Protective Effects Of Astragaloside Ⅳ On Hypertrophy Induced By Angiotensin Ⅱ In Primary Cultured Cardiomyocytes Of Neonatal Rat

Objective:To investigate protective effects and mechanism of astragalosideâ…£on hypertrophy induced by angiotensinâ…¡(Angâ…¡) in primary cultured cardiomyocytes of neonatal rats.Methods:Cardiomyocytes were normally cultured for 72 hours,then replaced medium with serum free.Twenty four hours later,different agents were added to medium for another 72 hours incubation.Cardiomyocytes were divided randomly into 6 groups: control group;Angâ…¡group:Angâ…¡10^-7mol/L;AST 10 group:AST 10 mg/L;AST 20 group:AST 20 mg/L;AST 10+Angâ…¡group:AST 10mg/L+Angâ…¡10^-7 mol/L and AST 20+Angâ…¡group:AST 20mg/L+Angâ…¡10^-7 mol/L.The diameter of cardiomyocyte was measured by image analytical system.The total protein content of cardiomyocytes was detected by bradford method.The activity of Sarcoplasmic reticulum Ca²⁺-ATPases (SERCA) was evaluated by determination of Ca²⁺-dependent p-nitrophenyl phosphatase(pNPPase).The Ca²⁺ was labeled by Fura-2 fluorescence probe and the fluorescent intensity produced by Fura-2 was measured,which reflected[Ca²⁺]_i.The activity of Calcineurin(CaN) was determined by enzyme reaction phosphorus measurement.Results:1.Diameter of cardiomyocyte:compared with control group,diameter of cardiomyoeyte was increased significantly in Angâ…¡group(16.1±1.1 vs 11.1±1.0μm, P＜0.01),AST 10+Angâ…¡group(12.8±1.0 vs 11.1±1.0μm,P＜0.01) and AST 20+Angâ…¡group(12.4±0.9 vs 11.1±1.0μm,P＜0.01);diameter of cardiomyocyte wasn't increased in AST 10 or AST 20 group(P＞0.05,respectively).Compared with Angâ…¡group,diameter of cardiomyocyte was decreased significantly in AST 10+Angâ…¡group (12.8±1.0 vs 16.1±1.1μm,P＜0.01) and AST 20+Angâ…¡group(12.4±0.9 vs 16.1±1.1μm,P＜0.01).2.Total protein content of cardiomyocyte:compared with control group,protein content was increased significantly in Angâ…¡group(146.3±25.2 vs 95.9±20.5 μg/well,P＜0.01),AST 10+Angâ…¡group(121.1±23.6 vs 95.9±20.5μg/well,P＜0.05); total protein content wasn't increased in AST 10,AST 20 group or AST 20+Angâ…¡group(P＞0.05,respectively).Compared with Angâ…¡group,total protein content was decreased significantly in AST 10+Angâ…¡group(121.1±23.6 vs 146.3±25.2μg/well, P＜0.05) and AST 20+Angâ…¡group(114.1±18.0 vs 146.3±25.2μg/well,P＜0.01).3.[Ca²⁺]_i:compared with control group,[Ca²⁺]_i was increased significantly in Angâ…¡group(283.9±40.7 vs 150.6±27.1 nmol/L,P＜0.01),AST 10+Angâ…¡group (204.8±33.9 vs 150.6±27.1 nmol/L,P＜0.01) and AST 20+Angâ…¡group(194.9±34.7 vs 150.6±27.1 nmol/L,P＜0.05);[Ca²⁺]_i wasn't increased significantly in AST 10 or AST 20 group(P＞0.05,respectively).Compared with Angâ…¡group,[Ca²⁺]_i was decreased significantly in AST 10+Angâ…¡group(204.8±33.9 vs 283.9±40.7 nmol/L,P＜0.01) and AST 20+Angâ…¡group(194.9±34.7 vs 283.9±40.7 nmol/L,P＜0.01).4.Activity of SERCA:compared with control group,activity of SERCA was decreased significantly in Angâ…¡group(2.07±0.45 vs 0.83±0.20μmol/min·g pro, P＜0.01),AST 10+Angâ…¡group(1.29±0.23 vs 2.07±0.45μmol/min·g pro,P＜0.01) and AST 20+Angâ…¡group(1.42±0.37 vs 2.07±0.45μmol/min·g pro,P＜0.01);activity of SERCA wasn't decreased significantly in AST 10 group or AST 20 group(P＞0.05, respectively).Compared with Angâ…¡group,activity of SERCA was increased significantly in AST 10 mg/L+Angâ…¡group(1.29±0.23 vs 0.83±0.20μmol/min·g pro, P＜0.01) and AST 20 mg/L+Angâ…¡group(1.42±0.37 vs 0.83±0.20μmol/min·g pro, P＜0.01).5.Activity of CaN:compared with control group,activity of CaN was increased significantly in Angâ…¡group(0.490±0.080 vs 0.297±0.072μmol/mg pro,P＜0.01) and AST 10+Angâ…¡group(0.385±0.089 vs 0.297±0.072μmol/mg pro,P＜0.05);activity of CaN wasn't increased in AST 10,AST 20group or AST 20+Angâ…¡group(P＞0.05, respectively).Compared with Angâ…¡group,activity of CaN was decreased significantly in AST 10+Angâ…¡group(0.385±0.089 vs 0.490±0.080μmol/mg pro, P＜0.05) and AST 20+Angâ…¡group(0.355±0.085 vs 0.490±0.080μmol/mg pro, P＜0.01).Conclusion:It was first time reported that hypertrophic cardiomyocytes of neonatal rats induced by Angâ…¡were protected by AST,the mechanism was related to increasing the activity of SERCA,decreasing the[Ca²⁺]_i,inhibiting the overload of Calcium.At the same time,protective effects of AST on the hypertrophy may be relevant to decreasing the activity of CaN,inhibiting the Ca²⁺-CaN signaling pathway.