| Objective:To explore the mechanism and effect of excessive iron catalyzed oxidative stress on the proliferation, cell cycle, cell apoptosis and hematopoietic supportive capacity of BM-MSC and UC-MSC.Methods:1. Iron overload model of BM-MSC and UC-MSC was first established by adding FAC into the culture medium, the level of Labile iron pool(LIP) was measured to confirm the model.2. Changes in cell proliferation of BM-MSC, UC-MSC after iron overload was measured through population double time(DT). Cell cycle and apoptosis were analyzed by propidium Iodide (PI) staining method and Annexin V-PI double staining assay. Co-culture system was used to assess the hematopoietic support capacity of UC-MSC in different groups.3. Cellular ROS level was tested using fluorescent probe2’,7’-dichlorofluoresin diacetate (DCFH-DA). Finally, the expression of phosphorylated p38Mitogen activated protein kinase (P-p38MAPK), p38MAPK, protein kinase B(AKT), p53was determined through western blot to investigate which ROS-mediated signaling pathway took part in this process and whether the pathway can be inhibited by deferasirox (DFO)/N-acetyl-L-cysteine (NAC) treatment.Results:1. BM-MSC after passage3demonstrated a fibroblast-like, spindle-shaped morphology and the purity of BM-MSC analyzed by flow cyto meter was high. The LIP level of BM-MSC was significantly increased by adding400μmol/L FAC for24h. After treatment with400μmol/L FAC at different times (12,24,48h), the DT of MSCs was (35.28±2.64),(43.20±3.12),(48.96±3.36)h respectively which was signifcantly longer than control (28.80±1.25)h (P<0.05). The apoptosis rate was also observed to be higher in iron overload group [(3.51±1.17)%VS (0.66±0.62)%, P<0.05)]2. Level of intracellular ROS was positively correlated with the concentration of FAC and reached highest level when cultured at400μmol/L FAC (P<0.05). The expression of P-p38MAPK, p38MAPK and p53protein in iron overload group was significantly increased, while no significant difference was found in the expression of AKT.3. Similarly, UC-MSC treated with400μmol/L FAC for12h also showed increased intracellular LIP, and DT of UC-MSC in iron overload group was signifcantly longer than control (P<0.05), but the difference wasn’t significant after two passages (P>0.05). UC-MSC in iron overload group exerted G0/G1phase cell cycle arrest and increased cell apoptosis [(12.75±0.32%) VS (3.63±0.80%),(P<0.05)]. The colony forming capacity of MNC co-cultured with UC-MSC of iron overload group for one/two weeks was significantly decreased, which showed that the hematopoietic supportive capacity of UC-MSC in iron overload group was impaired.4. ROS level of UC-MSC with iron overload was higher than control which was time-and concentration-dependent and it reached high peak at400μmol/L of FAC forl2h. Western blot analysis of whole cell lysates from UC-MSC using antibodies recognizing known ROS-related signaling pathways revealed robust increases in phospho-p38MAPK, p53in response to FAC compared with control, with inhibition of these signaling pathways noted in response to DFO or NAC at suitable dose, suggesting that chelation therapy and antioxidant can inhibit ROS-induced signaling pathway after iron overload.Conclusion:Excessive iron could inhibit proliferation, induce apoptosis, arrest cell cycle and decrease function of BM-MSC and UC-MSC. And the mechanism of iron induced cell injury may due to the increased ROS level and subsequently stimulated p38-MAPK, p53signaling pathway, which could be partially reversed by DFO or NAC. Thus, Our finding indicates that iron overload can injure hematopoiesis by enhancing oxidative stress in MSC. It suggests creatively that antioxidant or cytotherapy may be an effective method in curing deficient hematopoiesis in iron overload. |
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