ROS activates JNK-mediated autophagy to counteract apoptosis in mouse mesenchymal stem cells in vitro

Aim:

Transplantation of mesenchymal stem cells (MSCs) for the treatment of diabetic erectile dysfunction (ED) is hampered by apoptosis of the transplanted cells. In diabetic ED, there is increased oxidative stress and decreased NO in the corpora cavernosa, and reactive oxygen species (ROS) induce apoptosis of the transplanted cells. In this study we examined whether and how autophagy was involved in ROS-induced apoptosis of MSCs.

Methods:

Mouse C3H10 MSCs were treated with H₂O₂ to simulate the high oxidative condition in diabetic ED. Cell viability was measured using MTT assay. Apoptosis was analyzed by flow cytometry. Apoptosis- and autophagy-related proteins were detected with Western blot assays. Intracellular autophagosome accumulation was studied using transmission electron microscopy.

Results:

Treatment of MSCs with H₂O₂ (50–400 μmol/L) inhibited the cell viability in concentration- and time-dependent manners. Furthermore, H₂O₂ (300 μmol/L) induced apoptosis, as well as activated autophagy in MSCs. Pretreatment with lysosome inhibitor chloroquine (10 μmol/L) or PI3K inhibitor 3-methyladenine (5 mmol/L) significantly enhanced H₂O₂-induced cell death. Pretreatment with JNK inhibitor SP600125 (10 μmol/L) abrogated H₂O₂-induced accumulation of LC3-II, and attenuated H₂O₂-induced reduction of Bcl-2 levels in MSCs.

Conclusion:

ROS induce autophagy to counteract apoptosis in MSCs by activation of JNK. Thus, augmentation of autophagy may reduce apoptosis, prolonging MSC survival and improving MSC-based therapeutic efficacy for diabetic ED.