鸢尾素通过激活3-磷酸磷脂酰肌醇激酶-蛋白激酶B-内皮型一氧化氮合酶信号转导途径抗高糖诱导的人脐静脉内皮细胞凋亡

Irisin prevents high glucose-induced human umbilical vein endothelial cell apoptosis via phosphatidylinositol-3-kinase-protein kinase B-endothelial nitric oxide synthase signaling pathway

目的：探索鸢尾素对高糖培养的人脐静脉内皮细胞（HUVECs）作用及其相关机制。方法 HUVECs加或不加磷脂酰肌醇-3-激酶（PI3K）抑制剂LY294002或（和）内皮型一氧化氮合酶（eNOS）抑制剂N-硝基-L-精氨酸甲酯（L-NAME）预培养30 min后，按是否加入鸢尾素分为6组：正糖（NG）组、高糖（HG）组、高糖＋鸢尾素组、高糖＋LY294002＋鸢尾素（LY）组、高糖＋L-NAME＋鸢尾素（L-NAME）组、高糖＋LY294002＋L-NAME＋鸢尾素（LY＋L-NAME）组，培养48 h后应用原位末端标记（TUNEL）法检测各组细胞凋亡率，活性氧试剂盒及实时-聚合酶链反应（RT-PCR）检测各组细胞氧化应激水平，Western blotting法检测各组细胞蛋白激酶B（Akt）、磷酸化Akt（P-Akt）、eNOS、磷酸化eNOS （P-eNOS）水平。HUVECs以随机序列小干扰RNA（Scr-siRNA）转染作对照或以eNOS干扰RNA （eNOS-siRNA）转染沉默eNOS基因后，分成4组：正糖＋Scr-siRNA（NG＋Scr-siRNA）组、高糖＋Scr-siRNA（HG＋Scr-siRNA）组、高糖＋Scr-siRNA＋鸢尾素（HG＋Scr-siRNA＋irisin）组、高糖＋eNOS-siRNA＋鸢尾素（HG＋eNOS-siRNA＋irisin）组，培养48 h后应用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）双染法检测各组细胞凋亡率。采用单因素方差分析（ANOVA）法比较各组数据。结果高糖组HUVECs氧化应激和凋亡率比正糖组明显升高（25.6%±3.2%比6.0%±1.5%，t=-13.55，P〈0.01），而鸢尾素组凋亡率较高糖组显著下降（19.8%±2.3%比25.6%±3.2%，t=-3.55，P〈0.01）。LY组、L-NAME组和LY＋L-NAME组HUVECs凋亡率均较鸢尾素组显著升高（t=-3.85、-5.19、-7.11，均P〈0.01）。HUVECs的eNOS基因沉默后，高糖＋Scr-siRNA组细胞凋亡率比正糖＋Scr-siRNA组显著增加（58.0%±7.8%比12.4%±2.8%，t=-13.48，P〈0.01），高糖＋Scr-siRNA＋鸢尾素组细胞凋亡率明显低于高糖＋Scr-siRNA组（27.8%±5.3%比58.0%±7.8%，t=-7.84，P〈0.01），而高糖＋eNOS-siRNA＋鸢尾素组细胞凋亡率显著高于高糖＋Scr-siRNA＋鸢尾素组（48.1%±5.5%比27.8%±5.3%，t=-6.48，P〈0.01）。高糖组P-Akt/Akt、P-eNOS/eNOS水平比正糖组显著降低（均P〈0.01），鸢尾素组P-Akt/Akt、P-eNOS/eNOS水平明显高于高糖组（均P〈0.01），而LY组、L-NAME组及LY＋L-NAME组P-Akt/Akt、P-eNOS/eNOS水平较鸢尾素组均显著降低（均P〈0.01）。结论鸢尾素可抗高糖诱导的HUVECs氧化应激和凋亡，其保护内皮细胞的机制与激活PI3K-Akt-eNOS信号转导途径有关。鸢尾素具有潜在的防治糖尿病血管并发症的临床价值。

Objective To explore the effects of irisin on endothelial cells cultured with high glucose and the possible mechanism-involved. Methods Human umbilical vein endothelial cells （HUVECs） were pre-incubated with inhibitors against phosphatidylinositol-3-kinase （PI3K） （LY294002） or endothelial nitric oxide synthase（eNOS） （L-NAME） for 30 min before irisin was added, then divided into 6 groups： normal glucose （NG） group, high glucose （HG） group, high glucose＋irisin （Irisin） group, high glucose＋LY294002＋irisin （LY） group, high glucose＋L-NAME＋irisin（L-NAME） group, high glucose＋LY294002＋L-NAME＋irisin （LY＋L-NAME） group, cultured for 48 h. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling （TUNEL） staining was applied to identify the apoptotic endothelial cells. Reactive oxygen species assay kit and reverse transcription polymerase chain reaction（RT-PCR） was used to determine the oxidative stress in HUVECs. The expression levels of protein kinase B（Akt）, phospheralyted Akt（P-Akt）, eNOS and phospheralyted eNOS（P-eNOS） were measured by Western blotting. Moreover, HUVECs were transfected with eNOS-siRNA, and cultured in high glucose and then divided into 4 groups：NG＋Scr-siRNA group, HG＋Scr-siRNA group, HG＋Scr-siRNA＋irisin group and HG＋eNOS-siRNA＋irisin group, cultured for 48 h. After treatment, the cells were double-stained with fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） and analyzed by flow cytometry. Data were compared using one-way analysis of variance （ANOVA）. Results The apoptotic rate of HUVECs in HG group were significantly higher than that in NG group （25.6%± 3.2% vs 6.0%± 1.5%, t=-13.55, P〈0.01）, while it significantly decreased in Irisin group than that in HG group （19.8%± 2.3% vs 25.6%± 3.2%, t=-3.55, P〈0.01）. The apoptotic rate of HUVECs in LY, L-NAME and LY＋L-NAME group were all significantly higher than that in Irisin group （t=-3.85,-5.19,-7.11, all P〈0.01）. After eNOS gene silencing in HUVECs, the cellular apoptotic rate significantly increased in HG＋Scr-siRNA group when compared with that in NG＋Scr-siRNA group （58.0%±7.8%vs 12.4%±2.8%, t=-13.48, P〈0.01）. The cellular apoptotic rate significantly decreased in HG＋Scr-siRNA＋irisin group when compared with that in HG＋Scr-siRNA group （27.8%±5.3%vs 58.0%± 7.8%, t=-7.84, P〈0.01）, while it significantly increased in HG＋eNOS-siRNA＋irisin group than that in HG＋Scr-siRNA＋irisin group （48.1%±5.5%vs 27.8%±5.3%, t=-6.48, P〈0.01）. The relative P-Akt/Akt, P-eNOS/eNOS levels in HG group were significantly lower than those in NG group （all P〈0.01）. The relative P-Akt/Akt, P-eNOS/eNOS levels in irisin group were significantly higher than those in HG group （all P〈0.01）. The relative P-Akt/Akt, P-eNOS/eNOS levels in LY, L-NAME and LY＋L-NAME group were significantly lower than those in Irisin group （all P〈0.01）. Conclusions Irisin prevents high glucose-induced endothelial oxidative stress and apoptosis. The protective effect of irisin on HUVECs apoptosis is mediated through the PI3K-Akt-eNOS signaling pathway. Irisin could be a potential therapeutic approach for diabetic vascular complications.