| Background:Myocardial ischemia reperfusion injury (MIRI) refers to the tissue of ischemicmyocardium after restoring blood flow could be injured heavier and even produce thephenomenon of irreversible damage. Many important treatments to ischemic myocardiumsuch as coronary artery bypass graft surgery, acute myocardial infarction fibrinolytictherapy, and revascularization by percutaneous coronary intervention were restricted byMIRI, which has become the clinical problems to be solved. Large amounts of data showedthat apoptosis is the main form of cardiac cell death after MIRI. However, the researchabout cardiomycocyte apoptosis by MIRI mostly concentrated on the discovery ofapoptosis and tried to intervene it but the molecular mechanism. With the development ofthe research, mitochondrial regulation in the process of cardiomyocytes apoptosis wasrevealed gradually in MIRI, which was considered as one of the apoptotic signaltransduction of the significant progress. In recent years, the study found that Smac/Diablowas released from mitochondria into the cytoplasm to promote apoptosis after cell wasstimulated by apoptotic signal. Herbal fuzi, the monarch drug of chinese tradional formulasuch as sini decoction and shenfu decoction, displayed good resistance to cardiomyocytesapoptosis on the research of MIRI. At present, traditional Chinese medicine monomer has become a research focus, FPS, another monomer which is different from aconitine, wasextracted from aconite. It was being people’s attention gradually because of hypoglycemiceffect and lipid modulation, immunity-enhancing and anti-tumor assistance but theprotection of cardiomyocytes and mechanism about MIRI studies have not been reported.Objective:In this study, cultured neonatal rat cardiomyocytes was performed to prepared H/Rinjury in vitro, and was given with FPS to investigate the protective effect oncardiomyocytes apoptosis and to further explore the protective mechanism of FPS in theMIRI based on signal transduction pathway of mitochondrial apoptosis. The researchprovide a solid theoretical foundation and laboratory evidence for the FPS drugdevelopment.Methods:To establish the model of hypoxia/reoxygenation with primary culturedcardiomyocytes were obtained from neonatal rat. The cells were divided into groupsrandomly: Control group, H/R group (the reoxygenation6h after hypoxia3h), FPS groups(concentrations of0.1mg/mL,1mg/mL,10mg/mL, and20mg/mL FPS interventioncultivation for24h and then the same treatment with H/R group), the results are asfollows:1Cardiomyocytes morphology: The change of the cardiomyocytes morphology wasobserved by an inverted microscope to analyse the effect of hypoxia and reoxygenationand different concentrations of aconite polysaccharides on the morphology ofcardiomyocytes.2The determination of cardiomyocytes viability: The survival rate of cardiomyocyteswas measured by MTT assay to determine that whether FPS chould protectcardiomyocytes from hypoxia/reoxygenation.3Leakage volume of LDH and the activity of CK in cardiomyocytes: The damageddegree of cytomembrane and mitochondrial membrane were displayed by determinating the activity of LDH in cardiomyocytes medium and CK intracellular to analyse theeffect of FPS on the cytomembrane permeability change in the process of oxidativestress.4Contention of MDA, activity of SOD in cardiomyocytes: To determinate thecontention of MDA and SOD activity intracellular and judge the effect of FPS on lipidoxidation and antioxidant enzyme synthesis hypoxia/reoxygenation intracellular.5MT of cardiomyocytes: The expression level of cardiomyocytes metallothionein (MT)was detected by Enzyme-linked immunoassay (ELISA), which reflect the ability ofcardiomyocytes to oxygen free radicals, and analyse the effect of FPS anti-oxidativestress injury on cardiomyocytes.6The determination of cardiomyocytes apoptosis rate: Cardiomycocyte apoptosis wasdetected by Flow Cytometry to judge the effect of FPS on induced apoptosis ofhypoxia/reoxygenation cardiomyocytes.7The detection of Smac/Diablo in cytoplasmic: Western Blot was used to detectSmac/Diablo protein content of cardiomyocytes cytoplasmic released frommitochondria. To reflect the degree of openness of the mitochondrial permeabilitytransition pore (mPTP) to investigate the role of the FPS with mitochondrial apoptosissignal transduction pathway.8Bcl-2mRNA, Bcl-xl mRNA and Bcl-xs mRNA in cardiomyocytes: Quantitative PCRwas used to detect the effect of FPS on the levels of Bcl-2mRNA, Bcl-xl mRNA andBcl-xs mRNA to explore its role in cardiomyocytes protection from the genetic level.Results:1The effect of FPS on hypoxia/reoxygenation in neonatal rat cardiomyocytesmorphology: Cardiomyocytes grew well with morphology diversified (round, conical,or spindle). Clusters of cells increased with beating regularly and the cytoplasm budlike protuberances and grew radial around cell clusters. Cells beating and cytoplasmprotuberances reduced or even disappeared with a large number of dead cells floating inthe medium after hypoxia/reoxygenation. The above situation could be improved by FPS on the better concentration as10mg/mL.2Effect of FPS on cardiomyocytes vitality: The survival rate of cardiomyocytesreduced significantly. There are significant statistical difference as compared to Controlgroup(P<0.01). With the concentration increasing, FPS could improve the survival rateof cardiomyocytes. There are significant statistical difference from0.1mg/mL of FPSas compared to H/R group(P<0.05).3Effect of FPS on the leakage volume of LDH and CK: LDH activity in the mediumand CK vitality intracellular improved significantly compared to the Control group.There was statistical difference between them(P<0.01). When the concentration isbelow20mg/mL, FPS could reduce activity of LDH in the medium with theconcentration increasing. There also has statistical difference(P<0.05) from theconcentration of1mg/mL compared to H/R group. Meanwhile, FPS could reduce CKvitality intracellular but only the concentration is10mg/mL, the result is meaningful ascompared to H/R group(P<0.05).4Effect of FPS on the content of MDA and SOD vitality: The content of MDA wasincreased significantly in H/R group as compared with the Control group(P<0.01).When the concentration is below20mg/mL, FPS could reduce MDA contentintracellular with the concentration increasing,there are statistical difference (P<0.05)from the concentration of1mg/mL as compared to H/R group. SOD vitality decreasedsignificantly as compared to Control group(P<0.01). FPS could improve SOD vitalityintracellular but only the concentration is10mg/mL, the statistical difference ismeaningful as compared to H/R group(P<0.05).5The effect of FPS on the content of MT: Content of MT increased significantly in H/Rgroup was compared to Control group(P<0.01). With the concentration increasing from1mg/mL, the content of MT increased in FPS group as compared to H/Rgroup(P<0.05), the content of MT is the maximum while as10mg/mL.6Effect of FPS on the rate of cardiomycocyte apoptosis: The apoptosis rate ofcardiomycocyte was increased in H/R group compared to Control group(P<0.01). Whenthe concentration bellow20mg/mL, the higher concentration of FPS, the lower apoptosis rate of cardiomycocyte. The statistical difference is meaningful as comparedto H/R group(P<0.05) from the concentration of1mg/mL.7The effect of FPS on Smac/Diablo: The content of Smac/Diablo from cytosolicincreased significantly in H/R group compared with the Control group(P<0.01). FPScould reduce the content of Smac/Diablo with a dose-dependent manner when bellow20mg/mL, The statistical difference is meaningful as compared to H/R group(P<0.05)from the concentration of1mg/mL.8The effect of FPS on Bcl-2mRNA,Bcl-xl mRNA and Bcl-xs mRNA: Bcl-2mRNA,Bcl-xl mRNA and Bcl-xs mRNA decreased significantly in H/R group as compared toControl group(P<0.05). When the concentration of FPS is below20mg/mL, it couldimprove the level of Bcl-2mRNA and Bcl-xl mRNA. There are statistical differencebetween FPS group and H/R from the concentration of0.1mg/mL and1mg/mLrespectively(P<0.05). Bcl-xs mRNA increased in H/R group as compared to Controlgroup(P<0.05). The level of Bcl-xs mRNA is decreased gradually induced by FPS from1mg/mL, and get the minimum while10mg/mL. The statistical difference ismeaningful as compared to H/R group(P<0.01).Conclusion：1In this study, FPS with good protection of hypoxia/reoxygenation in cardiomycocytewas proved from the point of biochemicalâ†'proteinâ†'gene.2FPS on the protection of hypoxia/reoxygenation was related to its mitochondrialapoptosis signal transduction pathway of anti-oxidative stress, mitochondrial protection,inhibition of apoptosis.3The optimal concentration of FPS to play a protective effect to hypoxia/reoxygenationin cardiomycocyte is10mg/mL. |
PDF Full Text Request