Experimental Study Of Protective Effects Of The Aconite And Glycyrrhiza Component Compatibility On The Aconitine-induced Passaged Rat Myocardial Cell Injury

Objective:to observe the protective effects of the component compatibility between Aconite and Glycyrrhiza on the aconitine-induced passaged rat myocardial cell injury and to explore the mechanism.Methods:Using rats passage myocardial cell lines as an object of study,to explain the relative mechanisms of the protective effect of different activity components compatibilities between Aconite and Glycyrrhiza on the aconitine-reduced rat myocardial cell damage from the view of the cellular and molecular,and to explore the "attenuated efficiency" mechanism of Aconite and Glycyrrhiza compatibility.Then select the best effective compatibility group to provide a theoretical basis for guiding drugs containing aconitine and licorice compatible use.Using MTT,ultraviolet spectrophotometry detection,Western-blotting and other study methods of detect relative genes technology to explain effect mechanism from different levels and angles.Set up twelve groups as follows:the blank control group,aconitine group,nine groups of Aconite and Glycyrrhiza main active components combination,and verapamil group.By culturing passaged myocardial cell lines to observe the change of the myocardial cell morphology after the aconitine and the Aconite Glycyrrhiza activity components combination action from the view of morphology;Using MTT to detect the change of the survival rate of each group,and to study the effects of aconitine and the Aconite Glycyrrhiza activity components combination on myocardial energy metabolism of SDH,Na+/K+-ATPase,Ca2+/Mg2+-ATPase;Using Western-blotting immunoblotting to analyze the expressing level of aconitine and different compatibility of Aconite Glycyrrhiza main active components on myocardial calcium regulation protein(DHPR-a1、NCX1、RyR2).Results:(1)myocardial cell morphology observation:after four hours of the aconitine acting,myocardial cell pseudopodia shortened,the cells became round,with the increasing of functional time,the cells volume became small,and a few of myocardial cells became vacuole or particles,spontaneous rhythm pulsating reduced and the rhythm was irregular,fibrillation irregular,myocardial cell shrinkage,myocardial cell contraction in late period is not obviously.After four hours of groups that compatibility between Aconite and Glycyrrhiza acting,the condition was improved than the aconitine group in morphology and shrinkage aspects,the condition of the pseudopodia shortened,the cytoplasm cavitation and the particles matter were all decreased than the aconitine group,in which the Aconite Glycyrrhiza fifth group(that 60μM aconitine,60μM hypaconitine,60pM glycyrrhetinic acid,60μM Liquiritin)is the most obviously improved.(2)Effect on myocardial cell activity:the survival rate of the 120μM concentration aconitine group decreased obviously(P<0.01),while the different compatibility groups of Aconite Glycyrrhiza main activity components could significantly increase the cell survival rate(P<0.01 或(P<0.05).By using orthogonal method,the cell survival rate in the best compatibility group was the third group of Aconite Glycyrrhiza(that 30μM aconitine,120μM hypaconitine,60μM of glycyrrhetinic acid and 240μM liquiritin)(P<0.01),which with no statistically difference(P>0.05)comparing with the verapamil group.(3)Effect on energy metabolism of myocardial cell:the SDH activity,Na+-K+ATPase,and Ca2+-Mg2+ATPase activity of the 120μM concentration aconitine group were all obviously decreased,while the SDH activity,Na+-K+ ATPase,and Ca2+-Mg2+ ATPase activity of the different compatibility groups of Aconite Glycyrrhiza activity components were all significantly recovered(P<0.05 or P<0.01).The activity of SDH in fifth group of Aconite Glycyrrhiza(that 60μM aconitine,60μM hypaconitine,60μM glycyrrhetinic acid,60μM Liquiritin))improved the most obvious(P<0.01),Na+-K+ATPase and the activity of Ca2+-Mg2+ATPase in fourth group of Aconite Glycyrrhiza(that 60μM aconitine,30μM hypaconitine,30μM of glycyrrhetinic acid and 240μM liquiritin)improved the most significantly(P<0.01),which partly with significant difference(P<0.05 or P<0.01)comparing with the verapamil group.(4)Effect on the expressing level of myocardial calcium regulation protein:the aconitine with the 120μM concentration affected myocardial cell,made myocardial cell injury,increased the RyR2 expressing obviously,and distinct decreased the NCX1,DHPR-a1 expressing.The different compatibility groups of Aconite Glycyrrhiza main activity components had antagonistic action to the above function of aconitine when affect on myocardial cell.The third group of Aconite Glycyrrhiza(that 30μM aconitine,120μM hypaconitine,60μM of glycyrrhetinic acid and 240μM liquiritin)decreased the most clearly on the aspect of RyR2(P<0.01),the fifth group of Aconite Glycyrrhiza(that 60pM aconitine,60μM hypaconitine,60μM of glycyrrhetinic acid and 60μM liquiritin)increased the most apparently on the aspect of NCX1 and DHPR-al(P<0.01),which with no statistically difference(P>0.05)comparing with the verapamil group.On the aspect of antagonism aconitine,the functions were the most clearly of liquiritin to increase RyR2 and glycyrrhetinic acid to decrease NCX1 and DHPR-al.Conclusions:the results all above show that the function of the different compatibility groups of Aconite Glycyrrhiza activity components to the decreasing activity of myocardial cell affected by antagonism aconitine and the function of improving the decreasing SDH activity,Na+-K+ATPase,Ca2+-Mg2+ATPase activity,improving the increasing RyR2 expressing,decreasing NCX1、DHPR-al expressing affected by aconitine were all obviously.Studies have found that,liquiritin and glycyrrhetinic acid are all play a role in detoxification of aconitine.The affection of Aconite Glycyrrhiza compatibility groups with low and middle concentration on reducing the myocardial cell injury by aconitine are the best(the third,fourth and fifth group of Aconite Glycyrrhiza).Inhibiting the aconitine toxicity of myocardial cell by regulating the expressing level of calcium regulation protein(DHPR-a1、NCX1、RyR2)may be the mechanism to achieve attenuated synergistic effect.Which may be one of the molecular mechanisms of Glycyrrhiza decreasing aconite toxicity,and also provide a certain experimental basis for guiding the using of compatibility between Aconite and Glycyrrhiza.