胍丁胺通过AMPK途径诱导间充质干细胞的凋亡

Agmatine Induces Apoptosis of MSCs Through the AMPK Pathway

间充质干细胞(mesenchymal stem cells,MSCs)具有强大的组织再生修复及免疫调节功能,广泛应用于损伤性和免疫相关性疾病的治疗,但其过早凋亡会降低临床疗效,且机制不明。研究发现,胍丁胺(agmatine,AGM)可通过抑制一氧化氮(nitric oxide,NO)生成发挥抗炎功能,对脓毒症小鼠具有保护效应。移植MSCs治疗脓毒症也是一种有效的治疗方式,但二者是否存在相互作用及AGM是否影响MSCs生存却未见报道。该研究旨在探讨AGM对MSCs生存的影响及其分子机制。研究MSCs之前,该研究通过检测细胞表面marker(CD29、CD34、CD44、CD45、CD90和CD105)及三系分化(成脂、成骨、成软骨)对其进行了鉴定。为了深入探究AGM对MSCs的作用,使用流式细胞术检测AGM处理后MSCs的凋亡率(Annexin V)以及Western blot检测经AGM处理后MSCs的凋亡相关信号通路蛋白p-AMPK、p-mTOR、p-S6K1、Cl-Caspase3的表达水平。之后进一步添加AMPK siRNA检测AGM诱导MSCs凋亡的分子机制。结果显示,AGM在体外以剂量依赖性的方式,通过抑制mTOR信号通路诱导AMP活化蛋白激酶(AMPK)活化,导致凋亡相关蛋白表达上调,从而介导MSCs凋亡。而AMPK siRNA处理能明显恢复mTOR通路,并抑制AMPK活化,使凋亡相关蛋白表达降低,进而减弱AGM诱导的凋亡效应。该文揭示了AGM可通过AMPK途径诱导MSCs的凋亡。

Mesenchymal stem cells(MSCs)are widely used in the treatment of injury and immune-related diseases because of their strong capacities of tissue regeneration and immunomodulation.However,the clinical efficacy of MSCs transplantation is limited by cell apoptosis and the mechanism is still unclear.It has been reported that agmatine(AGM)plays a protective role in septic mice through its anti-inflammatory function which inhibiting the production of nitric oxide(NO).Besides,MSCs transplantation is also an effective treatment for sepsis.However,whether AGM affects the survival of MSCs has not been reported,this study aims to investigate the effects of AGM on the survival of MSCs and its molecular mechanisms.Cell surface markers(CD29,CD34,CD44,CD45,CD90,CD105)and three lines of differentiation(adipogenic,osteogenesis and cartilage)had been identified before research.After treatment with AGM,MSCs’apoptosis and apoptosis-related signaling pathway markers were measured by flow cytometry and Western blot.It was found that AGM induced apoptosis of MSCs through AMPactivated protein kinase(AMPK)activation and mTOR signaling pathway inhibition in a dose-dependent manner which resulting in the up-regulation of apoptosis-related protein expression.However,AMPK siRNA treatment could significantly inhibit the AGM-induced apoptosis of MSCs through reversing mTOR inhibition,AMPK activation and decreasing the expression of apoptosis-related proteins.Our results demonstrated that AGM can induces MSCs apoptosis through the AMPK pathway.