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

Effects of ribosomal protein S6 kinase 1 gene silencing on the pathogenesis of non-alcoholic fatty liver disease in mice

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.

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.