2,5-Hexanedione Induced Apoptosis In Mesenchymal Stem Cells From Rat Bone Marrow

Objective To investigate apoptotic effect of 2,5-Hexanedione(2,5-HD) on bone marrow mesenchymal stem cells(BMSCs) of rats and its mechanism.Methods The cells were isolated from Bone marrows of rats and cultured. The morphology of cells were observed, their surface markers were determined by flow cytomerty. The cells cultured in osteogenesis induced liquid were identified by alizarin red staining; The cells were cultured in adipogenic induced liquid, and stained with Oil red; The cells were cultured in Neuroblast induced liquid, and they were identified by immunocytochemical staining using neural specific enolase(NSE) antibody. To confirmed the cells were BMSCs. Then, the fifth generation of rat BMSCs was exposed to 0 m M, 10 m M, 20 m M and 40 m M HD for 24 h. The cells viability was measured by MTT assay. The morphological changes of BMSCs were observed using HE staining and The apoptotic morphologic changes of BMSCs were observed by Hoechst 33342 staining and TUNEL-Hoechst double staining. The m RNA expression of Bax and Bcl-2 was determined by real-time RT-PCR. The protein expression of Bax, Bcl-2 and cytochrome C was determined by Western blot. The disruption of mitochondrial trans-membrane potential(MMP) was examined by JC-1 staining. The protein expression of cytochrome C in mitochondria and Cytoplasm was determined by Western blotting. And the caspase-3 activity were determined by Spectrophotometry. The experimental results were analyzed by usingSPSS 13.0 statistical package.Results The morphology of the BMSCs showed spindle-like or spindle-shaped and was almost uniform. The expression of the BMSCs surface markers CD29, CD90 and CD45 was 90.10%, 96.61% and 0.40%, respectively. After induction, BMSCs were differentiated into osteoblasts and adipocytes. The expression of NSE was 29.26%, 33.95% and 51.86% after 24 h, 48 h and 72 h respectively, while the control group was negative for their marker. It was indicated that BMSCs were differentiated into nerve cells. These results revealed that the cultivated cells were BMSCs. The viability of BMSCs in the exposed groups was significantly lower than that in control group(p < 0.05) and decreased in a dose-dependent manner. The morphological changes of BMSCs stained by HE showed that the BMSCs with pyknotic and darkly stained nuclei were observed in groups exposed to HD. Moreover, the cytoplasm dissolved and the polygonal apophysis became shorter or disappeared. The HD-exposed BMSCs showed crescent-shaped nuclei and fragmentation with heterogeneous intensity in the nuclei, after stained by Hoechst 33342. The apoptosis indices of BMSCs exposed to 10 m M, 20 m M and 40 m M HD were 3.00%, 4.12% and 18.63%, which is significantly higher than those in control group(p < 0.05). Real Time RT-PCR showed that HD significantly increased Bax expression(p < 0.05) and significantly decreased Bcl-2 expression(p < 0.05). Western Blotting showed that the expression of Bax protein in the HD-exposed BMSCs groups was significantly higher than the control group(p < 0.05). However, the expression of Bcl-2 protein in the HD-exposed BMSCs groups was significantly lower than the control group(p < 0.05). The ratio of Bax / Bcl-2 protein expression in the HD-exposed BMSCs groups significantly increased compared with the control group(p < 0.05). JC-1 staining results showed that the ratio of red to green fluorescence in the HD-exposed groups was significantly lower than that in control group(p < 0.05). The decreased ratio of red to green fluorescence suggests that HD induces MMP depolarization in the BMSCs. The activity of caspase-3 in the HD-exposed BMSCs groups was significantly higher than the control group(p < 0.05).Conclusion HD induces apoptosis in BMSCs; HD activates mitochondria-dependentcaspase-3 pathway; The activated mitochondria-dependent caspase-3 pathway may be involved in the HD-induced apoptosis.