低浓度H_2O_2预处理增强小鼠BMSCs抗氧化应激损伤能力

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(P0.05,P0.01);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。

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

Objective To investigate the effects of preconditioning with low-concentration hydrogen peroxide ( H2O2 ) on oxidative stress-induced bone marrow mesenchymal stem cells ( BMSCs ) apoptosis and its mecha-nism.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 H2O2 after preincubation with 50 μmol/L H2O2 or control medium.Apoptosis of these cells was measured by flow cytometry, and the expression of phos-phorylated PI3K, Akt and mTOR was analyzed by Western blot; BMSCs were also primed with PI3K inhibitor LY294002 for 30 min, then preincubated with 50 μmol/L H2O2 or control medium for 12 h before treatment with 300 μmol/L H2O2.Expression of apoptosis proteins Bcl-2, Bax, caspaase-3, cleaved-caspase-3 and the key pro-teins of the PI3K/Akt/mTOR pathway were detected by Western blot .Results H2O2 induced BMSCs apoptosis in a dose-dependent manner ,and pretreatment of BMSCs with low concentration of H2O2 significantly decreased H2O2-induced apoptosis of the BMSCs .Western blot results revealed that preconditioning with low-concentration H2O2 re-markably reversed the decrease in Bcl-2, total and phosphorylated PI3K, Akt and mTOR levels, and increased in Bax, cleaved-caspase-3 expression after high-dose H2O2 treatment.Such effects were antagonized by PI3K inhibitor LY294002 .Conclusions Preincubation with low-concentration H2O2 may indnce resistance of BMSC to oxidative stress, and such effect may be mediated by inhibition of pro-apoptotic proteins and activation of the PI 3K/Akt/mTOR pathway .