Abstract:

BACKGROUND: Previous studies demonstrated that ethanol can induce apoptosis in bone marrow mesenchymal stem cells (BMSCs), and decrease the number of osteoplasts and osteoclasts. However, the effect and mechanism of ethanol on apoptosis in BMSCs remains unclear.
OBJECTIVE: To investigate the effect of ethanol on apoptosis in BMSCs of rats and their mitochondrial function and to evaluate the pathway associated with the regulation of Bcl-2 and Caspase-3.
METHODS: BMSCs were isolated from Sprague-Dawley rats were treated with ethanol at doses of 0, 100, 200, 300, 400, 500, 600, 700, 800, 900 mmol/L for 24 hours. cytotoxic drug experiment was performed with MTT assay. BMSCs were treated with ethanol at doses of 0, 100, 200, 300, 400, 500 mmol/L for 6, 12, 24 hours, AnnexinV/PI flow cytometry of double label method was performed to detect the apoptosis and mitochondrial membrane potential, BMSCs were treated with ethanol at doses of 0, 427 mmol/L for 24 hours, the levels of Bcl-2 and Caspase-3 mRNA expression were determined by RT-PCR method.
RESULTS AND CONCLUSION: MTT assay results showed that 50% concentration of inhibition (IC50) of BMSCs of rats grew in ethanol was 427 mmol/L. The results of Annexin V/PI assay indicated that the apoptosis rates of BMSCs and mitochondrial membrane potential were higher than that of untreated (0 mmol/L) group when time and dose of ethanol was increased (P < 0.05). Compared with 0 mmol/L group, the level of Bcl-2mRNA expression was decreased in 427 mmol/L group after 24 hours, but the caspase-3 mRNA expression was increased significantly by treatment at 427 mmol/L (P < 0.05). These results suggest that ethanol can induce apoptosis in BMSCs of SD rat, and the occurrence of apoptosis may be related to the mitochondrial membrane potential damage, mitochondrial dysfunction, bcl-2 and Caspase-3 activation.