Fas/Fas配体系统及其下游信号转导通路的激活促进酒精性肝炎及酒精性肝纤维化的进展
Fas/Fas배체계통급기하유신호전도통로적격활촉진주정성간염급주정성간섬유화적진전
Activation of Fas/FasL and its downstream signaling pathway promotes development of alcoholic steatohepatitis and liver fibrosis in mice
  • 万方数据
    中华肝脏病杂志 중화간장병잡지
    CHINESE JOURNAL OF HEPATOLOGY
    2013年 2期 129-133 ,共5页

    任伟光%孔令波%米红梅%赵素贤%张玉果%王荣琦%南月敏

    임위광%공령파%미홍매%조소현%장옥과%왕영기%남월민

    肝炎,酒精性%肝硬化,酒精性%细胞凋亡%炎症%Fas%Fas配体 肝炎,酒精性%肝硬化,酒精性%細胞凋亡%炎癥%Fas%Fas配體
    간염,주정성%간경화,주정성%세포조망%염증%Fas%Fas배체
    Hepatitis,alcoholic%Liver cirrhosis,alcoholic%Apoptosis%Inflammation%Fas%Fas ligand
    目的 探明Fas/Fas配体(FasL)系统及其主导信号转导通路在酒精性肝炎和肝纤维化发生、发展中的作用及其机制. 方法 C57BL/6J小鼠以含4％(V/V)乙醇的Lieber-DeCarli液体饲料喂养4周,自第5周起联合微量CCl4腹腔注射至第8周,分别建立酒精性肝炎和肝纤维化模型.脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测肝细胞凋亡.分别采用逆转录-聚合酶链反应、westem blot及免疫组织化学染色检测肝组织Fas、FasL、天冬氨酸特异性半胱氨酸蛋白酶3(caspase3)及细胞色素P450 2E1(CYP 2E1)的mRNA及蛋白质表达.多组样本均数间差异比较用单因素方差分析或Kruskal-Wallis H检验,组间比较用LSD-t检验或Mann-Whitney U检验. 结果 应用含4％(V/V)乙醇的Lieber-DeCarli液体饲料联合微量CCl4腹腔注射可快速建立小鼠酒精性肝炎和肝纤维化模型.肝细胞凋亡数随肝脏炎症及纤维化的加重而增高,并伴有凋亡相关基因表达增强.对照组、酒精性肝炎组、酒精性肝纤维化组小鼠细胞凋亡相关基因表达水平依次升高:Fas mRNA的相对表达量分别为0.50±0.05、0.61±0.10、0.76±0.03(H=12.137 P＜0.05),Fas蛋白的相对表达量分别为0.52±0.14、0.86±0.10、0.99±0.09(F=12.758P＜0.01)；FasL mRNA相对表达量分别为0.31±0.03、0.53±0.02、1.02±0.04(F=153.260 P＜0.01)；caspase3mRNA对表达量分别为0.86±0.11、0.85±0.05、1.33±0.16(F=8.740,P＜0.01),caspase 3蛋白相对表达量分别为0.40±0.03、0.69±0.06、1.02±0.10(F=90.785,P＜0.01);CYP 2E1 mRNA相对表达量分别为0.72±0.14、1.00±0.15、1.30±0.20(H=4.713,P＜ 0.01)；各组间比较,P值均＜0.05.免疫组织化学染色结果显示,FasL及CYP 2E1的蛋白与mRNA的表达趋势一致. 结论 Fas/FasL系统及其下游信号转导通路的激活可能是酒精性肝病中诱导肝细胞凋亡的主导因素,可进一步促进酒精性肝炎和肝纤维化的发生与进展.
    목적 탐명Fas/Fas배체(FasL)계통급기주도신호전도통로재주정성간염화간섬유화발생、발전중적작용급기궤제. 방법 C57BL/6J소서이함4％(V/V)을순적Lieber-DeCarli액체사료위양4주,자제5주기연합미량CCl4복강주사지제8주,분별건립주정성간염화간섬유화모형.탈양핵당핵감산말단전이매개도적결구말단표기법(TUNEL)검측간세포조망.분별채용역전록-취합매련반응、westem blot급면역조직화학염색검측간조직Fas、FasL、천동안산특이성반광안산단백매3(caspase3)급세포색소P450 2E1(CYP 2E1)적mRNA급단백질표체.다조양본균수간차이비교용단인소방차분석혹Kruskal-Wallis H검험,조간비교용LSD-t검험혹Mann-Whitney U검험. 결과 응용함4％(V/V)을순적Lieber-DeCarli액체사료연합미량CCl4복강주사가쾌속건립소서주정성간염화간섬유화모형.간세포조망수수간장염증급섬유화적가중이증고,병반유조망상관기인표체증강.대조조、주정성간염조、주정성간섬유화조소서세포조망상관기인표체수평의차승고:Fas mRNA적상대표체량분별위0.50±0.05、0.61±0.10、0.76±0.03(H=12.137 P＜0.05),Fas단백적상대표체량분별위0.52±0.14、0.86±0.10、0.99±0.09(F=12.758P＜0.01)；FasL mRNA상대표체량분별위0.31±0.03、0.53±0.02、1.02±0.04(F=153.260 P＜0.01)；caspase3mRNA대표체량분별위0.86±0.11、0.85±0.05、1.33±0.16(F=8.740,P＜0.01),caspase 3단백상대표체량분별위0.40±0.03、0.69±0.06、1.02±0.10(F=90.785,P＜0.01);CYP 2E1 mRNA상대표체량분별위0.72±0.14、1.00±0.15、1.30±0.20(H=4.713,P＜ 0.01)；각조간비교,P치균＜0.05.면역조직화학염색결과현시,FasL급CYP 2E1적단백여mRNA적표체추세일치. 결론 Fas/FasL계통급기하유신호전도통로적격활가능시주정성간병중유도간세포조망적주도인소,가진일보촉진주정성간염화간섬유화적발생여진전.
    Objective To explore the role and mechanism of the Fas/Fas ligand(FasL)system and its downstream signaling pathway related to the progression of alcoholic steatohepatitis and liver fibrosis.Methods Eighteen C57BL/6J mice were randomly divided into three groups:controls;alcoholic steatohepatitis model,given four-weeks of a 4％ ethanol-containing Lieber-DeCarli liquid diet;alcoholic steatohepatitis and liver fibrosis model,given the four-week alcohol diet followed by twice weekly intraperitoneal injections of carbon tetrachloride(5％ olive oil solution;2 mL/kg dose)during the fifth to eighth weeks.Mice in the model groups were sacrificed at the end of week 4 and 8,respectively,along with control mice for comparative analyses.Liver tissue sections were evaluated for hepatocellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)assay.The mRNA expression of Fas,FasL,cysteine aspartate-specific proteases 3(caspase 3),and cytochrome P450 2E1(CYP 2E1)in liver tissues was detected by reverse transcription(RT)-PCR,visualized by ethidium bromide staining,and normalized to the gray-value of GAPDH expression.The protein expression of Fas and caspase 3 were detected by western blotting(b-actin normalized),and of FasL and CYP 2E1 by immunohistochemistry staining.Intergroup differences and statistical significance were evaluated by single factor analysis of variance and the least squares difference-t test or the Kruskal-Wallis H test and the Mann-Whitney U test.Results The number of apoptotic cells in the liver sections was significantly higher in both model groups with alcoholic steatohepatitis(vs.controls)and the amount in the alcoholic steatohepatitis plus liver fibrosis model was significantly higher than that in the model with only alcoholic steatohepatitis.In addition,activation of Fas,FasL and its downstream signaling pathway showed an increasing trend with extent of liver injury.The hepatic mRNA(by RT-PCR)and protein(by western blotting)normalized expression levels in the controls,alcoholic steatohepatitis models,and alcoholic steatohepatitis plus liver fibrosis models were,respectively:Fas mRNA:0.50 ± 0.05,0.61 ± 0.10,0.76 ± 0.03(H=12.137,P ＜ 0.05),protein:0.52 ± 0.14,0.86 ± 0.10,0.99 ± 0.09(F=12.758,P ＜ 0.01);FasL mRNA:0.31 ± 0.03,0.53 ± 0.02,1.02 ± 0.04(F=153.260,P＜ 0.01);caspase 3 mRNA:0.86 ± 0.11,0.85 ± 0.05,1.33 ± 0.16(F=8.740,P＜ 0.01),protein:0.40 ± 0.03,0.69 ± 0.06,1.02 ± 0.10(F=90.785,P ＜ 0.01);CYP 2El mRNA:0.72 ± 0.14,1.00 ± 0.15,1.30 ± 0.20(H=4.713,P ＜ 0.01).The changes in hepatic FasL and CYP 2E 1 expression detected by immunohistochemistry were consistent with the mRNA expression.Conclusion Activation of Fas/FasL and its downstream signaling pathway,which induces hepatocellular apoptosis,contributes to the development of alcoholic steatohepatitis and liver fibrosis.
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