Wang Shuo, Zhang Chao, Niyazi Sidikejiang, Zheng Long, Li Jiawei, Zhang Weitao, Xu Ming, Rong Ruiming, Yang Cheng, Zhu Tongyu
Department of Urology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China.
Shanghai Key Laboratory of Organ Transplantation, Shanghai, China.
J Transl Med. 2017 Feb 15;15(1):33. doi: 10.1186/s12967-017-1144-5.
Mesenchymal stem cell (MSC) has been widely explored in the past decade as a cell-based treatment for various diseases. However, poor survival of adaptively transferred MSCs limits their clinical therapeutic potentials, which is largely ascribed to the nutrient starvation. In this study, we determined whether a novel kidney protective peptide CHBP could protect MSCs against starvation and invested the underlying mechanisms.
MSCs were subjected to serum deprivation and CHBP of graded concentrations was administered. Cell viability and apoptosis were detected by CCK-8, Annexin V/PI assay and Hoechst staining. ROS generation, mitochondrial membrane potential indicated by JC-1 and mitochondrial mass were measured by flow cytometry. The location of cytochrome c within cells was observed under fluorescence microscopy. Expressions of Nrf2, Sirt3, and FoxO3a were analyzed by western blot. In addition, preconditioning MSCs with CHBP was applied to test the possible protection against starvation. Finally, the effect of CHBP on the differentiation and self-renewal capacity of MSCs was also examined.
CHBP improved cell viability and suppressed apoptosis in a dose dependent manner. Starvation resulted in the mitochondrial dysfunction and treatment of CHBP could alleviate mitochondrial stress by diminishing oxidative injury of ROS, restoring mitochondrial membrane potential and maintaining mitochondrial membrane integrity. Importantly, Nrf2/Sirt3/FoxO3a pathway was activated by CHBP and Sirt3 knockdown partially abolished the protection of CHBP. Moreover, MSCs pretreated with CHBP were more resistant to starvation. Under normal condition, CHBP exerted little effects on the differential and self-renewal capacity of MSCs.
The present study demonstrated the efficient protection of CHBP upon MSCs against starvation-induced mitochondrial dysfunction and apoptosis and indicated possible involvement of Nrf2/Sirt3/FoxO3a pathway in the protective effect.
在过去十年中，间充质干细胞（MSC）作为一种基于细胞的各种疾病治疗方法已被广泛研究。然而，适应性转移的间充质干细胞存活率低限制了其临床治疗潜力，这在很大程度上归因于营养饥饿。在本研究中，我们确定了一种新型肾保护肽CHBP是否能保护间充质干细胞免受饥饿影响，并探究其潜在机制。
对间充质干细胞进行血清饥饿处理，并给予不同浓度的CHBP。通过CCK-8、Annexin V/PI检测和Hoechst染色检测细胞活力和凋亡情况。通过流式细胞术检测活性氧（ROS）生成、由JC-1指示的线粒体膜电位以及线粒体质量。在荧光显微镜下观察细胞色素c在细胞内的定位。通过蛋白质免疫印迹法分析Nrf2、Sirt3和FoxO3a的表达。此外，用CHBP预处理间充质干细胞以测试其对饥饿的可能保护作用。最后，还检测了CHBP对间充质干细胞分化和自我更新能力的影响。
CHBP以剂量依赖的方式提高细胞活力并抑制凋亡。饥饿导致线粒体功能障碍，而CHBP处理可通过减少ROS的氧化损伤、恢复线粒体膜电位和维持线粒体膜完整性来减轻线粒体应激。重要的是，CHBP激活了Nrf2/Sirt3/FoxO3a通路，而Sirt3基因敲低部分消除了CHBP的保护作用。此外，用CHBP预处理的间充质干细胞对饥饿更具抵抗力。在正常条件下，CHBP对间充质干细胞的分化和自我更新能力影响很小。
本研究证明了CHBP对间充质干细胞免受饥饿诱导的线粒体功能障碍和凋亡具有有效保护作用，并表明Nrf2/Sirt3/FoxO3a通路可能参与了这种保护作用。
