Background: Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is one of the new therapeutic strategies for treating ischemic brain and heart tissues. However, the poor survival rate of transplanted BMSCs in ischemic tissue, due to high levels of reactive oxygen species (ROS), limits the therapeutic efficacy of this approach. Considering that BMSC survival may greatly enhance the effectiveness of transplantation therapy, development of effective therapeutics capable of mitigating oxidative stress-induced BMSC apoptosis is an important unmet clinical need.
Methods: BMSCs were isolated from the 4-week-old male Sprague Dawley rats by whole bone marrow adherent culturing, and the characteristics were verified by morphology, immunophenotype, adipogenic, and osteogenic differentiation potential. BMSCs were pretreated with artemisinin, and H2O2 was used to induce apoptosis. Cell viability was detected by MTT, FACS, LDH, and Hoechst 33342 staining assays. Mitochondrial membrane potential (ΔΨm) was measured by JC-1 assay. The apoptosis was analyzed by Annexin V-FITC/PI and Caspase 3 Activity Assay kits. ROS level was evaluated by using CellROX® Deep Red Reagent. SOD, CAT, and GPx enzymatic activities were assessed separately using Cu/Zn-SOD and Mn-SOD Assay Kit with WST-8, Catalase Assay Kit, and Total Glutathione Peroxidase Assay Kit. The effects of artemisinin on protein expression of BMSCs including p-Erk1/2, t-Erk1/2, p-c-Raf, p-p90rsk, p-CREB, BCL-2, Bax, p-Akt, t-Akt, β-actin, and GAPDH were measured by western blotting.
Results: We characterized for the first time the protective effect of artemisinin, an anti-malaria drug, using oxidative stress-induced apoptosis in vitro, in rat BMSC cultures. We found that artemisinin, at clinically relevant concentrations, improved BMSC survival by reduction of ROS production, increase of antioxidant enzyme activities including SOD, CAT, and GPx, in correlation with decreased Caspase 3 activation, lactate dehydrogenase (LDH) release and apoptosis, all induced by H2O2. Artemisinin significantly increased extracellular-signal-regulated kinase 1/2 (Erk1/2) phosphorylation, in a concentration- and time-dependent manner. PD98059, the specific inhibitor of the Erk1/2 pathway, blocked Erk1/2 phosphorylation and artemisinin protection. Similarly, decreased expression of Erk1/2 by siRNA attenuated the protective effect of artemisinin. Additionally, when the upstream activator KRAS was knocked down by siRNA, the protective effect of artemisinin was also blocked. These data strongly indicated the involvement of the Erk1/2 pathway. Consistent with this hypothesis, artemisinin increased the phosphorylation of Erk1/2 upstream kinases proto-oncogene c-RAF serine/threonine-protein kinase (c-Raf) and of Erk1/2 downstream targets p90 ribosomal s6 kinase (p90rsk) and cAMP response element binding protein (CREB). In addition, we found that the expression of anti-apoptotic protein B cell lymphoma 2 protein (BcL-2) was also upregulated by artemisinin.
Conclusion: These studies demonstrate the proof of concept of artemisinin therapeutic potential to improve survival in vitro of BMSCs exposed to ROS-induced apoptosis and suggest that artemisinin-mediated protection occurs via the activation of c-Raf-Erk1/2-p90rsk-CREB signaling pathway.
Keywords: Apoptosis; Artemisinin; Bone marrow mesenchymal stem cells; CREB; Erk1/2; Phosphorylation; Protection; ROS; Raf; p90rsk.