2,5-hexanedione induced apoptosis of rat bone marrow mesenchymal stem cells by reactive oxygen species

Abstract

Objectives: n-Hexane, a common industrial organic solvent, causes multiple organ damage, especially neurotoxicity, which is proved to be caused by its metabolite 2,5-hexanedione (2,5-HD). We previously showed that 2,5-HD induced apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). In the current study, we explored the mechanism of 2,5-HD-induced apoptosis, especially the role played by reactive oxygen species (ROS).

Methods: Intracellular ROS levels after 2,5-HD treatment were measured by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method, and the antioxidant N-acetyl cysteine (NAC) was used to scavenge ROS. Apoptosis, mitochondrial membrane potential (MMP), and caspase-3 activity were measured after 2,5-HD exposure with or without NAC pretreatment.

Results: In rat BMSCs, 20 mM 2,5-HD significantly increased ROS levels and apoptosis. In addition, MMP activity was decreased and caspase-3 activity was increased. With NAC pretreatment, ROS increases were prevented, cells were rescued from apoptosis, and both MMP and caspase-3 activity returned to normal levels. Western blotting analysis of malondialdehyde-modified proteins and superoxide dismutase (SOD) 1 showed that after 2,5-HD exposure, BMSCs had oxidative damage and abnormal SOD1 expression. These returned to normal when cells were pretreated with NAC in addition to 20 mM 2,5-HD. Furthermore, the expressions of NF-κB p65/RelA and phospho-NF-κB p65/RelA (Ser536) were suppressed after 2,5-HD exposure and restored by NAC pretreatment.

Conclusions: 2,5-HD-induced apoptosis in rat BMSCs is potentially mediated by excessive ROS production.

Fig. 1.. Effects of 2,5-HD and N-acetyl cysteine (NAC) on ROS levels. Intracellular ROS were measured by the DCFH-DA method. Results are expressed as fluorescent intensity per 5×10⁵cells. Means ± SEM. n=3, Student's unpaired t test or one-way ANOVA with post hoc LSD test. **p<0.01.

Fig. 2.. Effects of 2,5-HD and N-acetyl cysteine (NAC) on TUNEL-positive cells. Apoptosis was detected by the TUNEL assay, and samples were mounted with fluorescence mounting medium and visualized under the confocal microscope. To determine percentage of apoptotic cells, 300-500 cells were counted for each sample. Means ± SEM. n=3, Student's unpaired t test or one-way ANOVA with post hoc LSD test. **p<0.01.

Fig. 3.. Effects of 2,5-HD and N-acetyl cysteine (NAC) on mitochondrial membrane potential (MMP). Cells were incubated with JC-1 in L-DMEM at 37°C for 30 min and analyzed with a confocal microscope. The mean optical density (OD) value of each sample from at least six random fields was obtained using Image-Pro Plus 6.0, and the OD ratio of green to red fluorescence was calculated. (A) Representative JC-1 staining results. Control cells with normal MMP are primarily stained red (indicating JC-1 aggregates). Apoptotic cells with low MMP are stained green (indicating JC-1 monomers). Scale bar=50µm. (B) The OD ratio of green-to-red fluorescence with JC-1 staining. Means ± SEM. n=6-12, Student’s unpaired t test or one-way ANOVA with post hoc LSD test. **p<0.01.

Fig. 4.. Effects of 2,5-HD and N-acetyl cysteine (NAC) on caspase-3 activity. Caspase-3 activity was determined by measuring levels of p-NA cleaved from the substrate N-Ac-DEVD-pNA. Reactions were incubated at 37°C for 4 h. The optical density (OD) values were measured, and the caspase-3 activity expressed as μM pNA/mg protein. Means ± SEM. n=3, Student's unpaired t test or one-way ANOVA with post hoc LSD test. **p<0.01.

Fig. 5.. Effects of 2,5-HD and N-acetyl cysteine (NAC) on levels of MDA and SOD1. (A) Representative Western blotting images showing MDA and SOD1. (B) Quantitative densitometric analysis of MDA and SOD1 using Image Lab 4.1. All densitometry values were normalized to that of actin, and the ratio of the control group was defined as 1.00. Means ± SEM. n=3, Student's unpaired t test or one-way ANOVA with post hoc LSD test. *p<0.05, **p<0.01.

Fig. 6.. Effects of 2,5-HD and N-acetyl cysteine (NAC) on levels of NF-κB p65/RelA and phospho-NF-κB p65/RelA (Ser536). (A) Representative Western blotting images showing NF-κB p65/RelA and phospho-NF-κB p65/RelA (Ser536). (B) Quantitative densitometric analysis of NF-κB p65/RelA and phospho-NF-κB p65/RelA (Ser536) using Image Lab 4.1. All densitometry values were normalized to that of actin, and the ratio of the control group was defined as 1.00. Means ± SEM. n=3, Student’s unpaired t test or one-way ANOVA with post hoc LSD test. *p<0.05, **p<0.01.