中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
10期
747-752
,共6页
杜仲燕%高佳%王伟茜%于晨辉%窦晓兵
杜仲燕%高佳%王偉茜%于晨輝%竇曉兵
두중연%고가%왕위천%우신휘%두효병
肝疾病,酒精性%NF-κB%肿瘤坏死因子α%4-羟基壬烯酸
肝疾病,酒精性%NF-κB%腫瘤壞死因子α%4-羥基壬烯痠
간질병,주정성%NF-κB%종류배사인자α%4-간기임희산
Liver diseases,alcoholic%NF-kappa B%Tumor necrosis factor-α%4-Hydroxynonenal
目的 通过体内动物实验和体外细胞实验,探讨4-羟基壬烯酸(4-HNE)对肿瘤坏死因子(TNF)介导的肝细胞内核因子κB (NF-κ B)抗凋亡通路的抑制作用及其机制,进一步从细胞信号通路及蛋白质翻译后修饰水平揭示4-HNE促TNF介导酒精性肝病(ALD)的发病机制.方法 用Lieber-DeCarli小鼠模型、人肝癌细胞株HepG2和小鼠原代肝细胞,通过四甲基偶氮唑盐法、乳酸脱氢酶测定、Westem blot、免疫荧光染色等检测4-HNE对TNF介导的肝细胞损伤作用及4-HNE表达水平；通过酶联免疫吸附实验、实时荧光定量PCR检测肝细胞内NF-κ B的含量、其与DNA的结合活性及其所调控的抗凋亡基因的表达情况；用Western blot及免疫共沉淀技术检测NF-κB抑制蛋白(IκBα)及其激酶(IKK)的磷酸化以及4-HNE对IκBα、IKK的翻译后修饰水平.计量资料两个样本均数间比较用t检验,多个样本均数间比较用方差分析. 结果 (1)体内动物实验:模型组小鼠肝内甘油三酯水平(36.1±6.9) mg/g、血浆ALT水平(39.8±8.9) U/L和肝内TNF水平(650.8±28.9) pg/g明显高于对照组[分别为(13.6±3.2) mg/g、(18.3±4.1) U/L和TNF (320.2±14.1)pg/pg,差异有统计学意义,JP值均＜0.01.与对照组相比,模型组小鼠4-HNE与蛋白质形成的加合物增多,肝细胞脂质变性、细胞凋亡增多,肝细胞核内NF-κ B(p65)与DNA的结合活性(0.13±0.01)也较对照组(0.17±0.03)明显下降(t=4.178,P＜0.05),NF-κB(p65)蛋白表达水平降低,IκBα蛋白磷酸化水平降低,4-HNE-I κ B α加合物增多.(2)体外细胞实验:①在HepG2及小鼠原代肝细胞中,与TNF组相比,4-HNE/TNF组肝细胞增殖活性下降,随着4-HNE、TNF浓度的增加,增殖活性呈剂量依赖性降低,细胞凋亡增多,细胞释放的乳酸脱氢酶活性升高(F=7.262,P＜0.05),4-HNE/TNF组明显诱导聚腺苷二磷酸核糖聚合酶蛋白剪切.②在HepG2细胞和小鼠原代肝细胞中,随着4-HNE浓度升高,细胞内4-HNE与蛋白质形成的加合物增多,与TNF组相比,4-HNE/TNF组肝细胞内4-HNE与IκBα形成的蛋白质加合物增多,I κB α的蛋白磷酸化水平降低.③随着4-HNE浓度升高,HepG2细胞核内NF-κB (p65)与DNA的结合活性下降,NF-κB调控基因(cFLIP、cIAP、Bcl-2)的mRNA表达水平下降.④4-HNE对TNF诱导的IKK无明显作用.结论 长期摄入乙醇导致的肝损伤与体内4-HNE和TNF水平均有关.4-HNE可以通过与肝细胞内IκBα形成蛋白质加合物降低IκBα的磷酸化水平,抑制肝细胞内TNF介导的NF-κB抗凋亡信号通路,从而显著增加肝细胞对TNF杀伤作用的敏感性,诱导肝细胞死亡.
目的 通過體內動物實驗和體外細胞實驗,探討4-羥基壬烯痠(4-HNE)對腫瘤壞死因子(TNF)介導的肝細胞內覈因子κB (NF-κ B)抗凋亡通路的抑製作用及其機製,進一步從細胞信號通路及蛋白質翻譯後脩飾水平揭示4-HNE促TNF介導酒精性肝病(ALD)的髮病機製.方法 用Lieber-DeCarli小鼠模型、人肝癌細胞株HepG2和小鼠原代肝細胞,通過四甲基偶氮唑鹽法、乳痠脫氫酶測定、Westem blot、免疫熒光染色等檢測4-HNE對TNF介導的肝細胞損傷作用及4-HNE錶達水平；通過酶聯免疫吸附實驗、實時熒光定量PCR檢測肝細胞內NF-κ B的含量、其與DNA的結閤活性及其所調控的抗凋亡基因的錶達情況；用Western blot及免疫共沉澱技術檢測NF-κB抑製蛋白(IκBα)及其激酶(IKK)的燐痠化以及4-HNE對IκBα、IKK的翻譯後脩飾水平.計量資料兩箇樣本均數間比較用t檢驗,多箇樣本均數間比較用方差分析. 結果 (1)體內動物實驗:模型組小鼠肝內甘油三酯水平(36.1±6.9) mg/g、血漿ALT水平(39.8±8.9) U/L和肝內TNF水平(650.8±28.9) pg/g明顯高于對照組[分彆為(13.6±3.2) mg/g、(18.3±4.1) U/L和TNF (320.2±14.1)pg/pg,差異有統計學意義,JP值均＜0.01.與對照組相比,模型組小鼠4-HNE與蛋白質形成的加閤物增多,肝細胞脂質變性、細胞凋亡增多,肝細胞覈內NF-κ B(p65)與DNA的結閤活性(0.13±0.01)也較對照組(0.17±0.03)明顯下降(t=4.178,P＜0.05),NF-κB(p65)蛋白錶達水平降低,IκBα蛋白燐痠化水平降低,4-HNE-I κ B α加閤物增多.(2)體外細胞實驗:①在HepG2及小鼠原代肝細胞中,與TNF組相比,4-HNE/TNF組肝細胞增殖活性下降,隨著4-HNE、TNF濃度的增加,增殖活性呈劑量依賴性降低,細胞凋亡增多,細胞釋放的乳痠脫氫酶活性升高(F=7.262,P＜0.05),4-HNE/TNF組明顯誘導聚腺苷二燐痠覈糖聚閤酶蛋白剪切.②在HepG2細胞和小鼠原代肝細胞中,隨著4-HNE濃度升高,細胞內4-HNE與蛋白質形成的加閤物增多,與TNF組相比,4-HNE/TNF組肝細胞內4-HNE與IκBα形成的蛋白質加閤物增多,I κB α的蛋白燐痠化水平降低.③隨著4-HNE濃度升高,HepG2細胞覈內NF-κB (p65)與DNA的結閤活性下降,NF-κB調控基因(cFLIP、cIAP、Bcl-2)的mRNA錶達水平下降.④4-HNE對TNF誘導的IKK無明顯作用.結論 長期攝入乙醇導緻的肝損傷與體內4-HNE和TNF水平均有關.4-HNE可以通過與肝細胞內IκBα形成蛋白質加閤物降低IκBα的燐痠化水平,抑製肝細胞內TNF介導的NF-κB抗凋亡信號通路,從而顯著增加肝細胞對TNF殺傷作用的敏感性,誘導肝細胞死亡.
목적 통과체내동물실험화체외세포실험,탐토4-간기임희산(4-HNE)대종류배사인자(TNF)개도적간세포내핵인자κB (NF-κ B)항조망통로적억제작용급기궤제,진일보종세포신호통로급단백질번역후수식수평게시4-HNE촉TNF개도주정성간병(ALD)적발병궤제.방법 용Lieber-DeCarli소서모형、인간암세포주HepG2화소서원대간세포,통과사갑기우담서염법、유산탈경매측정、Westem blot、면역형광염색등검측4-HNE대TNF개도적간세포손상작용급4-HNE표체수평；통과매련면역흡부실험、실시형광정량PCR검측간세포내NF-κ B적함량、기여DNA적결합활성급기소조공적항조망기인적표체정황；용Western blot급면역공침정기술검측NF-κB억제단백(IκBα)급기격매(IKK)적린산화이급4-HNE대IκBα、IKK적번역후수식수평.계량자료량개양본균수간비교용t검험,다개양본균수간비교용방차분석. 결과 (1)체내동물실험:모형조소서간내감유삼지수평(36.1±6.9) mg/g、혈장ALT수평(39.8±8.9) U/L화간내TNF수평(650.8±28.9) pg/g명현고우대조조[분별위(13.6±3.2) mg/g、(18.3±4.1) U/L화TNF (320.2±14.1)pg/pg,차이유통계학의의,JP치균＜0.01.여대조조상비,모형조소서4-HNE여단백질형성적가합물증다,간세포지질변성、세포조망증다,간세포핵내NF-κ B(p65)여DNA적결합활성(0.13±0.01)야교대조조(0.17±0.03)명현하강(t=4.178,P＜0.05),NF-κB(p65)단백표체수평강저,IκBα단백린산화수평강저,4-HNE-I κ B α가합물증다.(2)체외세포실험:①재HepG2급소서원대간세포중,여TNF조상비,4-HNE/TNF조간세포증식활성하강,수착4-HNE、TNF농도적증가,증식활성정제량의뢰성강저,세포조망증다,세포석방적유산탈경매활성승고(F=7.262,P＜0.05),4-HNE/TNF조명현유도취선감이린산핵당취합매단백전절.②재HepG2세포화소서원대간세포중,수착4-HNE농도승고,세포내4-HNE여단백질형성적가합물증다,여TNF조상비,4-HNE/TNF조간세포내4-HNE여IκBα형성적단백질가합물증다,I κB α적단백린산화수평강저.③수착4-HNE농도승고,HepG2세포핵내NF-κB (p65)여DNA적결합활성하강,NF-κB조공기인(cFLIP、cIAP、Bcl-2)적mRNA표체수평하강.④4-HNE대TNF유도적IKK무명현작용.결론 장기섭입을순도치적간손상여체내4-HNE화TNF수평균유관.4-HNE가이통과여간세포내IκBα형성단백질가합물강저IκBα적린산화수평,억제간세포내TNF개도적NF-κB항조망신호통로,종이현저증가간세포대TNF살상작용적민감성,유도간세포사망.
Objective To investigate the effects and mechanism of intracellular 4-hydroxynonenal (4-HNE) accumulation on tumor necrosis factor (TNF)-induced hepatotoxicity in alcoholic liver disease (ALD).Methods An ALD model was established in male C57BL/6 mice (6-8 weeks old) by feeding an ethanol-containing diet for 5 weeks; mice given regular (non-ethanol) diet served as controls.ALD-related changes in 4-HNE and TNF levels were detected by western blotting.The underlying mechanisms of this molecular effect were examined by pre-treating HepG2 cells with 4-HNE followed by exposure to various concentrations of TNF.Effects on cell death were evaluated by MTT assay.Effects on TNF-mediated upstream factors' expression were detected by ELISA,western blotting,and real-time PCR.Effects on the TNF-induced inhibitor of NF-κB (Iκ Bα) activity (phosphorylation status) and its formation of adducts were detected by western blotting and immunoprecipitation.Results ALD mice showed increased hepatic 4-HNE and TNF levels,and the increases were associated with extent of liver injury.Cell culture studies revealed that 4-HNE,at non-toxic concentrations,sensitized hepatocytes to TNF killing,which was associated with suppressed NF-κB transactivity.Furthermore,4-HNE prevented phosphorylation of IκBα without affecting upstream IκB kinase activity.The ALD-enhanced 4-HNE content was found to associated with increased formation of 4-HNE-Iκ Bα adduction for both the 4-HNE-treated hepatocytes in culture and in the livers of ALD mice.Conclusion Alcohol-induced increase in 4-HNE accumulation represents a potent and clinically relevant mechanism of sensitizing hepatocytes to TNF-induced toxicity.These data support the notion that decreasing or eliminating accumulated intracellular 4-HNE can serve as a potential therapeutic option for ALD.
