促红细胞生成素对棕榈酸诱导肝癌HepG2细胞胰岛素信号通路活性的作用

Role for erythropoietin on insulin signaling of palmitate treated HepG2 cells

目的：研究促红细胞生成素（EPO）改善棕榈酸（PA）诱导肝癌HepG2细胞胰岛素信号通路活性的作用及机制。方法以PA（250 nmol/L）及EPO（10 U）或烟酰胺腺嘌呤二核苷酸依赖的组蛋白去乙酰化酶（SIRT1）激动剂白藜芦醇（Rsv,10 nmol/L）干预HepG2细胞，分为6组：对照组、胰岛素组（Ins）、PA组、PA＋Ins组、PA＋EPO组、PA＋Rsv组；将含SIRT1的小干扰RNA（siRNA）转染HepG2细胞，再分为对照组、EPO组、EPO＋阴性siRNA组、EPO＋siSIRT1组及相应的PA干预组。Western blotting法检测胰岛素受体底物2（IRS-2）、磷酯酰肌醇3激酶/蛋白激酶B（PI3K/AKT）及SIRT1蛋白水平，定量逆转录多聚酶链反应（qRT-PCR）检测SIRT1基因变化。采用独立样本t检验进行两组间均数比较。结果与对照组比较（1.00±0.03），PA组的pIRS-2（Ser731）蛋白升高，pAKT（Ser473）、SIRT1蛋白水平降低（分别为1.25±0.06、0.77±0.02、0.74±0.03，t=6.048、-13.686、-10.649，均P〈0.05）。经EPO干预后，与PA组相比，pIRS-2（Ser731）蛋白降低，pAKT（Ser473）、SIRT1蛋白水平升高（分别为0.70±0.03比1.00±0.11、1.60±0.14比1.00±0.08、1.39±0.03比1.00±0.03，t=-4.853、6.330、25.868，均P〈0.05）。当使用siRNA下调SIRT1后，生理及PA状态下EPO激活HepG2细胞胰岛素信号通路的作用均被阻断。结论在PA处理的HepG2细胞中，EPO能够通过促进SIRT1蛋白表达，从而激活受损的胰岛素信号通路。

Objective To investigate the role for erythropoietin（EPO）on insulin signaling of palmitate （PA） induced HepG2 cells. Methods HepG2 cells were intervened with palmitic acid （250 nmol/L）, EPO （10 U） and resveratrol （Rsv, 10 nmol/L）, activator of nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1 （SIRT1）, respectively. Cells were randomly assigned into： control, insulin （Ins, 100 pmol/L）, palmitic acid （PA）, PA＋EPO （10 U） and PA＋resveratrol （10 nmol/L）, respectively. Then HepG2 cells under both normal and palmitic acid were transfected with SIRT1 RNAi mixed with lipofacfectamine 2000 and were divided into following four groups：control,EPO,EPO ＋ Scramble,EPO ＋ siSIRT1. The expression of insulin receptor substrate 2 （IRS-2）, phosphatidylinositol 3 kinase/ protein kinase B（PI3K/AKT） and SIRT1 were detected by Western blotting. The expression of SIRT1 mRNA level was determined by quantitative reverse transcription polymerase chain reaction（qRT-PCR）. Independent samples t-test was used for data analysis. Results Compared with control group （1.00 ± 0.03）, levels of pIRS-2（Ser731） increased and pAKT（Ser473） ,SIRT1 decreased significantly in palmitate induced HepG2 cells （1.25±0.06, 0.77±0.02, 0.74±0.03, t=6.048,-13.686,-10.649, respectively, all P〈0.05）. After EPO treatment, levels of pAKT（Ser473） and SIRT1 under insulin resistance conditions were significantly enhanced while pIRS-2 （Ser731） were significantly decreased （1.60±0.14 vs 1.00±0.08, 1.39±0.03 vs 1.00±0.03,0.70±0.03 vs 1.00± 0.11, t=6.330, 25.868,-4.853, all P〈0.05）. However, when SIRT1 expression was inhibited by small interfering RNA （SiRNA）, neither PI3Kp85 nor pAKT（Ser473） levels were altered in response to EPO under both normal and insulin resistant conditions. Conclusion EPO positively regulates impaired PI3K/AKT signaling caused by PA through SIRT1 in HepG2 cells.