| IntroductionNaVH+ exchanger (NHE) is one of the pathways which extrudes protons and its activity will lead to a Na+ influx. As to ischemia - reperfusion injury , especially in the period of ischemia and at the start of reperfusion, its activity contribute to Ca"+ entering into cardiomyocytes and to [ Ca^+ I, overload which will lead to damage of the cells.A series of studies have shown that manipulations that attenuate the activity of NHE by adding NHE inhibitor can reduce effectively ischemia reperfusion injury and protect cardiomyocytes such as reducing the area of infarction, anti - arrhythmia, improving the function of en-dodermis and decreasing apoptosis and so on. However, the mechanisms of the protection of NHE inhibitors are unclear; and it is debated whether addition of NHE inhibitors at the start of reperfusion is protective. So, in the study the cultured neonatal rat cardiomyocytes were used for the experiments of hypoxia/reoxygenation and utilized to simulate ischemia - reperfusion injury . In order to demonstrate the mechanisms for the EIPA cardioprotection , [ Ca2+ ]; in single cardiomyocytes and the affect of EIPA added in different period was measured ; At the same time, the change of the cells'area and survival rate was observed.Materials and Methods1. Material:1. 1 Experimental animals; neonatal Wistar rat born for 1 -2 days1. 2 Medicine in experiment :5 - ( N - ethyl - N - isopropyl) -amiloride (EIPA)2. Methods2.1 The establishment of I/R injury model:For hypoxia, the culture media were replaced for 180 min by a modified glucose -free -Tyrodes solution (no sugar) which was saturated for no less than 30 min with 5% C02 -95%N2. For reoxygen-ation for TOmin, the solution was changed to DMEM saturated with 5% CO2 - 95% atmosphere.2.2 Grouping; Those were;Control ( experimental model without any treatments) ;EIPA - I ( EIPA added during the whole period of hypoxia /reox-ygenation) ;EIPA - R1(1 mol/L EIPA added only just 5 min before reoxy-genation) ;EIPA - R2(4 mol/L EIPA added only just 5 min before reoxy-genation) ;3. Assay indexes3. 1 Measure of [Ca2+ ];: a customized inverted microscope was used t measure epifluorescence of cells loaded with Fura -2. The myocytes to be studied was illuminated with a xenon lamp at 490 -510nm, and the wavelengths of emitted light were 340nm and 380nm. Its image directed to the high - sensibility cool CCD camera. The signal was analyzed with Meta - fluo software. [ Ca + ]; was calculated with the standard equation supported by Gynkiewicz.3.2 Measure of the individual cell' s area: the area of a single cell skinned with the Meta - fluo software was measured.3.3 The survival rate of cardiac myocytes4. Statistics: data are given as mean values SD . Statistics analyses were performed using Student's t - test or analysis of variance (ANOVA) or analysis of linear regression. From all experiments, differences between groups were considered significant if the probability value was p <0.05.Results1. Effect on [ Ca2+], when EIPA were add during hypoxia /reox-ygenation: The changes in [ Ca2 + ] i from EIPA - I were significantly lower than that from control and EIPA - R,and EIPA - R2 during the ISOmin -period of hypoxia. The increasing extant of [Ca2+ ]; from EIPA -1 and EIPA - R2 was significantly lower than that from control ,however, there was no difference between EIPA - R,and control during /reoxygenation.2. The changes of the area of myocytes and effect of EIPA added during hypoxia /reoxygenation on its area; the myocytes from control underwent contracture after 30min - period hypoxia, which was significantly different versus EIPA -1. Contracture of the myocytes from EIPA -1 postponed to 90min after hypoxia. Comparing the extent of contracture of all groups, myocytes from EIPA -1 underwent contracture with (47. 63 12.98) % at the end of reoxygenation, EIPA - R, with (57.63 8.16)% ,EIPA -R2 with(56.00 14. 27)% , which had significantly difference versus control with ( 7... |
PDF Full Text Request