯降低(P<0.05),見圖3A。隨著4-HNE濃度的增高，NF-κB(p65)的活性進(jìn)一步降低，對照組NF-κB(p65)的活性是4-HNE 40 μmol/L組的2.02倍(P<0.05)，見圖3B。

Western blotting結(jié)果顯示，隨著4-HNE濃度的增高，HepG2細(xì)胞核內(nèi)NF-κB(p65)蛋白的表達(dá)明顯降低，見圖3C。

4乙醇對小鼠肝組織內(nèi)4-HNE、TNF-α、甘油三酯(triglyceride,TG)和血漿丙氨酸氨基轉(zhuǎn)移酶(alanineaminotransferase,ALT)的影響

與正常喂食組小鼠相比，實(shí)驗(yàn)組小鼠肝臟內(nèi)TG的含量顯著升高(圖4A),并且肝臟出現(xiàn)脂肪水解現(xiàn)象(圖4B)。同時(shí)發(fā)現(xiàn)，攝入乙醇提高了實(shí)驗(yàn)組小鼠血漿內(nèi)ALT的活性(圖4C)。TUNEL檢測顯示，實(shí)驗(yàn)組小鼠比正常組小鼠表現(xiàn)出明顯的肝細(xì)胞凋亡現(xiàn)象(圖4D)。肝臟內(nèi)TNF-α水平也明顯升高(圖4E)。此外，用含乙醇的食物喂養(yǎng)5周后小鼠肝細(xì)胞內(nèi)4-HNE-蛋白質(zhì)加合物水平明顯提高(圖4F)。

Figure 3. 4-HNE inhibitied TNF-α-mediated NF-κB activation in HepG2 cells. A: HepG2 cells were pretreated with 20 μmol/L 4-HNE for 2 h, followed by 100 μg/L TNF-α incubation for 4 h, and then intranuclear NF-κB (p65) DNA binding activity were measured by ELISA; B: HepG2 cells were pretreated with 4-HNE (20 or 40 μmol/L) for 2 h, followed by 40 μg/L TNF-α incubation for 4 h, and then intranuclear NF-κB (p65) DNA binding activity were measured by ELISA; C: HepG2 cells were pretreated with 4-HNE (20 or 40 μmol/L) for 2 h, followed by 100 μg/L TNF-α incubation for 4 h, and then NF-κB (p65) protein expression was detected by Western blotting. Mean±SD.n=3.*P<0.05,**P<0.01vscontrol (0 μmol/L 4-HNE+0 μg/L TNF-α);△P<0.05vs40 μg/L TNF-α alone;#P<0.05vs20 or 40 μmol/L 4-HNE alone;▲P<0.05vs20 μmol/L 4-HNE + 40 μg/L TNF-α.

圖34-HNE抑制HepG2肝細(xì)胞內(nèi)TNF-α介導(dǎo)的NF-κB活化

討 論

肝臟是酒精代謝的主要部位，長期攝入酒精會(huì)導(dǎo)致肝細(xì)胞發(fā)生氧化應(yīng)激和脂質(zhì)過氧化。血漿中4-HNE的生理濃度是0.3～0.7 μmol/L[9-10]，ALD患者的血漿中4-HNE水平顯著增加，肝炎早期4-HNE水平是生理濃度的2.8倍，肝硬化時(shí)期的4-HNE水平是生理濃度的8.4倍[11]，肝臟的局部細(xì)胞為了應(yīng)對氧化應(yīng)激，濃度甚至可高達(dá)100 μmol/L[12-13]。本研究中我們用20～60 μmol/L 4-HNE來模擬早期ALD的體內(nèi)4-HNE水平。雖然4-HNE對許多類型細(xì)胞的毒性作用已被廣泛證實(shí)，但在病理狀態(tài)下，肝細(xì)胞對4-HNE的耐受力將增強(qiáng)，這是因?yàn)楦渭?xì)胞對4-HNE具有較強(qiáng)的降解代謝功能[14]。本研究體外實(shí)驗(yàn)數(shù)據(jù)表明，20～40 μmol/L 4-HNE單獨(dú)作用于人肝細(xì)胞HepG2和小鼠原代肝細(xì)胞并沒有引起細(xì)胞死亡或僅引起很小程度死亡，但TNF-α可以誘導(dǎo)4-HNE致敏的肝細(xì)胞顯著殺傷；體內(nèi)實(shí)驗(yàn)也證實(shí)，長期攝入酒精可以引起肝組織內(nèi)TNF-α和4-HNE水平顯著升高，從而誘導(dǎo)ALD。上述結(jié)果均表明TNF-α誘導(dǎo)4-HNE致敏的肝細(xì)胞產(chǎn)生細(xì)胞毒作用可能是導(dǎo)致ALD患者肝損傷的一個(gè)新機(jī)制。

TNF-α水平過高是ALD的另一個(gè)病理因素。乙醇會(huì)增加腸的通透性，引起巨噬細(xì)胞吞噬脂多糖增加，導(dǎo)致TNF-α水平升高。NF-κB是用以應(yīng)對TNF-α刺激的細(xì)胞生死存亡之間一個(gè)重要的轉(zhuǎn)換樞紐。在TNF-α的誘導(dǎo)下，缺乏NF-κB p65亞基或IKK復(fù)合物重要組成部分的小鼠在妊娠中期就會(huì)因肝細(xì)胞凋亡而死亡[15]。此外，IκBα突變的肝細(xì)胞對TNF-α誘導(dǎo)的殺傷作用更敏感[16]。本研究結(jié)果表明，4-HNE可以抑制TNF-α誘導(dǎo)的肝細(xì)胞內(nèi)NF-κB的激活，從而增加肝細(xì)胞對TNF-α的敏感性，誘導(dǎo)肝細(xì)胞死亡，這個(gè)結(jié)論被以下2個(gè)實(shí)驗(yàn)結(jié)果所支持：(1)4-HNE降低了p65與DNA的結(jié)合活性； (2)4-HNE減少了細(xì)胞核內(nèi)p65的含量。雖然前期研究有報(bào)道4-HNE對NF-κB活化的影響，但本研究結(jié)果與以前的結(jié)果存在不一致之處。4-HNE具有明顯的細(xì)胞類型和刺激因素依賴性，4-HNE可以激活血管平滑肌細(xì)胞NF-κB的活性[17]，而在THP-1單核細(xì)胞中，4-HNE通過抑制IκBα的磷酸化來抑制NF-κB的活性，這種抑制作用受到脂多糖、白細(xì)胞介素1β和佛波酯的刺激，而不是TNF-α的刺激[18]。目前為止，本文首次報(bào)道4-HNE可以抑制TNF-α誘導(dǎo)的肝細(xì)胞內(nèi)NF-κB活性。

Figure 4. Effects of long-term (5 weeks) alcohol intake on the morphology and functions of mouse hepatic tissues, and the hepatic levels TNF-α and 4-HNE-protein adducts. A: hepatic triglyceride (TG) content; B: histological changes of hepatic tissues (HE staining, ×100); C: plasma alanine aminotransferase (ALT) activity; D: hepatocyte apoptosis in hepatic tissues (TUNEL, ×100); E: hepatic TNF-α level; F: Western blotting analysis of hepatic 4-HNE-protein adduct formation. PF: pair fed; AF: alcohol fed. Mean±SD.n=3.*P<0.05vsPF.

圖4長期攝入酒精對小鼠肝組織形態(tài)、功能、TNF-α水平和4-HNE-蛋白質(zhì)加合物形成的影響

4-HNE影響蛋白質(zhì)功能的主要機(jī)制之一是通過與蛋白質(zhì)形成加合物，這一過程稱為蛋白質(zhì)的羰基化。本研究體內(nèi)和體外實(shí)驗(yàn)結(jié)果均表明，外源4-HNE的加入及慢性酒精攝入均可以引起肝細(xì)胞內(nèi)4-HNE與蛋白質(zhì)形成加合物水平的升高，這與前期報(bào)道相一致[19-21]。這些均表明4-HNE在ALD的發(fā)生發(fā)展過程中起著至關(guān)重要的作用。

本研究提示，長期攝入乙醇使肝細(xì)胞發(fā)生氧化應(yīng)激，其產(chǎn)物4-HNE可能作為肝細(xì)胞致敏因子，通過抑制肝細(xì)胞內(nèi)TNF--α介導(dǎo)的NF-κB抗細(xì)胞凋亡信號(hào)通路而誘導(dǎo)ALD。本文為脂質(zhì)過氧化作用和TNF-α誘導(dǎo)的肝細(xì)胞損傷作用之間潛在的相互聯(lián)系及ALD的發(fā)病機(jī)制提供了新的思路，為ALD的臨床治療提供新的策略，也為以4-HNE為靶點(diǎn)的新型藥物研發(fā)提供了重要的理論指導(dǎo)和依據(jù)，但4-HNE抑制NF-κB活性的確切機(jī)制和作用靶點(diǎn)仍需進(jìn)一步研究。

[1] 賈 青, 張維東,王朝霞,等. 不同劑量乙醇對小鼠早期肝纖維化的影響及機(jī)制研究[J]. 中國病理生理雜志, 2010, 26(9):1801-1806.

[2] 劉 陽, 遲寶榮. 酒精性肝硬化237例臨床分析[J]. 吉林醫(yī)學(xué), 2004, 25(4):40-42.

[3] 中華醫(yī)學(xué)會(huì)肝病學(xué)分會(huì)脂肪肝和酒精性肝病學(xué)組.酒精性肝病診療指南[J].胃腸病學(xué), 2010, 15(10):617-621.

[4] Wheeler MD, Kono H, Yin M, et al. Delivery of the Cu/Zn-superoxide dismutase gene with adenovirus reduces early alcohol-induced liver injury in rats [J]. Gastroenterology, 2001, 120(5):1241-1250.

[5] Videla LA. Oxidative stress signaling underlying liver disease and hepatoprotective mechanisms [J]. World J Hepatol, 2009, 1(1):72-78.

[6] Smathers RL, Galligan JJ, Stewart BJ, et al. Overview of lipid peroxidation products and hepatic protein modification in alcoholic liver disease [J]. Chem Biol Interact, 2011, 192(1-2):107-112.

[7] Goldblum SE, Hennig B, Jay M, et al. Tumor necrosis factor α-induced pulmonary vascular endothelial injury [J]. Infect Immun, 1989, 57(4):1218-1226.

[8] McClain CJ, Hill DB, Song Z, et al.S-Adenosylmethionine, cytokines, and alcoholic liver disease [J]. Alcohol, 2002, 27(3):185-192.

[9] Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes [J]. Free Radic Biol Med, 1991, 11(1):81-128.

[10] Strohmaier H, Hinghofer-Szalkay H, Schaur RJ. Detection of 4-hydroxynonenal (HNE) as a physiological component in human plasma [J]. J Lipid Mediat Cell Signal, 1995, 11(1):51- 61.

[11] Aleynik SI, Leo MA, Aleynik MK, et al. Increased circulating products of lipid peroxidation in patients with alcoholic liver disease [J]. Alcohol Clin Exp Res, 1998, 22(1):192-196.

[12] Tsukamoto H, Horne W, Kamimura S, et al. Experimental liver cirrhosis induced by alcohol and iron [J]. J Clin Invest, 1995, 96(1):620-630.

[13] Benedetti A, Comporti M, Fulceri R, et al. Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids: identification of 4,5-dihydroxydecenal [J]. Biochim Biophys Acta, 1984, 792(2):172-181.

[14] Sakuma S, Negoro M, Kifamura T, et al. Xanthine oxidase-derived reactive oxygen species mediate 4-oxo-2-nonenal-induced hepatocyte cell death [J]. Toxicol Appl Pharmacol, 2010, 249(2):127-131.

[15] Tanaka M, Fuentes ME, Yamaguchi K, et al. Embryonic lethality, liver degeneration, and impaired NF-κB activation in IKK-β-deficient mice [J]. Immunity, 1999, 10(4):421-429.

[16] Chaisson ML, Brooling JT, Ladiges W, et al. Hepatocyte-specific inhibition of NF-κB leads to apoptosis after TNF treatment,but not after partial hepatectomy [J]. J Clin Invest, 2002, 110(2):193-202.

[17] Lee JY, Je JH, Jung KJ,et al. Induction of endothelialiNOS by 4-hydroxyhexenal through NF-κB activation [J]. Free Radic Biol Med, 2004, 37(4):539-548.

[18] Page S, Fischer C, Baumgartner B, et al. 4-Hydroxynonenal prevents NF-κB activation and tumor necrosis factor expression by inhibiting IκB phosphorylation and subsequent proteolysis [J]. J Biol Chem, 1999, 274(17):11611-11618.

[19] Koteish A, Yang S, Lin H, et al. Chronic ethanol exposure potentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation [J]. J Biol Chem, 2002, 277(15):13037-13044.

[20] Diehl AM. Effect of ethanol on tumor necrosis factor signaling during liver regeneration [J]. Clin Biochem, 1999, 32(7):571-578.

[21] Mandrekar P, Catalano D, White B, et al. Moderate alcohol intake in humans attenuates monocyte inflammatory responses:inhibition of nuclear regulatory factor kappa B and induction of interleukin 10[J]. Alcohol Clin Exp Res, 2006, 30(1):135-139.

InhibitionofTNF-α-mediatedNF-κBactivationby4-hydroxynonenalcontributestoliverinjuryinalcoholicliverdisease

YU Chen-hui, DU Zhong-yan, GAO Jia, WANG Wei-xi, DOU Xiao-bing

(LifeSciencesCollegeofZhejiangChineseMedicalUniversity,Hangzhou310053,China.E-mail:xbdou77@163.com)

AIM: To study the role of 4-hydroxynonenal (4-HNE) in hepatocyte death induced by tumor necrosis factor α (TNF-α).METHODSHuman liver cell line HepG2 and primary mouse hepatocytes were used to establish the cell model. The effect of 4-HNE on TNF-α-induced cell death was determined by lactate dehydrogenase (LDH) release and MTT assays. The intracellular levels of 4-HNE-protein adducts were determined by Western blotting. The intranuclear NF-κB (p65) and its DNA binding activity were detected by Western blotting and ELISA, respectively. Long-term intake of alcohol in C57BL/6 mice was performed to establish the animal model. The histological changes of mouse hepatic tissues and the apoptosis of hepatocytes were observed by HE staining and TUNEL assay, respectively. The hepatic levels of triglyceride (TG), TNF-α and 4-HNE-protein adducts, and the plasma activity of alanine aminotransferase (ALT) were also detected.RESULTS(1) 4-HNE significantly increased the sensitivity of HepG2 cells and primary mouse hepatocytes to the killing effect of TNF-α. (2) 4-HNE significantly increased the intracellular levels of 4-HNE-protein adducts. (3) 4-HNE inhibited TNF-α-mediated NF-κB (p65) activation in HepG2 cells. (4) Long-term intake of alcohol in mice resulted in high hepatic levels of 4-HNE and TNF-α, accompanied with the increases in hepatic TG content, plasma ALT activity and hepatocyte death.CONCLUSIONLong-term intake of alcohol induces oxidative stress and produces 4-HNE as a hepatocyte-sensitizing factor, which inhibits TNF-α-mediated NF-κB anti-apoptotic signaling pathway in hepatocytes, thus inducing alcoholic liver damage.

Alcoholic liver disease; 4-Hydroxynonenal; Tumor necrosis factor alpha; Nuclear factor kappa B; Hepatocytes

R363

A

10.3969/j.issn.1000- 4718.2013.06.016

1000- 4718(2013)06- 1046- 07

2012- 12- 08

2013- 04- 17

國家自然科學(xué)基金資助項(xiàng)目(No.81241145);浙江省自然科學(xué)基金資助項(xiàng)目(No.LY12C07002);浙江省衛(wèi)生廳中醫(yī)藥科技計(jì)劃(No.2013KYA138)

△通訊作者Tel： 0571-86613724； E-mail： xbdou77@163.com