| OBJECTIVE: Plateau under the field study of Salidroside of H9C2 myocardial cell morphology and proliferation activity of influence, and the influence of the expression of LDH, MDA, SOD activity, and the influence on mitochondrial membrane potential and caspase expression, thus revealed low oxygen environment of rhodiola glucoside of oxygen, and for the treatment of many diseases in ischemia hypoxia provides new ideas and methods.Methods: 1.H9C2 hypobaric hypoxia culture, adherent cells were cultured according to the conventional method were seeded in H-DMEM containing 15% fetal bovine serum, 5% CO2,37 ℃incubator, 2-3d was changed passaged once.2. inverted phase contrast microscope cell H9C2 cardiomyocytes mentality.3. CCK-8 detection H9C2 cardiomyocytes cell viability.4. activity were changed by LDH, MDA, SOD kit within H9C2 cardiomyocytes of LDH testing, the amount of MDA accumulation, SOD’s.5. JC-1 change detection H9C2 mitochondrial membrane potential situation.6. caspase-3 polyclonal antibody and ready-to-SABC-AP(rabbit Ig G) kit to detect changes in H9C2 cells caspase-3 protein expression.Results:1.When not dosing cell proliferation, for the first days of growth curves show a downward trend, growth curve trend within 7d is gently rising, and 7-11 d curve rises sharply, after 11 d the curve gradually becomes flat and trends decreased cell survival rate was 95.20%.2. When compared with without medicine,Salidroside acting on the H9C2 cardiomyocytes, the cell vitality strengthen, and enhance with the increase of doses of salidroside, strongest vitality to 100 ug/ml salidroside, the extension of time will also enhance the vitality of cells..3. 100μg/ml concentration of salidroside role in H9C2 cardiomyocytes, with the extension of the experimental group, the role of time in the medium LDH levels lower than the control group, and there is a statistically significant difference in 3d and 5d.4. With 100 μg / ml Salidroside role in H9C2 cells,compared with the control group, as the change of time, intracellular MDA expression level decreased, while the activity of SOD showed a trend of increase.5. The increase in oxygen free radicals cause damage mitochondrial membrane, so that the mitochondrial membrane potential, and in 1d dosing Salidroside(10μg/ml,50μg/ml,100μg/ml) after, 3d, 7d, respectively JC-1 staining, changes in mitochondrial membrane potential fluorescent microscope observation: dosing group and the control group had red fluorescence of mitochondrial membrane potential in the initial rapid response, with the extension of cell culture time, the red dosing group and the control group fluorescent contrast is obvious, and weakened, enhanced green fluorescent.6. Detection H9C2 cardiomyocytes of caspase-3 activity with caspase-3 polyclonal antibody and ready-to-use SABC- AP(Ig G) rabbit kits, found red fluorescence reduce gradually within 7d, and the intensity is abate, stating that caspase-3 protein expression levels drop.Conclusion: Salidroside by increasing SOD activity and reduce LDH and MDA content in order to maintain the balance of intracellular oxidation and antioxidant levels and the integrity of the membrane, shown to H9C2 cardiomyocytes have obvious resistance to hypoxia and protection, and mainly reflected in the high dose(> 10 μg/ml). By the rise of the mitochondrial membrane potential and the decrease of caspase-3 protein expression, display the salidroside has stable mitochondrial function and the ability of resistance to apoptosis. |
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