Effect Of Rosiglitazone On H9c2 Cardiomyocytes Under The Microenvironment Of High Concentration Of Glucose

Objective:To establish a microenvironment in vitro with a higher concentration of glucose,the effects of different concentrations of glucose on proliferation activity and oxidative stress in cardiomyocytes was investigated.The effects of rosiglitazone on oxidative stress,energy metabolism and apoptosis in H9c2cardiomyocytes were investigated under the microenvironment of high concentration of glucose.Methods:（1）To establish a microenvironment at a higher concentration of glucose:cardiomyocytes were cultured in the medium containing different concentrations of glucose for 24 hours,48 hours and 72 hours,respectively.The morphological changes of cardiomyocytes were observed by inverted microscope and cell viability were determined by MTT assay.The content of glutathione（GSH）and malondialdehyde（MDA）were detected by microplate reader.The activity of superoxide dismutase（SOD）was detected by Water-soluble tetrazolium（WST-1）assay.The level of reactive oxygen species（ROS）was detected by flow cytometer.Furthermore,the content of tumor necrosis factorα（TNF-α）and transforming growth factor beta 1（TGF-β1）were detected by enzyme linked immunosorbent assay（ELISA）.（2）To select the test concentrations and test time of rosiglitazone（RSG）:the low,medium and high concentration of test drug RSG and the optimum test time for the in vitro cardiotoxicity test were defined through determine cell viability by MTT assay and morphological changes of cells under microscope.（3）The effect of RSG on cardiotoxicity:under the microenvironment at a higher concentration of glucose,cardiomyocytes were cultured 48 hours in the medium containing RSG with low,medium and high concentration.The content of GSH and MDA were detected by microplate reader.The activity of SOD was detected by WST-1 assay.The level of ROS was detected by flow cytometer.The apoptosis rate of cardiotoxicity was detected by Annexin V-FITC/PI.The changes of mitochondrial membrane potential（MMP）was detected by rhodamine 123（Rh123）.Furthermore,the energy changes of cardiotoxicity was detected by reversed phase high performance liquid chromatography（RP-HPLC）.Results:（1）Cardiomyocytes were cultured in the medium containing 33.30 mmol·L^-11 of glucose for 24 hours,compared with the control group at a normal concentration(25 mmol·L^-1)of glucose,cell proliferation activity reduced significantly and the content of GSH and SOD activity reduced,whereas the content of MDA,the concentrations of TNF-aand TGF-β1 and ROS level increased（P<0.05）.Therefore,the toxic concentration of glucose and test time,which inhibit the proliferation of cardiomyocytes and trigger a series of oxidative stress reactions,were 33.30 mmol·L^-11 and 24 hours,respectively.（2）The experimental result of MTT showed that the cardiomyocytes cultured with RSG for 48 hours showed a good inhibition curve with R² 0.9861 and the concentration of RSG resulting in 50%viability of cardiomyocytes(IC₅₀)was 456.31μmol·L^-1.Therefore,the optimum test time was defined as 48 hours.At the same time,the low,medium and high concentrations of RSG were defined as 340.00μmol·L^-1,450.00μmol·L^-11 and560.00μmol·L^-1,respectively.Compared with the control group,the content of GSH,MMP,adenosine 5’-triphosphate（ATP）and SOD activity reduced,whereas the content of MDA,ROS level,adenosine 5’-diphosphate（ADP）,adenosine5’-monophosphate（AMP）and the apoptosis rate of cardiotoxicity increased（P<0.05）.（3）Detection of ATP,ADP and AMP by RP-HPLC analysis method:a mobile phase consisting of 0.2 mol·L^-11 phosphate buffer（consisted of 0.805 g tetrabutylammonium bromide）and methanol（95:5,v/v）,was pumped through a ZORBAX SB-C₁₈（4.6mm×150 mm,5μm）column at temperature 35°C at a flow rate of 1.0 m L·min^-1.The detection wavelength was set at 258 nm.Retention times of ATP,ADP and AMP were11.8 min,7.5 min,4.3 min,respectively.There is a good linear relationship between peak areas of ATP,ADP,AMP and concentrations.Linear range of ATP,ADP and AMP were 0.75⁴8μg·mL^-1,0.50³2.00μg·mL^-1,and 0.25¹6.00μg·mL^-1,respectively.Lower limit of quantification（LLOQ）of ATP,ADP and AMP were 0.75μg·mL^-1,0.50μg·m L^-11 and 0.25μg·mL^-1,respectively（S/N=10）.The extraction recovery rates of ATP,ADP and AMP were in the range of 65.28%⁸1.08%.Inter-day,intra-day and stability relative standard deviation（RSD）was less than 15%.Conclusion:（1）Microenvironment of high concentration of glucose was an important factor to inhibit the proliferation of cardiomyocytes and trigger a series of oxidative stress reactions.The optimum test concentration of glucose and test time for stimulating a microenvironment with a higher concentration of glucose causing in vitro cardiotoxicity were 33.30 mmol·L^-11 and 24 hours,respectively.（2）The analysis method of RP-HPLC was established to simultaneously determine the contents of ATP,ADP and AMP.The precision and accuracy of this method were up to the mustard and had good stability.（3）RSG concentration(which was greater than340.00μmol·L^-1)had a cardiotoxic effects on H9c2 cells by triggering a series of oxidative stress reactions,disordering the energy metabolism of cell and inducing apoptosis.