| ObjectiveShenFu injection(SFI)is derived from Shen Fu decoction,which has been widely applied clinically and been proved to be effective for various kinds of heart diseases,including myocardial ischemia and reperfusion injury.In this study,three important monomers in Shenfu injection,ginsenoside Re,Rgl and Aconine,were selected.The protecting effect of the three monomers and their compatibility in myocardial hypoxia/re-oxygenation injury in H9C2 cells was observed and the underlying mechanism of calcium homeostasis of this protection was explored with the detection of expression of NCX1,SERCA2a,Cav1.2,P25,This study will provide molecular level evidence for ingredients of ShenFu decoction to protect myocardial ischemia-reperfusion injury.Methods(1)The establishment of H9C2 myocardial hypoxia re-oxygenation(HR)injury model:In the process of hypoxia,H9C2 myocardial cell line was cultured in inert gas and sugar free and serum free radical media,in the process of re-oxygenation,H9C2 cells was cultured in normal cell culture box and normal medium to simulated myocardial ischemia reperfusion in vitro HR model.CCK-8 was used to detect the viability of cardiac myocytes,and the favorable experimental conditions were set when the cell survival rate was around 50%.(2)Detecting the protective effects of three monomers on the injury of H9C2 myocardial cells after hypoxia and re-oxygenation:Three monomers of different concentrations were used to pretreat H9C2 cardio-myocytes until the end of HR process.Cell viability was detected by CCK-8.(3)The regulating effect of three monomers on intracellular calcium concentration:H9C2 cells were pretreated with three monomers at different concentrations for 24h.After the hypoxia re-oxygenation process,Fura2-AM probe was used to detect the intracellular concentration of calcium in the normal group,model group and drug administration group.(4)Screening the best compatibility of the three monomers:According to the results of two experiments,the optimum concentration of each monomer was obtained.The optimum concentration was set as ED100,the SPSS statistical software was used to calculate the ED 10 and ED50 of the three components.Three factors and three level orthogonal design were applied for compatibility experiment.Then the cell survival rate was measured by CCK-8 kit,and the optimal compatibility was screened.(5)The effect of three monomers and optimal compatibility on the expression of NCX1,SERCA2a,Cav1.2 and P25:200?M Re,Rgland 100?M Aconine as well as their compatibility form were administrated to pretreat the cells.The proteins of each group were extracted.The expression of NCX1,SERCA2a,Cav1.2 and P25 in each group was analyzed by Western-Blot,so as to further explore the correlation between the single use and compatibility and its possible regulation pathway.Results(1)The establishment of H9C2 myocardial hypoxia re-oxygenation(HR)injury model:After different periods of hypoxia and re-oxygenation,the cell viability of H9C2 cells was significantly different.It was found that after cultured 4H in 100%N2 and sugar free serum free medium,and then re-oxygenation 3H under normal conditions,cell viability was about 53%,which was significantly different from that in normal culture group.(2)Detecting the protective effects of three monomers on the injury of H9C2 myocardial cells after hypoxia and re-oxygenation:Only 200?M Re pretreat can significantly enhance the cell survival rate about 12%(P<0.05).Rg1 pretreat can increase the survival rate about 26%(P<0.01),22%(P<0.01),17%(P<0.05)at 200?M,100?M and 50?M respectively.Aconine pretreat at 100?M and 200?M can increases the survival rate about 27%(P<0.01),15%(P<0.05)at 100?M and 200?M respectively(3)The regulating effect of three monomers on intracellular calcium concentration:As for the concentration of calcium ions,various concentration of the three monomers reduce the intracellular calcium concentration.It was found that 200?M Re pretreatment could reduce the Ratio value from 1.6687±0.03705 to 1.5204±0.10529(P<0.001),and 200?M Rg1 pretreatment could reduce the Ratio value from 1.6487±0.03705 to 1.3477±0.03667(P<0.001),1 00?M Aconine pretreatment could reduce the Ratio value from 1.6419±0.01496 to 1.3363±0.09447(P<0.001)(4)Screening the best compatibility of the three monomers:with overall consideration of the experimental results of different concentration monomer in cell survival experiment and intracellular calcium concentration detection,the optimum concentration of Re,Rgl and Aconine is 200?M,200?M and 100?M respectively.According to the statistics software,the best concentration of each effective component is ED 100,and ED50 and ED 10 of each effective component are calculated.It is concluded that the three levels of Re are 80?M,120?M and 200?M,and the three levels of Rg1 are 25?M,50?M,200?M,and Aconine are 25?M,50?M,and 100?M.The results of three factor and three level orthogonal test showed that the favorable compatability of the three monomers of Shenfu decoction to improve the H9C2 cell survival rate is the combination of 25?M Re,200?M Rgl and 100?M Aconine.(5)The effect of three monomers and optimal compatibility on the expression of NCX1,SERCA2a,Cav1.2,and the possible molecular regulation pathway:The results of Western-Blot showed that the three monomers had an effect on the expression of different calcium homeostasis related proteins.200?M Re can increase the expression of SERCA2a;200?M Rg1 can increase the expression of SERCA2a protein and decrease the expression of NCX1;100?M Aconine can reduce the expression of NCX1.The combination of the three monomers had a good influence on the expression of the three proteins.At the same time,we found that the three effective components and the optimal compatibility can reduce the expression of P25 protein.Calcium overload can activate the hydrolysis activity of calpain,and P25 is the hydrolysate of calpain.The decrease of P25 protein expression represents the decrease of calpain hydrolysis activity,which indicates that the three effective components and the optimal compatibility can inhibit the activity of calpain,thus reducing the hydrolysis of the cell structure by calpain and then exerting the protective effect of cell,thus enhancing the auxiliary effect of the effective component and compatibility of the Shenfu Decoction on the regulation of calcium homeostasis.ConclusionThe three effective components of SFD,Re,Rgl and Aconine,can increase the survival rate of H9C2 cardiomyocytes in hypoxia re-oxygenation model.The effect of compatibility on cell survival rate is more significant than that of three effective components alone.Pretreatment of SFD effective components,Re,Rgl and Aconine,can reduce the intracellular calcium concentration of H9C2 hypoxia re-oxygenation cells.The effect of reducing calcium concentration after compatibility is more significant than that of single application.The effective components of SFD,Re and Rgl,can increase the expression of SERCA2a protein in H9C2 cells;Rg1 can also reduce the expression of NCX1 protein;Aconine can reduce the expression of Cav1.2 in H9C2 cells.The compatibility can increase the expression of protein SERCA2a,and reduce the expression of protein NCX1 and Cav1.2.At the same time,the three effective components,either alone or in combination,can reduce the expression of the hydrolysate P25 of calpain,and enhance the protective effect on the injury of H9C2 myocardial cells after hypoxia and reoxygenation.The results showed that three kinds of effective components and compatibility can reduce calcium overload by regulating calcium homeostasis related protein,inhibit calpain activity directly,reduce the cell damage induced by calcium overload,and play a protective role in the damage of cell hypoxia and reoxygenation.In this study,the key mechanism of the protective effect of SFD effective components and compatibility on myocardial ischemia reperfusion injury was preliminarily elucidated from the angle of regulating the homeostasis of calcium ion in cardiac myocytes.It provides microcosmic evidence for clinical application of SFD for myocardial ischemia reperfusion injury,and provides reference for optimizing the prescription of SFD. |
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