The Effect Of Ilex Pubescens Saponin E On Cardiomyocytes Apoptosis Induced By Hypoxia/reoxygenation And Mechanism Research

Objective:Ilex Pubescens saponin E is the typical element of medicinal material base in Mao-Dong-Qing,the purpose of this paper is to use primary cultured neonatal rat cardiomyocyte and establish the model of hypoxia/reoxygenation injury in vitro,at a cellular level simulate rats myocardial ischemia/reperfusion injury in vivo,study the effect of IIex Pubescens saponin E on cardiomyocytes apoptosis induced by hypoxia/reoxygenation and the action mechanism.Methods:1.The cultivation and identification of primitive neonatal rat cardiomyocyte.The myocardial tissues from neonatal rats born within 1 to 3 day were soaked with 0.25% trypsin overnight at 4℃,then digested with 0.08% colla-genaseII by magnetic stirring at 37℃,the cardiomyocytes were cultivated in DMEM medium containing 15% fetal bovine serum,purification the cardiomyocytes were done by differential adhesion and the supplement of 5-bromodeoxyuri dine.Cardiomyocytes were identified by immunohisto chemistry staining of a-actin.2.The establishment of hypoxia/reoxygenation injury model.Uniformdesign was used to determine the different combinations of hypoxia and reoxyg-enation time,the viability was regarded as the main index,detected by CCK-8,in order to preliminarily screen the best model conditions,the apoptosis rate was regarded as auxiliary index,detected by TUNEL,for validating the best model conditions,finally the hypoxia/reoxygenation injury model was success-fully established.3.The effect of Ilex Pubescens saponin E on cardiomyocytes apoptosisinduced by hypoxia/reoxygenation.The cardiomyocytes were incubated with IIexPubescens saponin E before modeling,cardiomyocytes were randomly divided into5 groups:Control group(Control,CON);Hypoxia/reoxygenation group(hypoxia/reoxygenation,H/R);the low、medium and high dose of intervention group of IIexPubescens saponin E(EL,EM,EH + H/R).After hypoxia for 4h and reoxygenationfor 4h,cardiomyocytes survival rate and apoptosis rate was determined by CCK-8and TUNEL.4.the action mechanism of Ilex Pubescens saponin E inhibiting cardio-myocytes apoptosis induced by hypoxia/reoxygenation.The dosing method and experimental group of cardiomyocytes were alexandrine,after hypoxia for 4h and reoxygenation for 4h,the concentration of reactive oxygen species in cardiomyocytes was detected by DHE fluorescent probe,the expression of apop-tosis protein caspase-3 was detected by Western Blot.Results:1.The cardiomyocytes morphological observation.After 4~6 hours,cells started adherent growth,after 12~24 hours,some single cardiomyocyte bagan to beat spontaneously,after 48 hours,cardiomyocytes formed cell monolayer or cell clusters and beat synchronously,it was so-called functionalsyncytium.Through immunohisto chemistry staining of a-actin,cardiomyocytes was strong positive reaction(brown granular change)and positive rate was more than 95%.2.The establishment condition of hypoxia/reoxygenation injury model.Through the screening,the best times of hypoxia/reoxygenation model we asc-ertained was hypoxia for 4h and reoxygenation for 4h,under the condition,the cardiomyocytes survival rate was 48.80%;the apoptosis rate(Integrated Op-tical Density value,IOD)was 9494.27,about 8 times that of control group.3.The effect of IIex Pubescens saponin E on the viability and apoptosis of hypoxia/reoxygenation cardiomyocytes.Compared with control group,in H/R group,cardiomyocytes survival rate decreased significantly(45.10 ± 3.10%vs.100.00±2.94%,P< 0.01),the apoptosis rate increased obviously(16.57 ±0.45%vs.0.60 ± 0.16%,P< 0.01);compared with H/R group,in different dosesintervention groups of IIex Pubescens saponin E,cardiomyocytes survival rate increased significantly,the results in turn were(56.09 ± 3.95)%,(64.60±4.16)%,(78.03±2.56)%;apoptosis also decreased obviously,the results in turwere(13.15±0.18)%,(11.49±0.27)%,(8.45±0.20)%.4.The effect of IIex Pubescens saponin E on the Reactive oxygen species and Caspase-3 protein in hypoxia/reoxygenation cardiomyocytes.Compared with control group,in H/R group,the concentration of ROS in cardiomyocytes inc-reased obviously[(145.17±9.65)IOD vs.(33.96±3.10)IOD,P<0.01],Caspase-3expression also increased significantly [(1.07±0.03)gray level ratio vs.(0.38±0.01)gray level ratio,P<0.01];compared with H/R group,in different doses intervention groups of IIex Pubescens saponin E,the content of ROS reduced in different degrees,the results in turn were 109.01 ±7.55,86.45±4.92,54.05 ± 7.10;the expression of Caspase-3 reduced significantly in high dose intervention group,the value was 0.62±0.04.Conclusion:1.The primary cardiomyocytes culture method adopted in this subject is simple,stable and reliable,high quantity and survival and number of cardiomyocytes of rats can be effectively obtained.We successfully establish cardiomyocytes hypoxia/reoxygenation injury model and use to evaluate the myocardial protection of drug.2.Ilex Pubescens saponin E as the representative element of Mao-DongQing could significantly enhance the survival rate of the hypoxia/reoxygenation cardiomyocytes,reduce the apoptosis rate and inhibit directly the apoptosis of cardiomyocytes;the action mechanism is related with the effect,which IIex Pubescens saponin E can significantly reduce the concentration of ROS and the expression of apoptosis protein Caspase-3.