核糖体蛋白S6激酶1基因沉默对高脂喂养小鼠肝脏炎性因子和肝脏胰岛素抵抗的影响

Ribosomal protein $6 kinase 1 partakes hepatic insulin resistance by regulating expression of inflammatory cytokines

目的研究核糖体蛋白S6激酶1（S6K1）基因沉默对肝脏炎性因子表达的影响，探讨S6K1在肝脏胰岛素抵抗中的作用机制。方法将48只体重12．6～14．8g的6周龄雄性C57BL／6J小鼠按随机数字表法分为4组：普食对照组[普通饲料（ND）＋含U6启动子空载体腺病毒组（pU6Ax组），即ND＋pU6Ax组]、普食实验组[ND＋S6K1短发夹RNA（shRNA）重组基因腺病毒组，即ND＋S6KIAx组]、高脂对照组[高脂饲料（HFD）’pU6Ax组，即HFD＋pU6Ax组]、高脂实验组（HFD＋S6K1Ax组），分别喂养16周。实验组和对照组尾静脉分别注射S6K1Ax、pU6Ax（均为普食实验组及对照组90ml，高脂实验组及对照组120ml，效价为2．0×10^10pfu／m1）。注射后第6天过夜饥饿后，4组各随机抽取6只行葡萄糖耐量实验，剩余的小鼠处死取肝脏，逆转录-聚合酶链反应（RT—PCR）检测肝脏炎性因子单核细胞趋化蛋白-1（MCP-1）、肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-6、IL-10mRNA的表达，Western blotting观察肝脏蛋白S6K1、S6K1苏氨酸389（S6K1-thr389）、胰岛素受体底物1（IRS1）丝氨酸1101（IRS1-ser1101）、IRS1丝氨酸636／639（IRS1-ser636／639）、蛋白激酶B丝氨酸473（Akt—ser473）、c—Jun氨基末端激苏氨酸183／酪氨酸185（JNK—thr183／tyr185）表达。两组比较采用t检验，多组问比较采用单因素方差分析。结果肝脏S6KI基因沉默后，与高脂对照组相比，HFD＋S6K1Ax组小鼠炎性因子MCP-1、TNF-α、IL-1βmRNA均表达下降（分别为0．549±0．016比0．871±0．081、0．635±0．079比0．905±0．059、0．642±0．042比1．327±0．025；t=9．55、6．71、34．07，均P〈0．05）。IL-6mRNA未表现出组间差异（P〉0．05），抗炎因子IL-10mRNA在HFD＋S6KIAx组肝脏S6K1基因沉默后升高（0．773±0．076比0．436±0．046，t=9．27，P〈0．05）。Western blotting显示与HFD＋pU6Ax组相比，HFD＋S6K1Ax组肝脏S6K1基因沉默后，IRS1－serll01、IRS1-ser636／639、S6K1-thr389、JNK—thr183／tyr185表达下降，Akt—ser473表达增强（t=6．59、8．44、5．37、3．15、6．52，均P〈0．05）。结论高脂喂养可引起肝脏低度炎症并介导胰岛素抵抗的发生，肝脏S6K1可能通过促进炎症因子、抑制抗炎因子表达参与了肝脏胰岛素抵抗发生。

Objective To study the effect of S6K1 silencing to hepatic inflammatory cytokines factors expression and explore the mechanism of S6K1 in the hepatic insulin resistance. Methods Forty eight male C57BL/6J mice （ six-week old ） with body weight 12.6 to 14. 8 g were randomly divided into four groups by using random number table： normal chow control group（ ND ± pU6Ax）, normal chow study group （ND ±S6K1Ax）, high fat diet control group （HFD ± pU6Ax） and high fat diet study group（ HFD ±S6K1Ax）. For the depletion of S6K1 in the liver, C57BL/6J male mice by high fat diet and normal chow feeding forl6 weeks were injected via the tail vein with S6K1 shRNA recombinant adenovirus （S6K1Ax） , U6 promoter recombinant adenovirus（pU6Ax） were injected into the control mice. Six days after virus injection, mice were killed and livers were obtained. Hepatic inflammatory cytokines expressions of Monocyte chemotactic protein 1 （ MCP-1 ）, Tumor Necrosis Factor-c~ （ TNF-ct）, Interleukin-1 [3 （ IL-113）, Interleukin-6 （IL-6）, Interleukin-10（IL-10） mRNA were examined by reverse transcription polymerase chain reaction, Hepatic protein expressions of S6K1, S6Kl-thr389, IRSl-serll01, IRSl-ser636/639, Akt-ser473, JNK- thr183/tyr185 were detected by western blotting. Results Comparing with the high fat diet control group, inflammatory factors of MCP-1, TNF-α, IL-113 mRNA in HFD ± S6K1Ax group showed lower expressions after hepatic S6K1 silencing（ MCP-1 0. 871 ±0. 081 vs 0. 549 ±0. 016,TNF-α 0. 905 ±0. 059 vs 0. 635 ±0. 079, IL-1β 1. 327 ± 0. 025 vs 0. 642 ± 0. 042, t = 9. 55,6. 71,34. 07, all P 〈 0.05 ）. There was no difference in IL-6 mRNA expressions between the two groups（ P 〉 0. 05 ）. Anti-inflammatory factor of IL-10 mRNA in the HFD ± S6K1Ax group increased after hepatic S6K1 silencing （0. 773 ± 0. 076 vs 0. 436 ± 0. 046, t = 9. 27, P 〈 0. 05 ）. Compared with the HFD ± pU6Ax group, the protein expressions of ISRl-ser1101, IRS1-ser636/ 639,S6K1-thr389,JNK-thr183/tyr185 decreased and protein expression of Akt-ser473 increased in the HFD ± S6K1Ax group by Western blotting detection after hepatic S6K1 silencing （ t = 6. 59,8.4g, 5.37,3.15, 6.52,all P 〈 0. 05 ）. Conclusions High fat diet feeding can cause low grade inflammation and medicate insulin resistance in the liver. Hepatic S6K1 maybe participate hepatic insulin resistance by promoting inflammatory cytokines and inhibiting anti-inflammatory cytokine production.