Study On The Mechanism Of Puerarin Metabolite M2 Against Cardiomyocyte Hypertrophy Induced By

Objectives:To investigate the antioxidation effect of metabolite M2of puerain: toinvestigate effect on AngⅡ (Angiotensin Ⅱ)induced cardiomyocytes hypertrophyand the change of oxidative stress parameters in the cardiomyocytes, we intervenedthe Ang Ⅱ stimulated cardiomyocytes with puerain. Furthermore, we tried toilluminate the mechanism between oxidative stress and cardiomyocytes hypertrophyand to provide evidence for the pathogenesis and clinical drug prevention andtherapy.Methods:Cardiomyocytes were isolated by trypsin digestion and differential attachmentmethods from neonatal Sprague Dawley (SD) rats, and then identified by invertedmicroscope and transmission electron microscope (TEM) observation. Thecardiomyocytes were treated with the xanthine/xanthine oxidase to establish theoxidative stress model of cardiomyocytes, and the level of superoxide anions wasmeasured by labeling cells with DHE. After the cardiomyocytes were treated withAng Ⅱ, puerain and its metabolite M2, the level of superoxide anions was measuredby labeling cells with DHE, the total antioxidant capacity was detected using theFRAP method, the total SOD activity was measured using the WST method. Thecardiomyocytes were labeled using phalloidin, and the pictures were captured tocalculate the cell surface. The expression of hypertrophy gene and DADPH oxidasegene were measured using quantitative RT-PCR.Results1. The effect of Puerain and its metabolite M2of puerain on oxidative stress1.1. the establishment of oxidative stressthe cardiomyocytes were treated with xanthine (500μmol/l)/xanthine oxidase(2U/L) for24-36hours, the the level of superoxide anions rised (P<0.05), thetotal antioxidant capacity and the total SOD activity have no significant change(P>0.05).1.2. the determination of the concentration of superoxide anions24-36hours after puerain and M2intervention, the cardiomyocytes were labeledwith DHE, the pictures were captured and the fluorescence intensity was analysedusing IPP6.0. Compared to the positive control group (xanthine/xanthine oxidase),the mean optical density significantly reduced (P<0.05); the fluorescenceintensity was measured using the fluorescence scanner, the fluorescence ID valueof M2treated group was lower than positive control group (P<0.05).1.3. the determination of total antioxidant capacity in cells 24-36hours after the M2intervention, the total antioxidant capacity in cells wasmeasured using FRAP method. The data showed that the total antioxidantcapacity in M2intervention group was significantly higher than the blank controlgroup and xanthine/xanthine oxidase group (P<0.05).1.4. the determination of total SOD activity24-36hours after the M2intervention, the total SOD activity was measured usingWST method, The data showed that the total SOD activity in M2interventiongroup was significantly higher than the blank control group andxanthine/xanthine oxidase group (P<0.05).2. The exploration of the mechanism by which M2antagonize thecardiomyocytes hypertrophy induced by AngⅡ2.1. The effect of AngⅡ on the cardiomyocytes hypertrophy2.1.1. The mRNA expression of hypertrophy gene ANF and β-MHC in thecardiomyocytes36hours after the Ang Ⅱ stimulated the cardiomyocytes, the mRNAexpression of hypertrophy gene ANF and β-MHC in the cardiomyocytes wereboth higher than the control group (P<0.05).2.1.2. The surface area measurement of cardiomyocytes36hours after the AngⅡ stimulated the cardiomyocytes, The cardiomyocyteswere labeled using phalloidin, and the pictures were captured to calculate thecell surface. The surface area increased significantly compared to controlgroup (P<0.05).2.2. the effect of AngⅡ on the oxidative stress in the cell2.2.1. the change of superoxide anion in cardiomyocytes36hours after the AngⅡ stimulated the cardiomyocytes, the cardiomyocyteswere labeled with DHE, the pictures were captured and the fluorescenceintensity was analysed using IPP6.0. Compared to the positive control group(xanthine/xanthine oxidase), the mean optical density significantly increased(P<0.05); the fluorescence intensity was measured using the fluorescencescanner, the fluorescence ID value of AngII treated group was higher thanpositive control group (P<0.05).2.2.2. the expression of NADPH oxidase P47phox in cardiomyocytes.36hours after the Ang Ⅱ stimulated the cardiomyocytes, the mRNAexpression of NADPH oxidase P47phox in the cardiomyocytes increasedsignificantly compared to the blank control group (P<0.05).2.2.3. the determination of the total antioxidant capacity and the total SOD activity36hours after the AngⅡ stimulated the cardiomyocytes, compared to blankcontrol group, the total antioxidant capacity and the total SOD activity risedslightly, but no significant difference was found (P>0.05).2.3. M2antagonize the cardiomyocytes hypertrophy induced by AngⅡ2.3.1. The mRNA expression of hypertrophy gene ANF and-MHC after the M2intervention.36hours after the co-stimulation of M2and AngⅡ, the mRNA expression ofhypertrophy gene ANF and-MHC significantly decreased significantly (P<0.05).2.3.2. The change of cardiomyocytes surface area after the M2intervetion36hours after the co-stimulation of M2and AngⅡ, the cardiomyocytessurface area of M2and AngⅡ co-stimulated group decreased significantlycompared to AngⅡ treated group (P<0.05).2.4. the effect of M2on the oxidative stress in the hypertrophic cardiomyocytesinduced by AngⅡ2.4.1. the change of superoxide anion in cardiomyocytes36hours after the co-stimulation of M2and AngⅡ, the cardiomyocytes werelabeled with DHE, the pictures were captured and the fluorescence intensitywas analysed using IPP6.0. Compared to the AngⅡ treated group, the meanoptical density significantly decreased (P<0.05); the fluorescence intensitywas measured using the fluorescence scanner, the fluorescence ID value of M2treated group was higher than AngⅡ treated group (P<0.05).2.4.2. the expression of NADPH oxidase P47phox in cardiomyocytes.36hours after the co-stimulation of M2and AngⅡ, the mRNA expression ofNADPH oxidase P47phox in the cardiomyocytes increased significantlycompared to the AngⅡ treated group (P<0.05).2.4.3. the determination of the total antioxidant capacity and the total SOD activity36hours after the co-stimulation of M2and AngⅡ, compared to the AngⅡtreated group, the total antioxidant capacity and the total SOD activity risedsignificantly (P<0.05).Conclusion1. The metabolite M2of puerain and puerain have equal antioxidant effect;2. oxidative stress is involved in the development of cardiomyocytes hypertrophy;3. M2privent the Ang Ⅱ induced cardiomyocytes hypertrophy through theantioxidant mechanism.