Effects Of Estrogen On Apoptosis Of Bone Marrow Mesenchymal Stem Cells Induced By Hydrogen Peroxide

ObjectiveTo investigate the effect of estrogen on the apoptosis of hydrogen peroxide (H₂O₂) induced bone marrow mesenchymal stem cell and explore its possible mechanism. To laid the scientific theoretical foundation for a better clinical therapy efficacy in treating coronary heart disease with bone marrow mesenchymal stem cells.MethodIsolated and cultured bone marrow mesenchymal stem cells(BMSC) from SD rats in density gradient centrifugation method, identify BMSC cells by flow cytometry phenotype. The cells in logarithmic growth phase were randomly divided into 6 groups: A group (control group), B group (H₂O₂ model group), C group (0.1% VitC positive control group), D group (2.5 nM estradiol), E group (5 nM estrogen group), F group (10 nM estradiol). C, D, E, F group pretreated with VitC or different concentrations of estrogen for 24 hours, and then cocultured with 1 nM of H₂O₂ for 1 hour, (1) MTT cells viability assay; (2) Observe BMSCS morphological changes with laser confocal microscope after dyed by Hoechst 33342 and Fluor -594; (3) Detect BMSCs mitochondrial membrane potential changes by flow cytometry after stained by Rhodamine-123; (4) Detect cells apoptosis by flow cytometry after AnnexinⅤ/ FITC-PI double staining ; (5) Observe the expression of Caspase-9 mRNA by real-time fluorescent quantitative RT-PCR; (6) Analysis c-Jun and JNK1 / 2 protein expression by immunocytochemistry.Results(1) Successfully isolated SD rat bone marrow mesenchymal stem cells in density gradient centrifugation method, and cells surface markers CD29, CD90 expression were positive, but CD34, CD45 expression were negative; (2) H₂O₂ promots BMSCs apoptosis, and estrogen can increase the activity of BMSCs; compared with the model group, MTT test showed the absorbance of each group increased; cells morphology observation notes nuclear condensation, margination phenomenon was significantly reduced compared with the model; flow cytometry found that estrogen can stabilize mitochondrial membrane potential and reduce the apoptosis rate; FQ-PCR results showed estrogen groups significantly reduced Caspase-9 mRNA expression; Immunocytochemical technic anasysis JNK1 / 2 and c-Jun protein expression decreasing in estrogen groups. The ability of Estrogen apoptosis inhibition presents in a concentration-dependentant relationship, and the most significant (P <0.01) anti-apoptotic effect is 10 nM estrogen, otherwise, the effect of 2.5 nM and 5 nM estrogen is weaker (P <0.05).ConclusionsDensity gradient centrifugation method can successfully isolate SD rat bone marrow mesenchymal stem cells; estrogen can inhibit H₂O₂-induced BMSCs apoptosis, and this anti-apoptosis effect is concentration-dependent in a certain range of estrogen concentration, 10 nM appears the most significant protection; its mechanism may prevent ion current through PT channels, so stable mitochondrial membrane potential, interrupt MAPK signaling pathway, then inhibit JNK and C-Jun activation, and at last inhibit the action of caspases cascade.