Study On The Attenuating Effect Of Licorice And Aconite Based On Mitochondrial Energy Metabolism In Cardiomyocytes

Objects:To explore the relationship between the detoxification effect of Gancao combined with Fuzi and the regulation of mitochondrial function and energy metabolism.Methods:1.Exploration of concentration of drug administration of Fuzi and Gancao components:Taking the survival rate of H9c2 cardiomyocytes as the index,the concentration and gradient of aconitine,glycyrrhizin,glycyrrhizin and glycyrrhetinic acid were determined.MTT method was used to determine the influence of four components on the survival rate of H9c2 cardiomyocytes,so as to determine the drug concentration of each group and set the concentration gradient.2.Screening of attenuant active ingredients of licorice:Taking the survival rate of H9c2 cardiomyocytes as the index,MTT assay was used to determine the effects of aconitine,glycyrrhizin,glycyrrhetinic acid before and after compatibility on the survival rate of H9c2 cardiomyocytes,so as to screen out the attenuant active ingredients of licorice.3.Research on the attenuation effect of glycyrrhetinic acid combined with aconitine in regulating mitochondrial energy metabolism and calcium homeostasis:culture H9c2 cardiomyocytes and divide them into blank group,60?mol/L glycyrrhetinic acid group,120,240,480,720,960?mol/L aconitine group,and 120,240,480,720,960?mol/L aconitine each compatible with 60?mol/L glycyrrhetinic acid group.Detect the ATP content,Ca²⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase activity in the cardiomyocytes of the different administration groups through the kit,and detect the mitochondrial membrane potential,ROS and calcium ion levels in mitochondria of the cardiomyocytes in the different administration groups through fluorescent probes,to study the relationship between the attenuation of glycyrrhetinic acid and aconitine and the improvement of energy metabolism of cardiomyocytes,the reduction of mitochondrial damage,and the regulation of mitochondrial calcium homeostasis.Result:1.The results of the exploratory experiment on the concentration of the components of Radix Aconiti and Glycyrrhizae:The effect of aconitine,glycyrrhizin,glycyrrhizin,and glycyrrhetinic acid on the survival rate of H9c2 cardiomyocytes was determined by MTT method.Compared with the blank group,the effect of cell survival rate is extremely significant(P<0.01),so five gradients of 120?mol/L,240?mol/L,480?mol/L,720?mol/L,960?mol/L are set as the initial concentration;and 40?mol/L glycyrrhizin,120?mol/L glycyrrhizin,60?mol/L glycyrrhetinic acid are the highest concentrations that have no significant difference in the effect of cardiomyocyte viability compared with the blank group(P>0.05).In this study,the glycyrrhizin group The component should express its protective effect on cardiomyocytes,so the highest safe concentration of licorice component is taken as the administration concentration to carry out subsequent screening experiments of attenuating active components of the licorice component of aconite.2.Screening test results of attenuating active ingredients of licorice:the MTT method was used to determine the effect of aconitine and licorice on the survival rate of H9c2 cardiomyocytes.The results showed that 40?mol/L glycyrrhizin,120?mol/L glycyrrhizin and different concentrations The compatibility of gradient aconitine can not improve the survival rate of cardiomyocytes(P>0.05),while the compatibility of60?mol/L glycyrrhetinic acid and different concentration gradients of aconitine can significantly improve the survival rate of cardiomyocytes(P<0.01)Therefore,it is believed that glycyrrhetinic acid is the attenuating active ingredient of licorice,and further research on its attenuation mechanism will be carried out.3.Research results of the attenuation of glycyrrhetinic acid and aconitine and the regulation of mitochondrial energy metabolism and calcium homeostasis:Compared with the blank group,120,240,480,720,960?mol/L aconitine can significantly reduce the myocardium Intracellular ATP content,reduce mitochondrial membrane potential,increase the content of ROS in cardiomyocytes,inhibit Ca²⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase activity in cardiomyocytes and increase Ca²⁺level in mitochondria(P<0.05),the above effects were dose-dependent;while 120,240,480,720,960?mol/L aconitine was combined with 60?mol/L glycyrrhetinic acid,and the ATP content in myocardial cells was significantly increased compared to before the combination(P<0.01),mitochondrial membrane potential increased significantly(P<0.05),ROS content in cardiomyocytes decreased significantly(P<0.05),Ca²⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase in cardiomyocytes The activity was significantly increased(P<0.05),and the level of Ca²⁺in the mitochondria was significantly reduced(P<0.05).Conclusion:1.Aconitine reduces mitochondrial membrane potential and destroys mitochondrial function,promotes the production of ROS and damages mitochondria,thereby reducing energy metabolism of cardiomyocytes and causing cardiomyocyte cytotoxicity.2.Aconitine reduces the activity of ATPase(Ca²⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase)in cardiomyocytes so that Ca²⁺in cardiomyocytes cannot normally take in sarcoplasmic reticulum or transfer out of the cell,resulting in excessive Ca²⁺transfer into mitochondria,causing mitochondrial calcium overload to damage mitochondrial function.3.Glycyrrhetinic acid in combination with aconitine can reduce mitochondrial damage by increasing the mitochondrial membrane potential of cardiomyocytes,reducing the production of ROS,and increasing ATPase in cardiomyocytes(Ca²⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase)activity promotes the uptake of Ca²⁺in the cardiomyocytes into the sarcoplasmic reticulum or transfers out of the cell to relieve mitochondrial calcium overload,improve the function of cardiomyocytes mitochondria,and increase the energy metabolism of cardiomyocytes to play an attenuating effect.