The Protective Effect Of Dioscin On Cultured Cardiomyocytes Treated By Hypoxia/Reoxygenation

OBJECTIVE:To investigate the protective effects of dioscin(D)on cultured neonatal Wistar rats cardiomyocytes treated by Hypoxia/Reoxygenation(H/R)and its mechanisms.METHODS:Cultured cardiomyocytes of neonatal Wistar rats were exposed to H/R and randomly divided into five groups:Control group;H/R group;dioscin L,M,H group. At the end of experiments,cells beating rate were determined by manual & viability were determined by MTT assay.Mitochondria were labeled by Rhodamine123 (Rh123)fluorescent probe,fluorescent intensity which showed the change of mitochondrial transmembrane potential of cardiomyocytes(△Ψm)was determine by Wallace Victory³;apoptosis percentage of cardiomyocytes were assayed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL); cardiomyocytes were loaded by Fluo-3,alteration of[Ca²⁺]i on cardiomyocytes were determined by laser scanning confocal microscope(LSCM),the concentrations of nitric oxide(NO)in cardiomyocytes cultured fluid were determined with nitrate reductase.RESULTS:1.Cell beating rate:Compared with normal group,the beating rate in H/R group decreased significantly(34.07±5.42 vs.99.20±5.51 beat /min, P＜0.01);compared with H/R group,the beating rate in dioscin groups(L\M\H) increased significantly(34.07±5.42 vs.90.45±5.40,34.07±5.42 vs.91.18±5.91,34.07±5.42 vs.97.27±5.64 beat/min,P＜0.01);The beating rates in dioscin groups were same as that in C group.2.Cell viability:Compared with normal group,cell viability in H/R group decreased significantly(32.83±5.00 vs.98.50±0.80%,P＜0.01);compared with H/R group,cell viability in dioscin groups(L\M\H)increased significantly(32.83±5.00vs.84.41±2.77,32.83±5.00 vs.85.54±2.57,32.83±5.00 vs.93.83±3.65 %,P＜0.01);Cell viability in dioscin groups was same as that in C group.3.△Ψm:Compared with normal group,△Ψm in H/R group decreased significantly (3.0±1.1 vs.5.7±0.5×10⁵,P＜0.01);compared with H/R group,△Ψm in dioscin groups(L\M\H)increased significantly(3.0±1.1 vs.5.3±1.3,3.0±1.1 vs.5.5± 1.7,3.0±1.1 vs.5.5±1.6×10⁵,P＜0.01);△Ψm in dioscin groups were same as that in C group.4.Apoptosis index(A/I):Compared with normal group,A/I in H/R group increased significantly,from 28.51±3.88%to 88.71±3.51%(P＜0.01);compared with H/R group,A/I in dioscin groups(L\M\H)decreased significantly(28.51±3.88 vs. 47.8±4.5,28.51±3.88 vs.39.19±2.56,28.51±3.88 vs.33.62±4.08%,P＜0.01); A/I in dioscin groups were same as that in C group.5.[Ca²⁺]i:Compared with normal group,[Ca²⁺]i in H/R group increased significantly(1887.21±692.51 vs,681.58±260.94,P＜0.01);compared with H/R group,[Ca²⁺]i in H dioscin group decreased significantly(1887.21±692.51 vs. 907.40±414.91,P＜0.01).6.Concentrations of NO:Cardiomyocytes can secrete NO spontaneously.The secretory volume of NO in cardiomyocytes did not increase significantly stimulated by H/R.2 hours after H/R,synthesis of NO increased significantly(48.37±12.85 vs.79.32±6.71μmol /L,P＜0.01);7 hours after H/R, synthesis of NO increased sharply by 3.9 times more than C group(48.37±12.85 vs.187.65±18.63μmol/L,P＜0.01).CONCLUSIONS:At the cellular level,it is proved that preventive dioscin treatment has important protective effects on H/R damaged cardiomyocytes.The protective mechanism may involve:1.△Ψm of damaged cardiomyocytes are heightened,the integrity of cellular membrane is protected.2.Calcium overload of cardiomyocytes is lightened,A/I is decreased.3.Elevated NO concentrations in damaged cardiomyocytes are inhibited,cellular toxic effects of high concentration of NO decreased.