| Objectiv：Investigate the protective effects and antiapoptotic of curcumin onhypoxia/reoxygenation cardiomyocyte cells. Clear the relationship between prote-ction and concentration of curcumin, and the relationship between curcumin anti-apoptotic mechanisms and HSP70expression. Investigate the relationship betweenthe dose of the drug-induced HSP70expression and phase change, Clear phase andeffect of the drug-induced protection.Methods：Primary culture the myocardial cells of1～3days SD neonatal rats.To synchronous pulsation of the primary cultured cells used in the experiment. UseMTT assay detect the toxic effects of curcumin on myocardial cells, then calculate theIC50. Find the best intervention concentration, which has no obvious side effects onmyocardial cells. Select the high, medium and low concentration used in theexperiment. Counting cells and inoculated plate, grouping and curcumin intervention.Then application of a continuous specteum multfunctional mircroplate readerdetected in each group at each time point supernatant lactate dehydrogenase (LDH)activity and malondialdehyde (MDA) content. Use Hoechst33342and propidiumiodide (PI) double staining the cells, then observation the cells apoptosis and necrosisby fluorescence microscope camera. Immunohistochemical method to observe theexpression of HSP70in myocardial cells in each group.Result：1.The toxic effect of curcumin in normal myocardial cells, the IC50was77.57μmol/L. In this study, grouping selected curcumin the high, medium and low concent-rations of50μmol/L,25μmol/L,12.5μmol/L.2.The comparison of LDH activity in each group cell culture fluid Eachgroup cells compared to the same time. Found LDH activity in normal control groupwas lowest. With curcumin concentration decreased, LDH activity increased. Eachgroup cells compared to different time. Found LDH activity in normal control groupware not significantly changed with time (p=0.292). Myocardial cells after hypoxia/reoxygenation injury with time, LDH activity increased. And groups of LDH activity with time was a decreasing trend after first increment, reached a peak at12h, decr-eased at24h.3. The comparison of MDA content in each group cell culture fluid Eachgroup cells compared to the same time. Found MDA content in normal control groupwas lowest. With curcumin concentration decreased, MDA content elevated different.Each group cells compared to different time. Found MDA content in normal controlgroup ware not significantly changed with time (p=0.122). Myocardial cells afterhypoxia/reoxygenation injury with time, MDA content increased different. Andgroups of MDA content with time showed an increasing trend in24h.4. The comparison of apoptosis and necrosis of the cells in each groupEach group cells compared to the same time. Hypoxia/reoxygenation0h, therewere no apparent apoptotic cells in each group. Hypoxia/reoxygenation2h,4h,12h,24h each treated groups cells compared with normal control group, the apoptosisincreased. But the group with curcumin apoptosis less than I/R group. Apoptosis andcurcumin concentration is inversely proportional. Each group cells compared todifferent time. Normal control group,cell apoptosis with the time change is notobvious. But myocardial cells after hypoxia/reoxygenation injury with time,apoptosis increased progressively. Compared with I/R group cells, apoptosis of thegroup with curcumin were less. Apoptosis is inversely proportional to cells withcurcumin concentration.5. The comparison of immunohistochemistry to detect the change of HSP70in each group cells Each group cells compared to the same time. Hypoxia/reoxygenation0h, cells were almost no HSP70expression. Hypoxia/reoxygenation2h,4h,12h,24h each treated cells compared with normal control group, HSP70expression increased. But the curcumin groups express increased more than the I/Rgroup. HSP70expression amount is proportional to the curcumin concentration. Eachgroup cells compared to different time. There were almost no HSP70expression innormal control group, and no significant change with time. Myocardial cells afterhypoxia/reoxygenation injury with time, HSP70expression was increased.Compared with I/R group, HSP70expression of the groups with curcumin wasincreased. HSP70expression is proportional to cells with curcumin concentration. Conclusion：1.Curcumin has protection and anti-apoptotic effects on in vitro hypoxia/reoxygenation cardiomyocyte cells, and the protective effect of curcumin concentr-ation showing a certain dose-dependent manner.2.With increasing curcumin concentration, the HSP70expression in hypoxia/reoxygenation cardiomyocyte cells increase. And intracellular HSP70expression ofhypoxia/reoxygenation cardiomyocyte cells increased with time. Curcumin protect-tive effect of hypoxia/reoxygenation cardiomyocyte cells may be associated with theincreased expression of HSP70. |
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