Effects Of Roudoukou-8 San On Adenylic Aid Content Of MIRI Rats In Myocardium And Cell Activity And PCR Identification Of Different Animal Cardiac Medicines

Objectives（1）To establish a method for the simultaneous determination of ATP,ADP and AMP in myocardial tissue of rats by high performance liquid chromatography（HPLC）,and to investigate the effect of Roudoukou-8 San on energy metabolism after myocardial ischemia reperfusion injury（MIRI）in rats.（2）To study the protective effect of Roudoukou-8 San on cardiomyocyte injury in neonatal rats induced by hydrogen peroxide.（3）To study the feasibility of PCR in the identification of different animal heart medicines,and to reveal the differences between different animal heart materials at the genetic level.Methods（1）The content of ATP,ADP and AMP in myocardium homogenate of sham operation group,MIRI model group,positive drug control group and different doses of Roudoukou-8 San groups（high,medium and low）.Chromatographic conditions:the column was Thermo Hypersil C₁₈column（250 mm×4.6 mm,5μm）with a column temperature of 25℃;the DAD detector with a detection wavelength of 259 nm;the mobile phase is methanol-50 mmol·L^-11 potassium phosphate Buffer（7:93）,pH 6.68;flow rate is 1.0 mL·min^-1.（2）Isolation and culture of rat neonatal cardiomyocytes,H₂O₂ was used to simulate ROS-induced cardiomyocyte injury in vivo.The morphological changes of cardiomyocytes were observed by an inverted microscope.The viability of cardiomyocytes was measured by MTT assay.The contents of LDH,CK and AST in cell culture fluid were detected by automatic biochemical analyzer.The content of MDA,SOD and NO in cells were detected by kit method.Hoechst fluorescence staining was used to observe the apoptotic morphology of cardiomyocytes.The apoptosis rate of cardiomyocytes was detected by flow cytometry.（3）According to the four EPO gene sequences,we designed four pairs of PCR primers that could accurately identify the four kinds of animal heart medicines,amplified them by PCR.Agarose gel electrophoresis analysis of the bands to verify the amplification products.Results（1）ATP,ADP and AMP were separated well.The concentrations of ATP,ADP and AMP were in the range of 2.5¹60.0μg·mL^-1,5.0³20.0μg·mL^-1,10.0⁶40.0μg·mL^-1,respectively.Peak area and concentration had a good linear relationship（r>0.9990）.The RSDs of intra-day and inter-day precision were less than 10.0%;the recoveries were 88.4%¹03.5%,and the RSDs of stability tests were0.54%⁹.48%.Different doses of Roudoukou-8 San groups have an impact on energy metabolism of MIRI rats.（2）The effect of 100μmol·L^-11 H₂O₂ for 2h obviously caused about50%injury of cardiomyocytes.Observed under an inverted optical microscope,the cells in the H₂O₂ model group exhibited obvious cell damage such as increased cell gap,decreased cell quantity,cytoplasm vacuoles,etc.Compared with H₂O₂ model group,the morphological changes of cardiomyocytes in different dose groups were improved to some extent.Roudoukou-8 San can obviously reduce the content of LDH,CK,AST in H₂O₂-injured cardiomyocytes;decrease the content of MDA and NO in H₂O₂-induced myocardial cells and increase the activity of SOD;（3）Four kinds of animal heart medicinal materials appeared corresponding bands in four different positions.The amplified products were purified by two-way sequencing and BLAST analysis was used to confirm that the experimental sequence was destination band.Conclusions（1）The determination the contents of ATP,ADP and AMP in myocardium by HPLC method can meet the requirements of biological samples,which is simple and easy to operate with accurate results.Roudoukou-8 San can obviously improve the energy metabolism of myocardium in MIRI rats and protect myocardium.The mechanism of oxygen metabolism may be related to promoting ATP synthesis,reducing its hydrolysis and improving energy metabolism.（2）Roudoukou-8 San can protect cardiomyocytes by improving cell survival,increasing cell viability,reducing oxidative stress,inhibiting cell apoptosis and reducing H₂O₂ damage of cardiomyocytes.（3）The method of PCR was used to identify four kinds of animal heart medicines.The method is simple,rapid and easy to operate and reveals the differences among the four kinds of animal heart medicines at the molecular level of DNA.