Protective Effects And Mechanisms Of Polyphenol Fraction Of Potentilla Anserine L. Against Hypoxia Injury In Cardiomyocytes

Objectives: To study the protective effects and mechanisms of polyphenol fraction of Potentilla anserine L. on cultured cardiomyocytes of neonatal rats in hypoxia injury.Methods: (1) Primary cultured SD neonatal rat (1-3d) cardiomyocytes were used to explore. Through MTT method and measuring Lactate dehdrogenase (LDH) we ascertained the best time of hypoxia and then established the model of hypoxia. (2) The cultured cardiomyocytes with growth in good condition were divided randomly into control group,model group,intervention group of polyphenol fraction of Potentilla anserine L. (0.44mg/ml,0.22mg/ml,0.11mg/ml ) and danshen root group (3mg/ml). After hypoxia for 6h,the effect of different polyphenol fraction of Potentilla anserine L. doses on hypoxia injury in cardiomyocytes were studied by HE staining,PI-Hoechst staining,Dapi staining,flow cytometry and biochemical indicators (LDH,CK). (3) The possible mechanisms on hypoxia injury in cardiomyocytes were approached by biochemical indicators (SOD,MDA,NO,NOS),RT-PCR (Caspase-3,Caspase-9,Bcl-2,Bax,P53) and Western Blot (Caspase-3,Cyt-c,Bcl-2,Bax,P53).Results: 1 The established model and determinatecon centration:?During six hours of cardiomyocytes hypoxia injury, the cells viability and the release of LDH in model group degraded and increased in the largest extent, so we ascertained six hours as the best time of hypoxia. Then through MTT method We ascertained the doses of high,medium and low guop respectively as 0.44mg/ml,0.22mg/ml,0.11mg/ml .2 The Morphological findings: After having been cultured 3d, the cell shape of myocardial cells showed diversification,?strong refractive index,?the number increasing of cell clusters. After being injured by hypoxia, refracting power of the cells mass declined. Cell pulse became weaker significantly.3. The protective effects of polyphenol fraction of Potentilla anserine L. on cultured cardiomyocytes of neonatal rats in hypoxia injury:(1) HE staining: the intervention group of polyphenol fraction of Potentilla anserine L. and dancen root group could significantly ameliorate shaps and Structures of cultured cardiomyocytes by hypoxia injury.(2) PI-Hoechst staining: the intervention group of polyphenol fraction of Potentilla anserine L. and dancen root group could remarkably ameliorate shaps and Structures of nucleus by hypoxia injury,also they may decrease Cell Number of red-dyeing cells.(3) Dapi staining: Postintervention with polyphenol fraction of Potentilla anserine L. and dancen root group, the shaps and Structures of cultured cardiomyocytes by hypoxia injury were remarkably ameliorated.(4) Flow cytometry: polyphenol fraction of Potentilla anserine L. (0.44mg/ml) could remarkably decrease the percentage of apoptotic cells induced by hypoxia (P<0.01).(5) Biochemical indicators: Each dose of of polyphenol fraction of Potentilla anserine L. could significantly reduce the release of LDH, CK (P<0.01).4 The mechanisms of protective effects as polyphenol fraction of Potentilla anserine L. on cultured cardiomyocytes of neonatal rats in hypoxia injury:(1) Biochemical indicator: The polyphenol fraction of Potentilla anserine L. significantly increase SOD activities (P<0.01) and inhibit MDA productions in cells (P<0.05) and heighten the content of NO (P<0.01) and the level of NOS (P<0.05) compared with model group during hypoxia injury.(2) RT-PCR: the mRNA level of Bcl-2 was decreased significantly by hypoxia (P<0.01).In contrast,the mRNA levels of Caspase-9,Caspase-3,Bax,P53 were increased in comparing with that of the control group(P<0.01).each dose of polyphenol fraction enhanced the express level of Bcl-2 mRNA (P<0.01) and depressed the express level of Caspase-9(the high and intermediate doses P<0.01, the low dose P<0.05),Caspase-3,Bax,P53 (P<0.01) mRNA on hypoxia injury myocardial cells compared with model group.(3) Western blot: the protein level of Bcl-2 was decreased significantly by hypoxia (P<0.01).In contrast,the protein levels of Caspase-3,Cyt-c,Bax,P53 were heightened in comparing with that of the control group(P<0.01).each dose of polyphenol fraction enhanced the express level of Bcl-2 protein (P<0.01) and depressed the express level of Caspase-3,Cyt-c,Bax,P53 (P<0.01) protein on hypoxia injury myocardial cells compared with model group.Conclusions: The polyphenol fraction of Potentilla anserine L. could significantly improve the injury of cardiomyocytes induced by hypoxia, lessen the degree of ultramicrostructure changes, and make the Structures and functions to be normal, indicating its protection effect on cultured cardiomyocytes of neonatal rats in hypoxia injury. The possible mechanisms of this protection were: to elevate the cleaning ability to oxygen free radical, depress P53,Bax and enhanced Bcl-2 protein level, improve the function of mitochondria, depress Cyt-c level, then weaken Caspase cascade reaction and intercept the happeness of apoptosis.