Effects Of Glycyrrhizic Acid And Glycyrrhetic Acid On Nox4 And Nox2 NADPH Oxidase In Rat Cardiomyocytes And Their Mechanism

Objective:To explore the effect and mechanism of Glycyrrhizic acid(GCA)and Glycyrrhetinic acid(GA)to Nox4 and Nox2 on the H9c2 cell line.Method:The experimental subject is H9c2 cell line.1.Explore the toxicity of GCA and GA to the H9c2 by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT).Grouping design:the control group,solvent control,concentration gradient group(10-6-10-1μg/μL).H9c2 is cultured in 96-well plate,5×104cells/ml,100μL/well.After synchronization,inject drugs wait another 48h to analyze the groups.2.By 2’,7’-dichlorofluorescein diacetate(DCFH-DA)to test ROS(Reactive Oxygen Species),under the laser scanning confocal microscope.The grouping design of model verification:the control group,10-5-10-7mol/L Angiotensin Ⅱ(Ang Ⅱ)group.The grouping design of drug function:the control group,10-5mol/L AngⅡ,posictive control group(Apocynin,APO,100μmol/L),10-6μg/μL drugs group.The cell is cultuered in the small dish,5×104cells/ml,1mL/dish.After synchronization,inject drugs wait another 24h to analyze the groups.3.By Western blotting,to explore the protein expression of Nox4,Nox2 and the related proteins.The grouping design:the control group,10-5mol/L AngⅡ,posictive control group(Apocynin,100μmol/L),inhibitor group(20μmol/L),10-6μg/μL drugs group.The cell is cultuered in the φ10cm dish,5×104cells/ml,l0mL/dish.After synchronization,inject drugs wait another 24h to analyze the groups.Result:1.The toxicity of GA is found under the designed max.concentration 0.1μg/μL(P<0.01).The toxicity of GCA is not found.10-6-10-1μg/μL GCA,10-6-10-2μg/μL GA improve the cell concentration(P<0.01,P<0.05).2.10-5mol/L AngⅡ increase the ROS level in H9c2(P<0.01).10-6μg/μL GCA and GA decrease the ROS level induced by Ang Ⅱ(P<0.01).The effect is similar with the positive control group(APO)(P>0.05)。3.The expression average value of P47phox,Phospho-P47phox,Glucocorticoid Receptor(GCR)is 37.3%,26.5%,86%higher than the control group,respectively,induced by 10-5mol/L Ang Ⅱ.But the expression of Nox4,Peroxisome proliferative activated receptors(PPAR-gamma),Signal transducers and activators of transcription 3(Stat3),GCR,P47phox,Phospho-P47phox,Nox2,Neutrophil cytosolic factor 2(NCF2)(P67phox)are no difference between control and model group by current model method(P>0.05).Conclution:GCA and GA may be the important active ingredients of glycyrrhiza to intervene the heart failure.The decreasing of ROS may be the mechanisms of GCA and GA to relieve the heart failure.The molecular mechanisms needed more experiments to explain.