| Objective (1) To study the protective on mitochondria activity and antioxidising effect of Lovastatin in hypoxia-reoxygenated cardiomyocytes, (2) To study the effect of Lovastatin on activation of the nuclear factor-kappa B (NF-kB) of cardiomyocytes induced by the hypoxia-reoxygenation and serum from patient who received percutaneous coronary intervention (PCI). And (3) To study its roles in regulating gene expression and protein transcription of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible Nitric Oxide Synthase (iNOS).Methods (1) Lumps of myocardial tissue from neonatal Wister rat (24hr) were cultured and purified by treatment with differential enzymes and 5-Bromodeoxyuridine. The cardiomyocytes were cultured in hypoxic condition made with nitrogen to establish a model of hypoxia-reoxygenated cardiomyocytes. The cardiomyocytes and hypoxia-reoxygenated cardiomyocytes were identified by means of phase contrast microscopy, transmission and scanning electron microscopy, HE staining, Trypan blue staining, immunohistochemistry, Periodic acid Schiff reaction (PAS), CK-MB, Myoglobin (Mb), Troponin I (cTnI) by examining the culture supernatant. The immunohistochemistry and PAS were analyzed by the use of Image-Pro Plus software. (2) The mitochondria activity of cultured cardiomyocytes were examined with different doses of LOV: 5, 10, 20, 40 μmol/L, respectively, bymeans of MTT (3-2, 5-diphenyl tetrazolium bromide) test. Effective doses of LOV 5 and 10 nmol/L were chosen to test its effects on mitochondria activity and CK-MB, LDH1, Malondialdehyde (MDA), Superoxide Dismutase (SOD) in the supernatant of the cultured cells by means of MTT test, biochemistry and colorimetric measurement. (3) We examined the effect of Lovastatin on the model of hypoxia-reoxygenated cardiomyocytes or serum from patient who received PCI on activation of NF-kB, on expression of TNF-ou IL-^ iNOS and release of NO by mean of Immunohistochemistry, Laser Scanning Confocal Microscope (LSCM), reverse transcription-polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA). RT-PCR signals were analyzed by using Gel-Pro software.Results (1) We observed that many cells migrated from the lumps of myocardial tissue after 10 hours, and adherent to the culture plate. And formed a single cell layer after 3~5 days. After split of the myocardial cells after 24 hr, they growed rapidly with automaticity pulsating of single and clumps of cells at frequencies from 90 to 150 beat per minute (bpm). When the model of hypoxia-reoxygenated cardiomyocytes was formed, automaticity pulsating frequencies of clumps of cells were down to 30-90 bpm. HE staining showed multi-angle morphology of cardiomyocytes with intact membrane. In the model of hypoxia-reoxygenated cardiomyocytes, some cells were detached with broken membrane and nuclear shed off. Ultracellular structure shown by transmission electron microscope (TEM) indicated that the myocardial cells were in bundles and in good shape, rich in intact mitochodria. Scanning electron microscope (SEM) showed that the myocardial cells were multi-angle with fine, clear membrane, and cell junction was apparent. However, in the model of hypoxia-reoxygenated cardiomyocytes, the cells became round instead of multi-angle, and the membranes were rough, perforated and without clear junctions between cells. The viability of the cell was up to 94.6% asdetermined by Trypan blue staining. However, the viability decreased to 81.4% (p<0.05) when the model of hypoxia-reoxygenated cardiomyocytes was formed. The cell population was homogenous up to 96.4% determined by Actin HHF35 of Streptavidin Peroxidase conjunction method. The IOD was 3185.3 ± 9876.56. PAS showed that glycogen in the model of hypoxia-reoxygenated cardiomyocytes (IOD: 6210.05 ± 2875.11) was significantly decreased compared with normal cardiomyocytes (1548.06 ± 914.36). CK-MB, Mb and cTnl of the culture supernatant was increased significantly in the model of hypoxia-reoxygenated cardiomyocytes. (2) Optical Density (OD) readings in MTT test of different doses of LOV 5, 10, 20, and 40 umol/L and control group were 0.169 ± 0.022, 0.179 ± 0.019, 0.147 ± 0.021, 0.089 ± 0.023 and 0.173 ± 0.027, respectively. MTT OD readings of 5 and 10 umol/L: 0.134 ± 0.022, 0.152 ± 0.027, and the control 0.097 ± 0.018, showed significant difference compared with the control group. These two doses were found to increase the mitochondria activity of cultured cardiomyocytes, decrease the level of CK-MB, LDHi, MDA, and protect SOD in the culture supernatant. (3) The hypoxia-reoxygenation and serum from patient received PCI could stimulate a translocation of NF-KBp65 activity from cytoplasm to nucleus. Similar to pyrrolidine dithiocarbamate (PDTC), LOV can partially block this translocation. Both hypoxia-reoxygenated cardiomyocytes and serum from patient received PCI can upregulate expression of TNF-a, IL-lp and iNOS, and increase TNF-a, IL-lp and NO in the supernatant whereas LOV and PDTC down regulate them. LOV reduced the production of TNF-a, IL-ip, iNOS.Conclusion Our results have demonstrated that (1) the cardiomyocytes in our model system growed well, were highly viable and homogenous. The established model of hypoxia-reoxygenated cardiomyocytes was simple and reliable. (2) The effective doses of LOV protected the mitochondria activity of...
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