| Objective:To investigate the protective effects and mechanism of dioscin on rat H9c2 cardiac myocytes exposed to simulated hypoxia/reoxygenation.Methods:The hypoxia/reoxygenation injury model was established in H9c2 cell strain with or without dioscin treated. H9c2 cardiocytes were randomly divided into five groups:control group(group C); hypoxia/reoxygenation model group(group H/R); H/R+Dio low concentration group(10mg/L, group H/R+L), H/R+Dio middle concentration group(20mg/L, group H/R+M), H/R+Dio high concentration group(40 mg/L, group H/R+H).1. The survival rates of H9c2 cardiocytes were evaluated by methyl thiazolyl tetrazolium (MTT) colorimetry.2. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdehyde (MDA) was respectively measured in cellular supernate.3. DCFH-DA is a fluorescent explorer which can combine to reactive oxygen species (ROS) located in cells. The levels of ROS in different groups were obtained through the fluorescence microscopy.4. The apoptosis rates of H9c2 cardiocytes were assayed by flow cytometry using propidium iodide (PI).5. Mitochondrial membrane potential (△ΨPm) changing was estimated using Rhodamine 123 (Rho123) which is a fluorescent explorer. Fluorescence intensity was detected by multifunctional labeling apparatus.6. Cytochrome c released from mitochondria to cytoplasm was detected by enzyme linked immunosorbent assay (ELISA).7. The expressions of mRNA for apoptosis-related gene, such as Bax, Bcl-2, caspase-9 and caspase-3 were measured by RT-PCR.8. The expression of heat-shock protein 70(HSP70) was determined by Western blot analysis. Results:1. Cell survival rate:compared with group H/R, the result of group C was significant in statistics (34.50±8.50% vs 100.00±00.00%, P<0.01), the results of the cell survival rate of groups H/R+L. H/R+M and H/R+H were increased significantly (34.50±8.50% vs 66.47±10.56%,76.19±7.57%,93.38±6.09%, P<0.01); and compared with group H/R+H, the results of groups H/R+L and H/R+M were significant in statistics (P<0.05).2. LDH activity:compared with group H/R, the result of group C was significant in statistics (1287.68±58.97 U/L vs 975.29±35.66 U/L, P<0.01), the results of LDH activity of groups H/R+L, H/R+M and H/R+H were decreased significantly (1287.68±58.97 U/L vs 1150.85±40.67 U/L,1078.79±48.94 U/L, 1001.65±53.05 U/L, P<0.01); and compared with group H/R+H, the results of groups H/R+L and H/R+M were significant in statistics (P<0.05).SOD activity:compared with group H/R, the result of group C was significant in statistics (17.36±1.12 U/ml vs 23.55±1.19 U/ml, P<0.01), the results of SOD activity of groups H/R+L, H/R+M and H/R+H were increased significantly (17.36±1.12 U/ml vs 19.59±0.60 U/ml,21.08±0.63 U/ml,22.56±0.59 U/ml, P<0.01); and compared with group H/R+H, the result of group H/R+L was significant in statistics (P<0.05).MDA content:compared with group H/R, the result of group C was significant in statistics (2.88±0.04 nmol/ml vs 2.49±0.03 nmol/ml, P<0.01), the results of MDA content of groups H/R+L, H/R+M and H/R+H were decreased significantly (2.88±0.04 nmol/ml vs 2.72±0.06 nmol/ml,2.66±0.05 nmol/ml, 2.59±0.03 nmol/ml,P<0.01); and compared with group H/R+H, the results of groups H/R+L and H/R+M were significant in statistics (P<0.05).3. ROS:The result from fluorescence microscopy indicated:the fluorescence intensity of Rosup ROS positive control group was highest than other groups, and the fluorescence intensity of group H/R was higher. Compared with group H/R, the fluorescence intensity of groups H/R+L, H/R+M, and H/R+H were lower. It demonstrated that dioscin could reduce effectively the produce of ROS in cells.4. Cell apoptosis rate:compared with group H/R, the result of group C was significant in statistics (10.61±1.00% vs 0.15±0.04%, P<0.01), the results of the cell apoptosis rate of groups H/R+L,H/R+M and H/R+H were decreased significantly (10.61±1.00% vs 2.03±0.06%,1.80±0.06%,1.67±0.04%, P<0.01); and compared with group H/R+H, the results of groups H/R+L and H/R+M were significant in statistics (P<0.05, P<0.01).5.△Ψm fluorescence intensity:compared with group H/R, the result of group C was significant in statistics (106547±5907 vs 205486±4653, P<0.01), the results of fluorescence intensity of groups H/R+L, H/R+M and H/R+H were increased significantly (106547±5907 vs 160835±3521,186350±3353, 194694±3043, P<0.01); and compared with group H/R+H, the results of groups H/R+L and H/R+M were significant in statistics (P<0.01).6. Cyt-c release:compared with group H/R, the result of group C was significant in statistics (P<0.01), the results of the Cyt-c release from cell mitochondria of groups H/R+L, H/R+M and H/R+H were decreased significantly (P<0.01); and compared with group H/R+H, the results of groups H/R+L was significant in statistics (P<0.05).7. Bax,Bcl-2 mRNA:compared with group H/R, the result of group C was significant in statistics (P<0.05), the results of the expression of Bax mRNA in groups H/R+L, H/R+M and H/R+H were decreased significantly (P<0.01), and the results of the expression of Bcl-2 mRNA of groups H/R+L, H/R+M and H/R+H were increased significantly (P<0.05); and compared with group H/R+H, the results of groups H/R+L was significant in statistics (P<0.05).Caspase-9,caspase-3 mRNA:compared with group H/R, the result of group C was significant in statistics (P<0.01), the results of the expressions of caspase-9 and caspase-3 mRNA of groups H/R+L, H/R+M and H/R+H were decreased significantly (P<0.01); and compared with group H/R+H, the results of groups H/R+L was significant in statistics (P<0.01).8. HSP70:compared with group H/R, the result of group C was significant in statistics (100.±0.00% vs 134.29±8.58%, P<0.05), the results of the express of HSP70 of groups H/R+L, H/R+M and H/R+H were increased significantly (174.03±23.17% vs 276.25±21.87%,287.63±23.48%, P<0.05); and compared with group H/R+H, the result of group H/R+L was significant in statistics (P<0.01).Conclusion:1. Dioscin could decrease LDH leakage of rat H9c2 cardiac cell caused by hypoxia/reoxygenation injury, and increase the cell survival rate.2. Dioscin could protect rat H9c2 cardiac cell caused by hypoxia/reoxygenation injury against oxidative damage.3. Dioscin could protect rat H9c2 cardiac cell caused by hypoxia/reoxygenation injury against apoptosis.4. Dioscin could increase the expression of HSP70 of rat H9c2 cardiac cell caused by hypoxia/reoxygenation injury, and protected effectively cell. |
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