铁过载催化的氧化应激对骨髓间充质干细胞的影响及其作用机制

Effect and Mechanism of Iron-catalyzed Oxidative Stress on Mesenchymal Stem Cells

目的探讨铁过载催化的活性氧(ROS)生成对骨髓间充质干细胞(MSCs)增殖和凋亡的影响及其可能的作用机制。方法体外培养MSCs,加入不同浓度(100、200、400μmol/L)的枸橼酸铁铵(FAC),培养12、24、48 h,建立铁过载模型,测定铁过载前后细胞内的不稳定铁池(LIP)和ROS水平,并采用群体倍增时间(DT)检测MSCs的细胞增殖能力,采用Annexin V-PI双标法检测细胞凋亡率,进行蛋白质印迹实验检测磷酸化p38丝裂原活化蛋白激酶(P-p38MAPK)、p38MAPK、蛋白激酶B(AKT)及p53蛋白的表达情况。结果铁过载组MSCs的LIP水平明显高于对照组(平均荧光强度482.49±20.96比303.88±23.37,P<0.05);ROS的水平随FAC浓度增加而升高,在加入FAC浓度为400μmol/L时最高(P<0.05)。用400μmol/L的FAC分别处理12、24、48 h的MSCs DT分别为(1.47±0.11)、(1.80±0.13)、(2.04±0.14)d,均明显长于对照组的(1.20±0.05)d(P均<0.05)。铁过载组MSCs的凋亡率也明显高于对照组[(3.51±1.17)%比(0.66±0.62)%,P<0.05]。与对照组比较,铁过载组P-p38MAPK、p38MAPK、p53蛋白的表达增加,而AKT的表达无明显差异。结论铁过载可通过提高骨髓MSCs的ROS水平来抑制其增殖、诱导其凋亡,此过程可能与p38MAPK-p53信号通路的激活有关。

Objective To explore effect of iron overload on the proliferation and apoptosis of mesenchymal stem cell （MSCs） and the possible mechanism. Methods Iron overload model of MSCs was established by adding ferric ammonium citrae （ FAC ） into the culture medium at different concentrations （ 100, 200, 400 μmol/L） and incubated for different lengths of time （ 12, 24, 48 h）. The levels of labile iron pool （LIP） and reactive oxygen species （ROS） were measured to confirm oxidative stress state in the model. Changes in cell proliferation and apoptosis after iron overload were measured through population double time （DT） and annexin V-PI assay. Finally, the expressions of phosphorylated p38 mitogen activated protein kinase （P- p38 M APK） , p38 MAPK, protein kinase B （AKT） , and p53 were determined through Western blot analysis to investigate which ROS-mediated signaling pathway was involved in this process. Results The LIP level of MSCs was significantly increased by FAC treatment at 400 p, mol/L （ mean fluorescence intensity 482.49 ：t： 20.96 vs. 303.88 ＋ 23.37, P 〈 0.05 ）. The level of intracellular ROS was positively correlated with the con- centration of FAC and reached a peak level when cultured with 400 p, mol/L FAC （ P 〈 0. 05 ）. After treatment with 400μmol/L FAC at different time points （ 12 h, 24 h, and 48 h）, the DT of MSCs was （ 1.47 ± 0. 11 ） d, （1.80 ± 0. 13 ） d, and （2.04 ＋ 0. 14） d, respectively, which was signifcantly longer than that of the control, which was （ 1.20± 0. 05 ） d （ P 〈 0. 05 ）. The apoptosis rate was also significantly higher in iron overload group [ （3.51 ± 1.17 ）% vs. （0. 66 _＋ 0. 62）%, P 〈 0.05 ] with consequent increase in the expressions of P- p38MAPK, p38MAPK, and io53 proteins in iron overload group, while no significant difference was found in the expression of AKT. Conclusion Iron overload can inhibit the proliferation of MSCs and induce their apoptosis through the generation of ROS, which is probably due to the stimulation of p38MAPK- p53 signaling pathway.