核糖体蛋白S6激酶1基因沉默对小鼠非酒精性脂肪性肝病的影响
핵당체단백S6격매1기인침묵대소서비주정성지방성간병적영향
Effects of ribosomal protein S6 kinase 1 gene silencing on the pathogenesis of non-alcoholic fatty liver disease in mice
  • 万方数据
    中华糖尿病杂志 중화당뇨병잡지
    CHINES JOURNAL OF DLABETES MELLITUS
    2011年 4期 328-332 ,共5页

    李树颖%于德民%小川涉%春日雅人

    리수영%우덕민%소천섭%춘일아인

    脂肪肝%小鼠%核糖体蛋白S6激酶类,70-kDa%基因沉默%胰岛素抵抗 脂肪肝%小鼠%覈糖體蛋白S6激酶類,70-kDa%基因沉默%胰島素牴抗
    지방간%소서%핵당체단백S6격매류,70-kDa%기인침묵%이도소저항
    Fatty liver%Mice%Ribosomal protein S6 kinases,70-kDa%Gene silencing%Insulin resistance
    目的 探讨核糖体蛋白S6激酶1基因沉默对db/db小鼠非酒精性脂肪性肝病的影响及其作用机制.方法 根据随机数字表将12只体质量44.5～48.2 g的9周龄雄性db/db小鼠随机分至对照组(n=6)和实验组(n=6).实验组db/db小鼠尾静脉注射制备的核糖体蛋白S6激酶1短发夹RNA重组基因腺病毒,对照组db/db小鼠尾静脉注射含U6启动子空载体腺病毒.在注射病毒后第6天处死小鼠获取肝脏,提取肝脏蛋白,利用Western blot法观察其中胰岛素受体底物1、胰岛素受体底物2、蛋白激酶B及其丝氨酸473蛋白的表达;提取肝脏总RNA用实时荧光定量逆转录聚合酶链反应法检测比较2组参与肝脏脂肪酸合成基因mRNA的表达.处死2组小鼠前1 d禁食16 h后经尾静脉取血,采用比色法检测游离脂肪酸、甘油三酯、总胆固醇水平.组间数据比较采用t检验.结果 db/db小鼠注射病毒后6 d,苏木素-伊红染色显示实验组肝细胞胞质含脂肪滴较对照组减少,脂肪肝得到改善.Western blot检测显示实验组核糖体蛋白S6激酶1蛋白表达被抑制,与对照组相比差异有统计学意义(分别为0.12±0.01和0.87±0.06,t=5.36,P＜0.05);实验组胰岛素受体底物1、胰岛素受体底物2、蛋白激酶B丝氨酸473的蛋白表达均较对照组增强(均P＜0.05).和对照组相比,实验组参与脂肪酸合成的基因固醇调节元件结合蛋白1c(分别为2.33±0.29和1.34±0.39,t=3.46,P＜0.01)、脂肪酸合成酶(分别为7.8±1.2和3.4±0.4,t=4.67,P＜0.01)、硬脂酰辅酶A去饱和酶1(764±116和535±54,t=6.12,P＜0.01)mRNA表达均明显下降.血清生化测定结果显示和对照组相比实验组脂肪酸下降(t=2.64,P＜0.05),胆固醇水平降低(t=4.25,P＜0.01),甘油三酯组间比较差异无统计学意义(P＞0.05).结论在营养过剩的条件下,肝脏核糖体蛋白S6激酶1过度激活,可能通过负反馈引起肝脏胰岛素信号传导下降、上调脂代谢关键调控基因固醇调节元件结合蛋白1c表达而参与了肝脏胰岛素抵抗和脂肪肝的发生.
    목적 탐토핵당체단백S6격매1기인침묵대db/db소서비주정성지방성간병적영향급기작용궤제.방법 근거수궤수자표장12지체질량44.5～48.2 g적9주령웅성db/db소서수궤분지대조조(n=6)화실험조(n=6).실험조db/db소서미정맥주사제비적핵당체단백S6격매1단발협RNA중조기인선병독,대조조db/db소서미정맥주사함U6계동자공재체선병독.재주사병독후제6천처사소서획취간장,제취간장단백,이용Western blot법관찰기중이도소수체저물1、이도소수체저물2、단백격매B급기사안산473단백적표체;제취간장총RNA용실시형광정량역전록취합매련반응법검측비교2조삼여간장지방산합성기인mRNA적표체.처사2조소서전1 d금식16 h후경미정맥취혈,채용비색법검측유리지방산、감유삼지、총담고순수평.조간수거비교채용t검험.결과 db/db소서주사병독후6 d,소목소-이홍염색현시실험조간세포포질함지방적교대조조감소,지방간득도개선.Western blot검측현시실험조핵당체단백S6격매1단백표체피억제,여대조조상비차이유통계학의의(분별위0.12±0.01화0.87±0.06,t=5.36,P＜0.05);실험조이도소수체저물1、이도소수체저물2、단백격매B사안산473적단백표체균교대조조증강(균P＜0.05).화대조조상비,실험조삼여지방산합성적기인고순조절원건결합단백1c(분별위2.33±0.29화1.34±0.39,t=3.46,P＜0.01)、지방산합성매(분별위7.8±1.2화3.4±0.4,t=4.67,P＜0.01)、경지선보매A거포화매1(764±116화535±54,t=6.12,P＜0.01)mRNA표체균명현하강.혈청생화측정결과현시화대조조상비실험조지방산하강(t=2.64,P＜0.05),담고순수평강저(t=4.25,P＜0.01),감유삼지조간비교차이무통계학의의(P＞0.05).결론재영양과잉적조건하,간장핵당체단백S6격매1과도격활,가능통과부반궤인기간장이도소신호전도하강、상조지대사관건조공기인고순조절원건결합단백1c표체이삼여료간장이도소저항화지방간적발생.
    Objective To investigate the effects of ribosomal protein S6 kinase 1 (S6K1) gene silencing on the pathogenesis of non-alcoholic fatty liver disease.Methods Twelve 9-week male db/db mice ( body weight 44.5 to 48.2 g) were randomly assigned to the normal control group ( n = 6) and the study group( n= 6 ).The mice in the study group were injected with S6K1 short hairpin RNA recombinant adenovirus (S6K1 Ax) via the tail vein, and the control group was given U6 promoter recombinant adenovirus(pU6Ax).Six days after virus injection, db/db mice were killed and livers were harvested.Hepatic protein expression of insulin receptor substrate 1 ( IRSI ), insulin receptor substrate 2(IRS2) and protein kinease B (Akt), Akt473 was determined by Western blot in the two groups.Total hepatic RNAs were extracted to analyze genes expression of fatty acids synthesis by using real-time quantitative reverse transcription polymerase chain reaction.Blood was collected after 16 h of fasting before db/db mice were killed.The serum free fatty acids, triglyceride and cholesterol levels were quantified by colorimetry.The data of the two groups were compared with t-test.Results Six days after virus injection, fat droplet in hepatocyte decreased in study group compared with that in control group under HE staining observation and the fatty liver in study group was improved.Protein expression of S6K1 in the study group was down-regulated significantly compared with that in control group (0.12 ± 0.01 vs 0.87 ± 0.06, t = 5.36, P ＜ 0.05 ); and the expression of IRS1, IRS2 and Akt473 were up-regulated in the study group than those in the control group (all P ＜0.05).Compared with those in control group, fatty acid synthesis genes of sterol regulatory element binding protein 1c (SREBP1c, 2.33 ±0.29 vs 1.34 ±0.39, t =3.46, P ＜0.01 ), fatty acid synthesis (7.8 ± 1.2 vs 3.4 ± 0.4, t = 4.67, P ＜ 0.01 ), stearoyl-CoA desaturase 1 ( SCD1, 764 ± 116 vs 535 ±54, t = 6.12, P ＜0.01 ) mRNA expression descended in the study group.Fasting blood FFA and cholesterol decreased in the study group compared with those in the normal group ( t = 2.64, P ＜ 0.05; t =4.25, P ＜0.01 ).No significant difference in serum triglycerol was detected between the two groups (P ＞0.05).Conclusions Given excess nutrient, over-activated hepatic ribosomal protein S6 kinase 1 maybe cause hepatic insulin resistance and fatty liver through negative feedback and up-regulating SREBP1c expression.
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