摘要目的 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.

abstractsObjective 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 β(TGF-β) receptor Ⅰinhibitor (SB431542) group. After cultured for 30 min, 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-11 group, GDF-11+AMPK inhibitor (Compound C) group, GDF-11+L-NAME group and GDF-11+Compound C+L-NAME group. After cultured for 30 min, 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-11 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 Bcl-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.