2,5-hexanedione induced apoptosis in mesenchymal stem cells from rat bone marrow via mitochondria-dependent caspase-3 pathway

Abstract

2,5-hexanedione (HD) induces apoptosis of nerve cells. However,the mechanism of HD-induced apoptosis remains unknown. Mesenchymal stem cells (MSCs) are multipotential stem cells with the ability to differentiate into various cell types. This study is designed to investigate the apoptosis induced by HD in rat bone marrow MSCs (BMSCs) and the related underlying mechanisms. The fifth generation of MSCs was treated with 0, 10, 20 and 40 mM HD respectively. The viability of BMSCs was observed by MTT. Apoptosis were estimated by Hoechst 33342 staining and TUNEL assay. The disruption of mitochondrial transmembrane potential (MMP) was examined by JC-1 staining. Moreover, the expression of Bax and Bcl-2, cytochrome c release, and caspase-3 activity were determined by real-time RT-PCR, Western blot and Spectrophotometry. Our results showed that HD induced apoptosis in MSCs in a dose dependent manner. Moreover, HD downregulated the Bcl-2 expression,upregulated the Bax expression and the Bax/Bcl-2 ratio, promoted the disruption of MMP, induced the release of cytochrome c from mitochondria to cytosol, and increased the activity of caspase-3 in MSCs. These results indicate that HD induces apoptosis in MSCs and the activated mitochondria-dependent caspase-3 pathway may be involved in the HD-induced apoptosis.

Fig. 1.

Morphology and qualification of BMSCs. Morphology and qualification of BMSCs were assessed by optic microscopy and flow cytometry. a: Morphology observation of the 5th generation BMSCs. b: Adipogenic induction using Oil red O staining. c: Osteogenous induction using alizarin red calcification nodule staining. d: Cells were cultured in Neuroblast induced liquid at 24, 48 and 72 h, respectively. Then the expression of NSE antibody was detected by immunocytochemistry. e: Expression of CD29,CD90 and CD45 as surface markets was determined using flow cytometry.

Fig. 2.

Effect of HD on viability of BMSCs. BMSCs were treated with 0, 10, 20 and 40 mM HD for 12, 24 and 48 h, respectively. Cell viability was assayed by MTT analysis. Data are presented as mean ± SD from three independent experiments. a: p<0.05, compared with control group; b: p<0.05, compared with 10 mM group; c: p< 0.05, compared with 20 mM group.

Fig. 3.

The morphology of BMSCs exposed to HD. BMSCs were treated with 0, 10, 20 and 40 mM HD for 24 h. a: Cell morphological changes were observed with optic microscopy after HE staining. Arrows indicated the BMSCs with pyknotic and darkly stained nuclei. The images are representative for three independent experiments. b: Apoptosis was observed with fluorescence microscopy after nuclei staining with Hoechst 33342. Yellow arrows indicated apoptotic cells with crescent-shaped nuclei; Red arrows indicated apoptosis cells with fragmentary nuclei. The images are representative for three independent experiments.

Fig. 4.

Apoptosis in BMSCs exposed to HD. BMSCs were treated with 0, 10, 20 and 40 mM HD for 24 h. a: Apoptosis in BMSCs was observed by TUNEL assay. Green color represents TUNEL-positive cells as apoptosis. Blue color represents cell nuclei counterstained with Hoechst 33342. Scale bar=50 µm. b: Data are presented as mean ± SD from three independent experiments. a: p<0.05, compared with control group; b: p<0.05, compared with 10 mM group; c: p<0.05, compared with 20 mM group.

Fig. 5.

Expression of Bax and Bcl-2 in BMSCs exposed to HD. BMSCs were treated with 0, 10, 20 and 40 mMHD for 24 h. Real-time quantitative PCR was used to detect Bax and Bcl-2 mRNA expression; Western blot analysis was used to detect Bcl-2 and Bax protein expression. a: Bax mRNA expression in BMSCs exposed to HD. b: Bcl-2 mRNA expression in BMSCs exposed to HD. c: The ratio of Bax/Bcl-2 mRNA expression in BMSCs exposed to HD. d: Bax protein expression in BMSCs exposed to HD. e: Bcl-2 protein expression in BMSCs exposed to HD. f: The ratio of Bax/Bcl-2 protein expression in BMSCs exposed to HD. Data are presented as mean ± SD from three independent experiments. a: p<0.05, compared with control group, b: p<0.05, compared with 10 mM group, c: p<0.05, compared with 20 mM group.

Fig. 6.

The loss of MMP and Cytochrome C protein expression in BMSCs exposed to HD. BMSCs were treated with 0, 10, 20 and 40 mM HD for 24 h. The confocal images of the MMP loss were shown by JC-1 fluorescence. Western blot analysis was used to detect Cytochrome C protein level. a: Red fluorescence represents the mitochondrial aggregate JC-1 and green fluorescence indicates the monomeric JC-1, Scale bar=50 µm. b: Expression of cytochrome c in the mitochondrial fraction in BMSCs exposed to HD. c: Expression of cytochrome c in the cytosolic fraction in BMSCs exposed to HD. d: Data are presented as mean ± SD from three independent experiments. a:p<0.05, compared with control group; b: p<0.05, compared with 10 mM group; c: p<0.05, compared with 20 mM group.

Fig. 7.

Caspase-3 activity in BMSCs exposed to HD. BMSCs were treated with 0, 10, 20 and 40 mM HD for 24 h. Data are presented as mean ± SD from three independent experiments. a: p<0.05, compared with control group; b: p<0.05, compared with 10 mM group; c: p<0.05, compared with 20 mM group.