低浓度H_2O_2预处理增强小鼠BMSCs抗氧化应激损伤能力

Preincubation with low dose of hydrogen peroxide enhances anti-oxidative stress potential of mouse BMSCs

目的探讨PI3K/Akt/m TOR信号通路介导的低浓度过氧化氢(H_2O_2)预处理增强骨髓间充质干细胞(BMSCs)抗氧化应激损伤的作用及机制。方法通过差速贴壁法分离培养小鼠BMSCs。BMSCs经或不经低浓度(50μmol/L)H_2O_2预处理12 h,再暴露于不同高浓度H_2O_2(200、250、300和500μmol/L)刺激24 h后,流式细胞术检测BMSCs凋亡;低浓度H_2O_2预处理12 h再暴露于300μmol/L H_2O_224 h后,Western blot检测凋亡相关蛋白Bcl-2、Bax、caspase-3和cleaved-caspase-3的表达以及对PI3K/Akt/m TOR信号通路的影响。结果 H_2O_2呈浓度依赖性诱导BMSCs凋亡,50μmol/L H_2O_2预处理可降低200~500μmol/L诱导的BMSCs凋亡率,以及能降低300μmol/L诱导的促凋亡蛋白Bax和cleaved-caspase-3的上调和抗凋亡蛋白Bcl-2及磷酸化Akt和m TOR蛋白的表达的下调(P<0.05,P<0.01);PI3K抑制剂LY294002可明显地阻断H_2O_2预处理引起的上述变化。结论低浓度H_2O_2预处理通过活化PI3K/Akt/m TOR信号通路增强骨髓间充质干细胞的抗氧化应激损伤能力。

Objective To investigate the effects of preconditioning with low-concentration hydrogen peroxide（ H_2O_2） on oxidative stress-induced bone marrow mesenchymal stem cells（ BMSCs） apoptosis and its mechanism. Methods Mouse bone marrow mesenchymal stem cells（ BMSCs） were isolated and purified by differential centrifugation,and were treated with 0,200,250,300,500 μmol / L H_2O_2 after preincubation with 50 μmol / L H_2O_2 or control medium. Apoptosis of these cells was measured by flow cytometry,and the expression of phosphorylated PI3 K,Akt and m TOR was analyzed by Western blot; BMSCs were also primed with PI3 K inhibitor LY294002 for 30 min,then preincubated with 50 μmol / L H_2O_2 or control medium for 12 h before treatment with300 μmol / L H_2O_2. Expression of apoptosis proteins Bcl-2,Bax,caspaase-3,cleaved-caspase-3 and the key proteins of the PI3 K / Akt / m TOR pathway were detected by Western blot. Results H_2O_2 induced BMSCs apoptosis ina dose-dependent manner,and pretreatment of BMSCs with low concentration of H_2O_2 significantly decreased H_2O_2-induced apoptosis of the BMSCs. Western blot results revealed that preconditioning with low-concentration H_2O_2 remarkably reversed the decrease in Bcl-2,total and phosphorylated PI3 K,Akt and m TOR levels,and increased in Bax,cleaved-caspase-3 expression after high-dose H_2O_2 treatment. Such effects were antagonized by PI3 K inhibitor LY294002. Conclusions Preincubation with low-concentration H_2O_2 may indnce resistance of BMSC to oxidative stress,and such effect may be mediated by inhibition of pro-apoptotic proteins and activation of the PI3 K / Akt /m TOR pathway.