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Objective: To observe the effect of hydroxysafflower yellow A (HSYA) on the apoptosis of human umbilical vein endothelial cells EA.hy926 induced by hypoxia-reoxygenation. Methods: MTT colorimetry method was used to detect the effects of different hypoxia time (8, 12 h) and different reoxygenation time (4, 8, 12 h) on the cell viability. And after the cell had been in the status of hypoxia for 12 h and reoxygenation for 8 h, this method was adapted once again to evaluate the effects of different concentrations of HSYA (0.1, 1, 10, and 100 μmol/L) on cell viability in different time stages. After the cell had been in the status of hypoxia for 12 h, reoxygenation for 8 h, Western blotting was used to test its effects on the expressions of the following proteins in different time stages, which contained Bcl-2, Bax, cleaved Caspase-3, and activated cleaved Caspase-9. This method was also used to detect whether it had an improvement effect on the above proteins after the pr-treat the cells with HSYA. Real-time PCR was used to evaluate the mRNA expressions of Bax, Bcl-2 after the cell had been in the status of hypoxia for 12 h, reoxygenation for 8 h, and this method was also used to test the effect of HSYA on the expressions of Bax, Bcl-2 after the same time stages. Hoechest staining and flow cytometry were used to detect the apoptosis situation of the cell after it was in the status of hypoxia for 12 h and reoxygenation for 8 h. And this method was also adapted to detect the effect of HSYA on apoptosis of the cell after the same time stage. Results: Compared with the control group, the EA.hy926 cell viability decreased significantly after hypoxia for 8, 12 h, reoxygenation for 4, 8, and 12 h (P < 0.01). The cell viability decreased the most significantly after hypoxia for 12 h and reoxygenation for 8 h (P < 0.01), and during this period, the expression of Bax, cleaved Caspase-9, cleaved Caspase-3 protein increased significantly, and Bcl-2 protein was decreased significantly. Compared with the H/R group, HSYA (10 μmol/L) significantly increased the cell viability (P < 0.01) after hypoxia-reoxygenation, and significantly up-regulated the protein expression of Bcl-2, and down-regulated the protein expressions of Bax, cleaved Caspase-9, and cleaved Caspase-3. Conclusion: Hydroxysafflor yellow A can effectively inhibit the apoptosis of EA.hy926 induced by hypoxia and reoxygenation. The mechanism may be related to the down-regulation of Bax, cleaved Caspase-9, cleaved Caspase-3 protein as well as the up-regulation of Bcl-2 protein.
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Objective To investigate optimal method of establishing hypoxia/reoxygenation(H/R) model of H9c2 cell by using hypoxia/anoxic workstation under different conditions in hypoxia.Methods H9c2 cell was placed into hypoxia/anoxic workstation and simultaneously cultured with complete medium, glucose-free DMEM and acidic hypoxic solution for 1,2,4,6 and 8 h respectively, and then reoxygenated with complete medium for 1 h in normoxic incubator.The level of ROS was measured by flow cytometry.The cell viability was detected by MTT assay.The cellular morphology was observed by inverted microscope.Results With the extension of cell hypoxia time, there were no significant differences in the ROS level and cell viability in complete medium-and glucose-free DMEM-treated H/R groups compared with control group(P<0.05).There was no obvious morphologic change observed with inverted microscope, either.Nevertheless, when H9c2 cells were treated with acidic hypoxic solution in hypoxia, the ROS level continuously increased and the cell viability decreased with the extension of cell hypoxia time ( P<0.01 ).Since H:1 h/R:1 h, some of the cells shrunk and a few necrotic cells floated in the media under the inverted microscope , and the damage was aggravated with the extension of hypoxia time.After the cells were exposed in hypoxia for 8 h, they wrinkled to be round and a large number of floating necrotic cells were observed.When the cells were reoxygenated for 1 h, the cytomembrane was not smooth and there were still a few necrotic cells floating in culture dish .Conclusion The H9c2 cell H/R model with good repeatability can be established successfully by using hypoxia/anoxic workstation combining with acidic hypoxic solution.
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Objective To build hypoxia/reoxygenation injury model in cultured neonatal rat cardiomyocyte and screen active components from Shexiang Baoxin Pill ( SBP) absorbed in blood against hypoxia/reoxygenation injury .Methods Cardiomyocytes were isolated and purified from hearts of neonatal Sprague Dawley rats (1~3 days old) and were used to build hypoxia/reoxygenation injury model.The components of SBP absorbed in blood were screened by methyl thiazolil tetracolium (MTT) colorimetic method.Results SBP showed significant protective effect against cardiomyocytes hypoxia /reoxygenation injury atthe concentration of 50 μg/ml.Ginsen-oside Rb1, Rb2, bufalin and muscone of twenty components from SBP absorbed in blood also possessed significant protective effect a -gainst cardiomyocytes hypoxia/reoxygenation injury .Conclusion SBP have the protective activity against cardiomyocytes hypoxia /reoxygenation injury , and ginsenoside Rb1, Rb2, bufalin, muscone are the main active components of SBP .This experiment offered basis for further pharmacodynamics and mechanism study of SBP .