In Vitro Experimental Study On Glycyrrhetinic Acid Antagonizing Aconitine Cardiotoxicit

Objective:1.To replicate a failure cardiomyocyte model.2.To explore the relationship between toxic effects of aconitine on model cells and oxidative stress,mitochondrial energy metabolism disorder and calcium overload.3.To study the relationship between glycyrrhetinic acid antagonizing the toxic effect of aconitine on model cells and relieving oxidative stress,regulating mitochondrial energy metabolism and maintaining calcium homeostasis.4.To verify whether glycyrrhetinic acid can antagonize the toxic effect of aconitine on model cells by regulating the expression of AMPK,Ca MKⅡpathway.Methods:1.Replicate failure H9c2 cardiomyocyte model:H9c2 cardiomyocytes were treated with doxorubicin,pentobarbital sodium and hydrogen peroxide at different concentrations,and the survival rate of H9c2 cardiomyocytes was determined by CCK-8 method.2.Determine the optimal administration concentration of aconitine and glycyrrhetinic acid on failure H9c2 cardiomyocytes:H9c2 cardiomyocytes were divided into blank control group,model group,aconitine group（15,30,60,120,240,360,480,720μmol/L）and glycyrrhetinic acid group（15,30,60,120,240μmol/L）.Except blank control group,the other groups were firstly treated with 2μmol/L doxorubicin for 24 h,then treated with the corresponding concentration and volume of drugs for 24 h,and the survival rate of H9c2 cardiomyocytes was used as the index by CCK-8 method.3.Verify the effects of before and after compatibility of aconitine and glycyrrhetinic acid on oxidative stress,mitochondria and calcium in failure H9c2 cardiomyocytes:H9c2cardiomyocytes were divided into 15 groups,including blank control group,model group,30μmol/L glycyrrhetinic acid group,15,30,60,120,240,480μmol/L aconitine groups,and 30μmol/L glycyrrhetinic acid combined with 15,30,60,120,240 and 480μmol/L aconitine groups,respectively.Except blank control group,the other groups were treated with 2μmol/L doxorubicin for 24 h.After modeling,the other groups except blank control group and model group were treated with the corresponding concentration of drug for 24 h.Mitochondrial microstructure changes and cell morphology were based on microscopic observation method;the Cell survival was measured by CCK-8;lactic dehydrogenase（LDH）,ATPase activity,superoxide dismutase（SOD）,malondialdehyde（MDA）,catalase（CAT）,glutathione（GSH）,adenosine triphosphate（ATP）content were used in enzymatic immunoassay（EIA）;Fluorescence probe method was based on reactive oxygen species（ROS）,membrane potential,and Ca²⁺content inside and outside mitochondria.4.Detecte the expression of AMPK,PGC-1αand Ca MKⅡ,SERCA2a and Ry R2 in AMPK,Ca MKⅡpathway in failure H9c2 cardiomyocytes after combined with aconitine and glycyrrhetinic acid:1)H9c2 cardiomyocytes were divided into 7 groups,blank control group,model group,AMPK inhibitor group（Compound c）,30μmol/L glycyrrhetinic acid group,480μmol/L aconitine group,30μmol/L glycyrrhetinic acid combined with aconitine group 480μmol/L,Compound c combined with aconitine glycyrrhetinate in intervention group.Except blank control group,the other groups were treated with 2μmol/L doxorubicin for 24 h,Compound c group,Compound c and aconitine glycyrrhetinate intervention group were treated with 5μmol/L Compound c for 2 h,then treated 30μmol/L glycyrrhetinic acid group,480μmol/L aconitine group,30μmol/L glycyrrhetinic acid combined with aconitine group 480μmol/L and Compound c combined with aconitine glycyrrhetinate in intervention group with the corresponding concentration of the drug for 24 h.The expression levels of AMPK and PGC-1αin AMPK pathway were used as indicators.2)H9c2 cardiomyocytes were divided into 7 groups:blank control group,model group,Ca MKⅡinhibitor group（KN-93）,30μmol/L glycyrrhetinic acid group,480μmol/L aconitine group,30μmol/L glycyrrhetinic acid combined with 480μmol/L aconitine group,KN-93 combined with aconitine glycyrrhetinate in intervention group.Except blank control group,the other groups were treated with 2μmol/L doxorubicin for 24 h,KN-93 group,KN-93 and aconitine glycyrrhetinate intervention group were treated with 5μmol/L KN-93 for 2 h,and other groups except blank control group,model group and KN-93 group were treated with the corresponding concentration of the drug for 24 h.The expression levels of Ca MKⅡ,SERCA2a and Ry R2proteins in Ca MKⅡpathway were used as indicators.Results:1.Doxorubicin,pentobarbital sodium,hydrogen peroxide can reduce H9c2 myocardial cell survival:CCK-8 assay showed that showed that the survival rate of H9c2 cardiomyocytes was about 50%when treated respectively by 2μmol/L doxorubicin,0.8%pentobarbital sodium and400μmol/L hydrogen peroxide.And presented extremely significant difference compared with blank group（P<0.01）.2.The concentrations of aconitine and glycyrrhetinic acid were determined to be 15,30,60,120,240,480μmol/L and 30μmol/L respectively on cardiomyocytes of failure H9c2.The survival rates of failure H9c2 cardiomyocytes in 15,30,60,120,240,360,480,720μmol/L aconitine groups and 15,30,60,120,240 glycyrrhetinic acid groups were detected by CCK-8 method.The results showed that compared with model group,the cell survival rate of 15,30 and 60μmol/L aconitine groups showed a decreasing trend,but there was no significant difference.There was significant difference in the cell survival rate of 120,240,360,480 and 720μmol/L aconitine groups compared with model group（P<0.01 or P<0.05）.The cell survival rate of 720μmol/L aconitine group was too low.Thus the dosages of aconitine were 15,30,60,120,240,480μmol/L according to the equal ratio of pharmacological dosages.There was no significant difference in the survival rate between the 15,30 and 60μmol/L glycyrrhetinic acid group and the model group.The survival rate of the 30μmol/L glycyrrhetinic acid group was the closest to that of the model group,and the concentration of glycyrrhetinic acid was 30μmol/L.3.The combination of aconitine and glycyrrhetinic acid could protect the structure of mitochondria,increase the mitochondrial membrane potential,SOD,CAT,GSH,ATP content and ATP transporter activity,and decrease the content of MDA,ROS and the concentration of Ca²⁺in mitochondria:Compared with the model group,aconitine damaged the mitochondrial structure of failure H9c2 cardiomyocytes,aconitine at different concentrations could reduce SOD,CAT,GSH and ATP contents,reduce mitochondrial membrane potential,inhibit ATP-related transportase activity,increase MDA and ROS contents,and increase Ca²⁺concentration in cell and mitochondria（P<0.01 or P<0.05）.All the above effects were dose-dependent（R²=0.5445～0.9794）.The results showed that aconitine at different concentrations combined with 30μmol/L glycyrrhetinic acid were compared with aconitine group alone.All of them could increase SOD,CAT,GSH and ATP contents,protect mitochondrial structure,improve mitochondrial membrane potential,increase ATP-related transportase activity,reduce MDA and ROS contents,and decrease Ca²⁺concentration in cells and mitochondria（P<0.01 or P<0.05）.4.The combination of aconitine and glycyrrhetinic acid affects the expression of AMPK,PGC-1α,Ca MKⅡ,SERCA2a and Ry R2 in failure H9c2 cardiomyocytes:Compared with model group,the protein expressions of AMPK,PGC-1αand SERCA2a were decreased,while the protein expressions of Ca MKⅡand Ry R2 were increased（P<0.01 or P<0.05）,Compared with aconitine alone group,the protein expressions of AMPK,PGC-1αand SERCA2a in aconitine glycyrrhetinic acid combined group were increased,while the protein expressions of Ca MKⅡand Ry R2 were decreased（P<0.01 or P<0.05）.Conclusion:1.2μmol/L doxorubicin,0.8%pentobarbital sodium and 400μmol/L hydrogen peroxide can successfully replicate the failure myocardial cell model.Combined with the results of the preliminary experiment on the 0.8%pentobarbital sodium and 400μmol/L hydrogen peroxide model,the survival rate of cells with aconitine administration concentration was studied.In this study,2μmol/L doxorubicin cardiomyocyte model was used to replicate failure myocardial cell model.2.The toxic effects of aconitine on model cells were related to oxidative stress,mitochondrial energy metabolism disorder and calcium overload.3.Glycyrrhetinic acid antagonized the toxic effect of aconitine on on model cells which was related to relieving oxidative stress,regulating mitochondrial energy metabolism and maintaining calcium homeostasis.4.Glycyrrhetinic acid may antagonize the toxic effect of moncetine on failure H9c2cardiomyocytes by regulating AMPK,PGC-1α,Ca MKⅡ,SERCA2a and Ry R2 proteins.