Inhibition And Mechanisms Of N-butanol Fraction Of Potentilla Anserine L. Against Hypoxia-induced Calcium Overload In Myocardial Cells

Objectives: To study the inhibition and mechanisms of n-butanol fraction of Potentilla anserine L. against hypoxia -induced calcium overload in myocardial cells.Methods: (1)primary cultured myocardial cell from SD neonatal rat (1-3d) was used in the model establishment of hypoxia.(2)the best time of hypoxia and the dose of n-butanol fraction of Potentilla anserine L were ascertained through measurment of the dissociative calcium concentration in myocardial cells.(3)The cultured cardiomyocytes were divided randomly into control group,model group,intervention group of n-butanol alcohol fraction of Potentilla anserine L. (0.25mg/ml,0.0625mg/ml,0.0156mg/ml) and Verapamil group (5nmol/L).After hypoxia for 3h,the dissociative calcium concentration in myocardial cells was measured with spectrofluorometer and confocal laser scanning microscopy(CLSM),to observe protective effect of the intervention group at different doses against hypoxia-induced calcium overload in myocardial cells.(4)The possible mechanisms of calcium overload in myocardial cells were approached by biochemical indicators(Ca²⁺-ATP),RT-PCR (SERCA2,PLB,Cav1.2,RyR,μ-Calpain),immuno- histochemistry stain and Western Blot (SERCA2,μ-Calpain).Results: 1The Morphological findings:After having been cultured 3d,the cell shape of myocardial cells showed diversification, strong refractive index, the number increasing of cell clusters.After being injured by hypoxia,refracting power of the cells mass declined.Cell pulse became weaker significantly.2The change of calcium concentration on cardiomyocytes in hypoxia injury(1)Determination of the best hypoxia time:in the hypoxia injury duration whin three hours,the dissociative calcium concentration of myocardial cells in model group increased to the largest extent,so as the best time of hypoxia was ascertained as three hour.(2)The change of calcium concentration after drug intervention:â‘ measurement of the dissociative calcium concentration in myocardial cells with spectrofluorometer: after 3 hours of hypoxia injury,the dissociative calcium concentration of the model group(474.614±22.161)was much higher than that of the control group(112.740±13.694)(P<0.01). Verapamil (162.804±11.171)and every dose of Potentilla anserine L lessened the calcium overload of cells significantly(P<0.01).â‘¡detection of cell intracellular calcium fluorescence signal with confocal laser scanning microscopy(CLSM):the fluorescence intensity of model grope(0.288±0.020) was higher than that of control grope (0.074±0.014)(P<0.01).Verapamil(0.076±0.010)and the high dose of n-butanol fraction of Potentilla anserine Lcan lessened the fluorescence intensity (P<0.01).3The change of the Ca²⁺-ATP activities:The Ca²⁺-ATP activities of model group(2.404±0.151) were obviously reduced compared with that of control group (5.381±0.271) (P<0.01).Verapamil (4.485±0.202)and each dose of n-butanol fraction of Potentilla anserine L.could significantly increase the Ca²⁺-ATP activities with the model group as control during hypoxia injury(P<0.01).4 The change of the calcium-related gene expression(1)RT-PCR:the mRNA level of SERCA2 was decreased significantly by hypoxia (P<0.01).In contrast,the mRNA levels of RyR,Cav1.2,μ-Calpain were increased in comparing with that of the control group(P<0.01).each dose of Potentilla anserine L enhanced the express level of SERCA2 mRNA (P<0.01)and depressed the express level of RyR(P<0.01),Cav1.2,μ-Calpain (the high and intermediate doses P<0.01, the low dose P<0.05)mRNA on hypoxia injury myocardial cells compared with model group.No significant difference of PLB mRNA was observed between control group,model group and intervention group(P>0.05).(2)immuno-histochemistry stain:each dose of Potentilla anserine L enhanced the express levels of SERCA2 (P<0.01) and depressedμ-Calpain expression compared with model group (the high and intermediate doses P<0.01,the low dose P<0.05).(3)Western blot:each dose of Potentilla anserine L depressed the protein level of SERCA2 on hypoxia injury myocardial cells in comparing with that of the model group (P<0.01).Potentilla anserine L depressed the express level ofμ-Calpain on hypoxia injury neuron compared with model group (the high and intermediate doses P<0.01).No significant difference was observed between model group and low dose group(P>0.05).Conclusions: n-butanol Fraction of Potentilla anserine L. attenuated the calcium overload and improved the injury of myocardial cells induced by hypoxia. The mechanism might be that n-butanol fraction of Potentilla anserine L. raise the activities of Ca²⁺ -ATP,increase of sarcoplasmic reticulum calcium re-uptake capacity,reduce calcium influx and reduce the resulting induced sarcoplasmic reticulum calcium release. Furthermore, n-butanol Fraction of Potentilla anserine L. through inhibiting the occurrence of calcium overload to reduceμ-Calpain expression and further protect the myocardial cells.