Cobalt Chloride Enhances the Anti-Inflammatory Potency of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells through the ERK-HIF-1α-MicroRNA-146a-Mediated Signaling Pathway

Figure 4

Cobalt chloride (CoCl₂) stimulates the immunomodulation of hUCB-MSCs through an ERK- and HIF-1α-dependent pathway. (a) hMSCs were treated with 100 μM CoCl₂ and then harvested at the indicated times. The protein level of HIF-1α and p-ERK was determined by Western blot analysis. (b) At 48 hr posttransfection with control siRNA (CTRL) or HIF-1α-specific siRNA (si-HIF-1α), hUCB-MSCs were treated with 100 μM CoCl₂ for 30 min or 3 hr. (c) hUCB-MSCs were treated with 100 μM CoCl₂ for 30 min or 3 hr in the absence or presence of 10 μM U0126. ERK phosphorylation and HIF-1α expression were determined by Western blot analysis. (d) hUCB-MSCs were transfected with control siRNA (si-control) or HIF-1α siRNA (si-HIF-1α) and then treated with 100 μM CoCl₂ for 72 h. (e) hUCB-MSCs were pretreated with the vehicle control (DMSO) or the ERK-specific inhibitor 10 μM U0126 for 60 min and then treated with 100 μM CoCl₂ for 72 h. The expression level of miR-146a was determined by RT-qPCR. (f) Schematic illustration of the molecular mechanisms involved in the CoCl₂-enhanced anti-inflammatory effects. Data represent the , ; . P: hPBMCs; P: allogeneic hPBMCs as stimulator; H: phytohemagglutinin; MSC: naïve hUCB-MSCs; CoMSC: CoCl₂-pretreated hUCB-MSCs.

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