Naringin alleviates H2O2-induced apoptosis via the PI3K/Akt pathway in rat nucleus pulposus-derived mesenchymal stem cells.

ABSTRACT

Purpose: To investigate the protective effect of naringin (Nar) on H2O2-induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods: Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H2O2. Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results: The cells isolated from NP met the criteria for MSC. H2O2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 µM for 24 h. Nar exhibited protective effects against H2O2-induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H2O2-induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions: Our results demonstrated that Nar efficiently attenuated H2O2-induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.