Studies On The Protective Effect Of Artificial Musk On Neonatal Rat Ventricular Myocytes And Human Umbilical Vein Endothelial Cells(HUVEC)

As a substitute for natural musk,artificial musk was confirmed after years of modern pharmacological research in neuroendocrine system,cardiovascular system,anti-inflammatory and immune system is similar to natural musk of pharmacologic actions,exact clinical efficacy,safety and reliability.In clinically,musk as principal drug of multiple compound preparations rendering out more good at anti-myocardial ischemia and anti-myocardial infarction,our research group of the early pharmacological experiment study found that,artificial musk has effect of anti-myocardial ischemia of animals,protective effect of artificial musk preconditioning on rat myocardial ischemic and reperfusion injury,but its findings at the cellular level and its mechanisms are consistent with the overall level of research have not been reported.This research will develop a series of studies of protective effect about artificial musk on neonatal rat myocardial cells and human umbilical vein endothelial cells(HUVEC).The research is divided into four parts:Part one:Protective effect of artificial musk in H2O2-induced neonatal rat ventricular myocytes injuryPrimary cultured neonatal rat myocardial cells;Filter the suitable concentration of H2O2 damage and injury time,establish the myocardial cell injury model;Selecting the right concentration and solvent content;By observing the following indicators:morphology of myocardial cells,pulsating frequency of cardiac myocytes,cell viability,SOD?CK?LDH?MDA?NO level in cardiac myocytes,Hoechst 33258 fluorescent staining observation cell apoptosis,Bcl-2?Bax?Bad?Caspase-3 mRN A expression level in each group of myocardial cells,To reflect the protective effect of artificial musk in H2O2-induced cardiomyocytes injury.Part two:Protective effect of serum containing artificial musk in serum deprivation-induced neonatal rat ventricular myocytes injuryFirst of all,the serum containing artificial musk preparation;Primary cultured neonatal rat myocardial cells;Myocardial cells cultured in serum-free culture-based causes cell injury,filter suitable time for serum-free culture,establish the myocardial cell injury model;By observing the following indicators:morphology of myocardial cells,pulsating frequency of cardiac myocytes,cell viability,SOD?CK?LDH?MDA?NO level in cardiac myocytes,Bcl-2?Bax?Bad?Caspase-3 proteins expression level in each group of myocardial cells.To reflect the protective effect of serum containing artificial musk in serum deprivation-induced neonatal rat ventricular myocytes injury.Part three:Protective effects of artificial musk on ischemia-reperfusion injury in neonatal rat ventricular myocytesCultured neonatal rat myocardial cells;Preparation the ischemia-reperfusion injury model(I/R model);By observing the following indicators:morphology of myocardial cells,pulsating frequency of cardiac myocytes,cell viability,SOD?CK?LDH?MDA level in cardiac myocytes,intracellular Ca2+,apoptosis rate,MMP and Caspase-3 activity of cardiac myocytes,Bcl-2?Bax proteins expression level in each group of myocardial cells,To reflect the protective effect of artificial musk on ischemia-reperfusion injury in neonatal rat ventricular myocytes.Part four:Protective effect of artificial musk on HUVEC1.Protective effect of artificial musk in H2O2-induced HUVEC injuryCultured HUVEC,determine the concentration of artificial musk,establish the H2O2-induced HUVEC injury model;Determination the cell viability of HUVEC,Hoechst 33258 fluorescent staining observation cell apoptosis,flow cytometry detection of cell apoptosis,Determination of intracellular Ca2+,MMP and Caspase-3 activity of HUVEC,the expression of Bcl-2 protein in HUVEC.To reflect the protection effect of artificial musk on HUVEC.2.Proliferation and function of artificial musk on HUVECHUVEC cultured in vitro,MTT colorimetric method for the determination the proliferation effect of different concentrations artificial musk solution on HUVEC;Western Blot was used to detect expression levels of p-ERK and p-P38 proteins in HUVEC;RT-PCR determination the mRNA expression levels of eNOS,VEGFR1 and VEGFR2 in HUVEC.Observation the effects of proliferation and function of artificial musk on HUVEC.Conclusions:1.Artificial musk may through improve morphology of myocardial cells and pulsating frequency of cardiac myocytes,improve cell viability of myocardial cells,reduced the content of LDH?MDA?CK,recover the reduced SOD and NO enzyme activity,inhibit apoptosis of myocardial cells,increase anti-apoptosis factor Bcl-2 mRNA expression level,reduce mRNA expression levels of apoptosis factor Bax?Bad,also reduce mRNA expression levels of apoptosis executive factor Caspase-3.Demonstration the protective effect of artificial musk in H2O2-induced cardiomyocytes injury.2.Serum containing artificial musk through improve morphology of myocardial cells and pulsating frequency of cardiac myocytes,improve cell viability of myocardial cells,reduced the content of LDH?MDA?CK,recover the reduced SOD and NO enzyme activity,increase anti-apoptosis factor Bcl-2 protein expression level,reduce protein expression levels of apoptosis factor Bax?Bad,also reduce protein expression levels of apoptosis executive factor Caspase-3.Demonstration the protective effect of serum containing artificial musk in serum deprivation-induced neonatal rat ventricular myocytes injury.3.Artificial musk may through improve morphology of myocardial cells and pulsating frequency of cardiac myocytes,improve cell viability of myocardial cells,reduced the content of LDH?MDA?CK,recover the reduced SOD enzyme activity,reduce the intracellular concentration of Ca2+,stabilize mitochondrial membrane,down-regulation reduced Bcl-2 protein expression level and up-regulation increased Bax protein expression level.Demonstration the protective effect of artificial musk on ischemia-reperfusion injury in neonatal rat ventricular myocytes.4.Artificial musk may through inhibit apoptosis of HUVEC,stabilize mitochondrial membrane potential,down-regulation the intracellular concentration of Ca2+,reduce Caspase-3 activity,down-regulation reduced Bcl-2 protein expression level in HUVEC.Demonstration the protective effect of artificial musk in H2O2-induced HUVEC injury;Artificial musk through promoting proliferation of cultured HUVEC with respect to a certain extent to the compensatory effect of anti-myocardial ischemia.