The Protective Effect Of Different Polarity Compoents And Monomers From Ilex Pubescens On Neonatal Rat Cardiomyocytes Injury By Hypoxia/Reoxygenation

Objective:To culture cardiomyocytes from neonatal rats and establish a cardiomyocytes hypoxia/reoxygenation injury model. To observe the protective effect of different polarity compoents and monomers from Ilex pubescens on neonatal rat cardiomyocytes injury by hypoxia/reoxygenation.Methods:Myocardial tissues from neonatal rats born within1to3day were digested with0.1%trypsin and0.04%collagenase II. The cardiomyocytes were collected in DMEM medium, which contains10%horse serum and7%fetal bovine serum. Purification of cardiomyocytes was done by differential adhesion and the supplement of5-bromodeoxyuridi-ne. Cardiomyocytes were identified by immunohistochemistry staining of α-actin and we stained cardiomyocytes with trypan blue. We ascertained the best times of hypoxia/reoxygenation through combined detection of cell viability and RFU, detected by CCK-8and Caspase-3/7Assay, to established the model of hypoxia/reoxygenation. The cultured cardiocytes with growth in good condition were divided radmoly into seventeen groups:Control group; H/R group; different doses intervention group of low polarity compoents from Ilexpubescens(LL、LM、LH+H/R); different doses intervention group of middle polarity components from Ilex pubescens(ML、MM、HH+H/R); different doses intervention group of high polarity components from Ilex pubescens{HL、HM、HH+H/R); different doses intervention group of Ilexoside E(EL、EM、EH+H/R); different doses intervention group of Pubescenoside A(AL、AM、AH+H/R). After hypoxia for4h and reoxygenation for4h, cell viability and RFU, lactate dehdrogenase(LDH)、cratnie kniase(CK) and aspartate aminotransferase(AST) release, activities of supoerxide dismutase(SOD) and productions of malnodialdehyde(MDA) in cells were measured in each group.Results:1. After24hours some single cardiomyocytes began to beat spontaneously. After48hours, cardiomyocytes got together and beat synchronously. Cells used in experiments were confirmed as cardiomyocyte and95percent were alive.2. The best times of the model of hypoxia/reoxygenation we ascertained are hypoxia for4h and reoxygenation for4h. The correction measured is extremely closed to the theoretical maximum of uniform design.The model we established, to a certain extent, maximizes the cardiomyocytes apoptosis.3. Compared with control group, in H/R group, cell viability decreased significantly(29.33±1.55%vs.99.21±0.78%, p<0.01), apoptosis increased significantly (20866±875vs.80133±1532, p<0.01); compared with H/R group, in most of doses intervention groups of different polarity compoents and different monomers from Ilex pubescens, cell viability increased significantly, apoptosis decreased significantly.4. Compared with control group, in H/R group, the release of LDH、CK and AST increase significantly(0.1234±0.0063vs.0.3751±0.0005、0.2645±0.0046vs.0.4878±0.0047、0.1505±0.0064vs.0.4054±0.0053, P<0.01), the cardiomyocytes show significant injury; in cells, MDA proudetinos increased significantly(0.0087±0.0006vs.0.0198±0.0003, P<0.01), SOD activities decreased significantly(0.1204±0.0041vs.0.0373±0.0055, P<0.01), the antioxidation ability of the cardiomyocytes decreased. Compared with H/R group, most of doses intervention groups of different polarity compoents and monomers from Ilex pubescens could singifienatly reduce the release of LD^CK and AST (p<0.01)and increase SOD activities (p<0.01)and inhibit MDA proudetinos in cells(p<0.01).Conclusion:High quantity and survival cardiomyocytes of rat can be effectively and reliably cultured by this method. We successfully establish a cardiomyocytes hypoxia/reoxygenation injury model, which simulating myocardial ischemia/reperfusion injury, to a certain extent, maximizes the cardiomyocytes apoptosis, is conducive to screening the protective effect of the drugs and makes the efficacy is more intuitive and obvious. Different polarity compoents and Ilexoside E and Pubescenoside A from Ilex pubescens could singifienatly reduce the injury on neonatal rat cardiomyocytes by hypoxia/reoxygenation, reduce the cell membrane permeability, enhance the ability of cardiomyocytes to scanvenge oxygen free radicals, and effectively protect cardiomyocyte.