Xia Wenzheng, Zhang Fengyun, Xie Congying, Jiang Miaomiao, Hou Meng
Department of Neurosurgery, First Affiliated Hospital, Wenzhou Medical University, Wenzhou, 325000, PR China.
Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, 150086, PR China.
Stem Cell Res Ther. 2015 Apr 22;6(1):82. doi: 10.1186/s13287-015-0076-3.
Mesenchymal stem cells (MSCs)-based therapies have had positive outcomes in animal models of cardiovascular diseases. However, the number and function of MSCs decline with age, reducing their ability to contribute to endogenous injury repair. The potential of stem cells to restore damaged tissue in older individuals can be improved by specific pretreatment aimed at delaying senescence and improving their regenerative properties. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that modulates age-related signaling pathways, and hence is a good candidate for rejuvenative function.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated from young (6-month-old) or aged (24-month-old) male donor rats. Cell proliferation was measured using the CCK8 cell proliferation assay; secretion of VEGF, bFGF, HGF, and IGF was assessed by RT-qPCR and ELISA. Apoptosis was induced by hypoxia and serum deprivation (hypoxia/SD) for up to 6 hr, and examined by flow cytometry. Expression levels of AMP-activated protein kinase (AMPK) and forkhead box class O 3a (FOXO3a) were detected by Western blotting. CD74 expression was assayed using RT-qPCR, Western blotting, and immunofluorescence.
In this study, we found that MSCs isolated from the bone marrow of aged rats displayed reduced proliferative capacity, impaired ability to mediate paracrine signaling, and lower resistance to hypoxia/serum deprivation-induced apoptosis, when compared to younger MSCs. Interestingly, pretreatment of aged MSCs with MIF enhanced their growth, paracrine function and survival. We detected enhanced secretion of VEGF, bFGF, HGF, and IGF from MIF-treated MSCs using ELISA. Finally, we show that hypoxia/serum deprivation-induced apoptosis is inhibited in aged MSCs following MIF exposure. Next, we found that the mechanism underlying the rejuvenating function of MIF involves increased CD74-dependent phosphorylation of AMPK and FOXO3a. Furthermore, this effect was abolished when CD74, AMPK, or FOXO3a expression was silenced using small-interfering RNAs(siRNA).
MIF can rejuvenate MSCs from a state of age-induced senescence by interacting with CD74 and subsequently activating AMPK-FOXO3a signaling pathways. Pretreatment of MSCs with MIF may have important therapeutic implications in restoration or rejuvenation of endogenous bone marrow-MSCs in aged individuals.
基于间充质干细胞（MSC）的疗法在心血管疾病动物模型中已取得积极成果。然而，MSC的数量和功能会随着年龄增长而下降，从而降低其对内源性损伤修复的贡献能力。通过旨在延缓衰老并改善其再生特性的特定预处理，可以提高干细胞在老年个体中修复受损组织的潜力。巨噬细胞迁移抑制因子（MIF）是一种促炎细胞因子，可调节与年龄相关的信号通路，因此是具有恢复活力功能的良好候选因子。
从年轻（6个月大）或老年（24个月大）雄性供体大鼠中分离骨髓来源的间充质干细胞（BM-MSC）。使用CCK8细胞增殖测定法测量细胞增殖；通过RT-qPCR和ELISA评估VEGF、bFGF、HGF和IGF的分泌。通过缺氧和血清剥夺（缺氧/SD）诱导细胞凋亡长达6小时，并通过流式细胞术进行检测。通过蛋白质印迹法检测AMP活化蛋白激酶（AMPK）和叉头框O类3a（FOXO3a）的表达水平。使用RT-qPCR、蛋白质印迹法和免疫荧光法检测CD74的表达。
在本研究中，我们发现与年轻的MSC相比，从老年大鼠骨髓中分离的MSC显示出增殖能力降低、介导旁分泌信号的能力受损以及对缺氧/血清剥夺诱导的细胞凋亡的抵抗力降低。有趣的是，用MIF对老年MSC进行预处理可增强其生长、旁分泌功能和存活率。我们使用ELISA检测到MIF处理的MSC中VEGF、bFGF、HGF和IGF的分泌增加。最后，我们表明在MIF处理后，老年MSC中缺氧/血清剥夺诱导的细胞凋亡受到抑制。接下来，我们发现MIF恢复活力功能的潜在机制涉及增加CD74依赖性的AMPK和FOXO3a磷酸化。此外，当使用小干扰RNA（siRNA）使CD74、AMPK或FOXO3a表达沉默时，这种作用被消除。
MIF可以通过与CD74相互作用并随后激活AMPK-FOXO3a信号通路，使处于衰老状态的MSC恢复活力。用MIF对MSC进行预处理可能对老年个体内源性骨髓MSC的恢复或年轻化具有重要的治疗意义。
