生长分化因子11对软脂酸诱导小鼠主动脉内皮细胞损伤的保护作用

Protective effect of growth differentiation factor 11 on mice aorta endothelial cells cultured withpalmitate acids

目的探索生长分化因子11 （GDF-11）对软脂酸（PA）诱导的小鼠主动脉内皮细胞（MAECs）的作用及其相关机制。 方法通过加或不加内皮型一氧化氮合酶（eNOS）抑制剂N-硝基-L-精氨酸甲酯（L-NAME）或Smad3抑制剂SIS3研究eNOS和Smad通路在GDF-11保护软脂酸诱导的MAECs中的作用，将MAECs分为：（1）对照组；（2）PA组；（3）PA＋GDF-11组；（4）PA＋GDF-11＋L-NAME组；（5）PA＋GDF-11＋SIS3组，培养24 h后，活性氧试剂盒及实时-聚合酶链式反应（RT-PCR）检测各组细胞氧化应激水平；流式细胞术检测各组细胞凋亡率；Western blotting法检测各组抗凋亡蛋白Bcl-2、凋亡蛋白Bax及Cleaved-caspase3蛋白表达水平。研究GDF-11对MAECs Smad信号通路的影响，MAECs分为：（1）对照组；（2）GDF-11组；（3）GDF-11＋转化生长因子β（TGF-β）受体Ⅰ抑制剂组（GDF-11＋ SB431542组），培养30 min后，免疫荧光染色法检测各组MAECs细胞磷酸化Smad2/3 （P-Smad2/3）表达水平；研究GDF-11对MAECs AMPK/eNOS信号通路的影响，MAECs分为：（1）对照组；（2）GDF-11组；（3） GDF-11＋AMPK抑制剂Compound C组；（4）GDF-11＋L-NAME组；（5）GDF-11＋Compound C＋L-NAME组，培养30 min后，Western blotting法检测各组细胞AMPK、磷酸化AMPK （P-AMPK）、eNOS、磷酸化eNOS （P-eNOS）、蛋白激酶B（Akt）、磷酸化Akt （P-Akt）、蛋白激酶A （PKA）、磷酸化PKA （P-PKA）水平。多组之间比较采用单因素方差分析，组间多重比较用最小显著性差异t检验。 结果与对照组相比，PA组细胞氧化应激和凋亡率显著增加（28.9%±2.2％比7.9%±1.5%），PA＋GDF-11组细胞氧化应激和凋亡率明显低于PA组（12.6%±1.6％比28.9%±2.2%），而PA＋GDF-11＋L-NAME组和PA＋GDF-11＋SIS3组细胞氧化应激和凋亡率又明显低于PA＋GDF-11组（分别为21.8%±2.0%、20.1%±1.8%、12.6%±1.6%，F= 97.89，P〈0.05）。与对照组相比，GDF-11组P-Smad2/3表达水平明显增加，而GDF-11＋SB431542组P-Smad2/3表达水平明显降低（F=325.30 ，P〈0.05）。与对照组相比，GDF-11组P-AMPK/AMPK、P-eNOS/eNOS水平显著提高，P-Akt/Akt、P-PKA/PKA水平无明显差异，分别加入了Compound C、L-NAME或者Compound C联合L-NAME显著抑制了P-AMPK/AMPK、P-eNOS/eNOS表达水平（F=237.65、281.08，均P〈0.05），而对P-Akt/Akt、P-PKA-PKA水平无明显影响（F=1.94、1.48，均P〉0.05）。 结论GDF-11可抗软脂酸诱导的MAECs氧化应激和凋亡，增加Bcl-2表达水平、降低Bax、Cleaved-caspase3表达水平，其保护内皮细胞的机制与激活TGF-β/Smad和AMPK/eNOS信号途径有关。

Objective To explore the effects of growth differentiation factor 11 （GDF-11） on mice aorta endothelial cells （MAECs） cultured with palmitate acids （PA） and the possible mechanism involved.Methods To study the function of endothelial nitric oxide synthase（eNOS） and Smad pathways in GDF-11 protective effect by pre-incubated with inhibitors against eNOS（L-NAME） or Smad3 inhibitor（SIS3）. MAECs were divided into five groups：the control group, PA group, PA＋GDF-11 group, PA＋GDF-11 ＋L-NAME group and PA＋GDF-11 ＋SIS3 group, all the cells in the 5 groups were cultured for 24 h. Reactive oxygen species assay kit and reverse transcription polymerase chain reaction（RT-PCR） were used to determine the oxidative stress in MAECs and flow cytometry were used to assess the apoptosis of cells in the 5 groups. And Western blotting was used to measure the expression levels of Bcl-2, Bax and Cleaved-caspase3 protein. To study the effect of GDF-11 on the Smad signal pathway in MAECs. MAECs were divided into three groups： the control group, GDF-11 group and GDF-11 ＋transforming growth factor （TGF-15） receptor I inhibitor （SB431542） group. After cultured for 30 rain, the expression levels of phosphorylated Smad2/3 （P-Smad2/3） were measured by immunofluorescence in the three groups. In order to study the effect of GDF-11 on the adenosine monophosphate-activated protein kinase （AMPK）/eNOS pathway in MAECs, MAECs were divided into five groups： the control group, GDF-I 1 group, GDF-1 I＋AMPK inhibitor （Compound C） group, GDF-11＋ L-NAME group and GDF-11＋Compound C＋L-NAME group. After cultured for 30 rain, the expression levels of AMPK, phosphorylated AMPK（P-AMPK）, cAMP-dependent protein kinase （PKA）, phosphorylated PKA （P-PKA）, protein kinase B（Akt）, phosphorylated Akt（P-Akt）, eNOS and phosphorylated eNOS（P-eNOS） were measured by Western blotting in the 5 groups. Data among multiple groups were compared by using single factor variance analysis and the least significant difference t test. Results The apoptotic rate of MAECs in PA group was significantly higher than that in control group （28.9%± 2.2% vs 7.9% ±1.5% ）, while it decreased significantly in PA＋GDF-11 group （12.6%±1.6% vs 28.9%±2.2%）. The apoptotic rate of the cells in PA＋GDF-11 ＋L-NAME group and PA＋GDF-11 ＋SIS3 group were all significantly higher than that in PA＋ GDF-11 group （21.8%±2.0% vs 20.1%±1.8% vs 12.6%± 1.6%, F=97.89, P〈0.05）. Compared with control group, the expression of P-Smad2/3 increased significantly in GDF-I l group, but it decreased significantly in GDF-11 ＋SB431542 group（F=325.30, P〈0.05）. The relative P-AMPK/AMPK, P-eNOS/eNOS levels in GDF-11 group were significantly higher than those in control group. The relative P-AMPK/AMPK, P-eNOS/ eNOS levels in Compound C, L-NAME and Compound C＋L-NAME group were significantly lower than those in GDF-11 group （F=237.65, 281.08, both P〈0.05）. Whereas the relative P-Akt/Akt, P-PKA/PKA levels showed no difference among those groups （F=1.94, 1.48, both P〉0.05）. Conclusions GDF-11 prevents palmitate acids-induced endothelial oxidative stress and apoptosis, increases the expression of Bel-2 and decreases the levels of Bax, Cleaved-caspase3 in the MAECs. The possible mechanisms may involve the TGF-β/Smad and the AMPK/eNOS signaling pathways.