The Research Of Treatment Mechanism Of Compound Apocynum Tablet Protects Against Cardiac Hypertrophy In Mice

Objective1.Literature studies have well done before the research.We constructed a cardiac hypertrophy model by using transverse aortic constriction(TAC)surgery to impose pressure overload in C57BL/6 mice.Through mouse echocardiography after two weeks,we obtained echocardiography data and anatomy data.Cardiomyocytes from LV cross sections were stained with hematoxylin-eosin at the end of the experiments.2.Mechanism of Compound Apocynum Tablet(CAT/LBM)protection against pathological cardiac hypertrophy in mice.Record the survival status and mortality of four groups,estimate the anatomical data after 2 weeks,detecte mouse echocardiography before the surgery,1 week after surgery,two weeks after surgery,and Cardiomyocytes were stained with hematoxylin-eosin to evaluate cell size,stained with sirus to evaluate myocardial fibrosis using the difference in color(red fibrotic area as opposed to yellow myocardium).The protein expression of protein kinase B,phospho-extracellular regulated protein kinase,extracellular regulated protein kinase,phospho-extracellular regulated protein kinase,GATA binding protein 4,nuclear factor of activated T cells C3 and calcineurin N were detected.MethodsExperiment 1Male C57BL/6J mice were constructed a cardiac hypertrophy model by using transverse aortic constriction(TAC)surgery to impose pressure overload.Mice were assigned randomized to two groups:SHAM and TAC group.We observed the survival status and mortality in the two weeks'normal feeding.Through mouse echocardiography(2 weeks after the surgery),we obtained AoPg?HR?LVAWd?LVPWd?LVIDd?LVAWs?LVPWs?LVIDs?EDV?ESV,then counted EF%?FS%?LVW.Then the mice were anesthetized to death,we got BW,HW,LVW,LUW,LIW,TL,HW/BW,HW/TL,LVW/TL,LUW/BW and LIW/BW data,Cardiomyocytes were stained with hematoxylin-eosin to evaluate cell size to describe microscopic study.Experiment 2Male C57BL/6J mice were constructed a cardiac hypertrophy model by using transverse aortic constriction(TAC)surgery to impose pressure overload as Experiment 1.Mice were assigned randomized to four groups:NS+SHAM group,NS+TAC group,LBM+SHAM group,and LBM+TAC group.Mice in NS+SHAM received saline i.g and all the surgery except constricting the aorta;mice in NS+TAC were subjected to saline i.g and TAC surgery;LBM+SHAM mice received LBM i.g and all the surgery except constricting the aorta;LBM+TAC mice received LBM i.g and TAC surgery.We observed the survival status and mortality in the two weeks' feeding.Through mouse echocardiography,we obtained AoPg?HR?LVAWd?LVPWd?LVIDd?LVAWs?LVPWs?LVIDs?EDV?ESV,then counted EF%?FS%?LVW before the surgery,1 week after surgery,two weeks after surgery.Then the mice were anesthetized to death,we got data of BW,HW,LVW,LUW,LIW,TL,HW/BW,HW/TL,LVW/TL,LUW/BW and LIW/BW.Cardiomyocytes were stained with hematoxylin-eosin to evaluate cell size to describe microscopic study,stained with sirus to evaluate myocardial fibrosis ratio to count fibrosis.Experiment 3Male C57BL/6J mice accepted the same surgery to experiment 1.Mice were assigned randomized to four groups:NS+SHAM group,NS+TAC group,LBM+SHAM group,and LBM+TAC group.Then we extracted proteins from heart to detect expression of protein kinase B,phospho-extracellular regulated protein kinase,extracellular regulated protein kinase,phospho-extracellular regulated protein kinase,GATA binding protein 4,nuclear factor of activated T cells C3 and calcineurin N using Weston blot.ResultsExperiment 1Survival status in TAC group were better than SHAM group after two weeks normal feeding.TAC mice showed less activity,slower reaction,less intake,easier scared.AoPg,LVPWd,LVW and LVW(Corrected)of TAC mice were increased(P<0.001),ESV also increased(P<0.05);EF%,FS%were lower(P<0.05),all these data were statistically significant.The date of HR,LVAWs,LVPWs,LVIDd,LVIDs,EDV between two groups were basically the same.HW and LVW of TAC mice were increased(P<0.001),HW/BW(P<0.01),HW/TL(P<0.001)and LVW/TL(P<0.001)were statistically significant.The date of HW,TL,LUW,LIW between two groups were basically the same.There was no signs of heart failure in TAC mice.In summary,HW,LVW,HW/BW,HW/TL,LVW/TL in TAC mice were more deteriorated than SHAM group.HE stain pointed cardiomyocytes sizes were larger compared with SHAM group.Experiment constructed cardiac hypertrophy model by using transverse aortic constriction(TAC)surgery to impose pressure overload on mouse successfully.Experiment 21.Survival status NS+TAC group were worst in four groups after two weeks with some deaths.While LBM+TAC mice showed better than NS+TAC group.All the SHAM mice lived well.2.Heart function dataAoPg of NS+TAC mice were more than NS+SHAM mice and LBM+SHAM mice(P<0.001),LBM+TAC mice were more than NS+SHAM mice(P<0.01)and than LBM+SHAM mice(P<0.001);LBM +TAC mice were better than NS+TAC mice,but there was no statistically significant(P>0.05)LVAWd of NS+TAC mice were more than NS+SHAM mice and LBM+SHAM mice(P<0.001),LBM+TAC mice were more than NS+SHAM mice(P<0.01)and than LBM+SHAM mice(P<0.001);LBM +TAC mice were better than NS+TAC mice(P<0.01);EF%of NS+TAC mice were lower than NS+SHAM mice and LBM+SHAM mice(P<0.05),LBM+TAC mice were better than NS+TAC mice(P<0.05);FS%of NS+TAC mice were lower than NS+SHAM mice and LBM+SHAM mice(P<0.05),LBM+TAC mice were better than NS+TAC mice(P<0.05);LVW of NS+TAC mice were more than NS+SHAM mice and LBM+SHAM mice(P<0.001),LBM+TAC mice were more than NS+SHAM mice(P<0.05)and than LBM+SHAM mice(P<0.01);LBM +TAC mice were better than NS+TAC mice(P<0.01);LVW(Corrected)of NS+TAC mice were more than NS+SHAM mice and LBM+SHAM mice(P<0.001),LBM+TAC mice were more than NS+SHAM mice(P<0.05)and than LBM+SHAM mice(P<0.01);LBM +TAC mice were better than NS+TAC mice(P<0.01);Data of HR,LVAWs,LVPWd,LVPWs,LVIDd,LVIDs,EDVand ESV in four groups were no statistically significant(p>0.05).3.Anatomical dataHW?LVW and LVW/TL of NS+TAC mice were worse than NS+SHAM mice and LBM+SHAM mice(P<0.001).HW/BW and HW/TL of NS+TAC mice were worse than NS+SHAM mice(P<0.01)and than LBM+SHAM mice(P<0.001);LBM +TAC mice were better than NS+TAC mice on HW,LVW,HW/BW,HW/TL and LVW/TL(P<0.01);LBM+TAC mice were worse than LBM +SHAM mice on HW LVW,LVW/TL,HW/TL(P<0.01)and HW/BW(P<0.05);The date of HW,TL,LUW,LIW between four groups were basically the same(P>0.05).There were no signs of heart failure in TAC mice.4.H&E stainingThe LV crosssections of the four groups stained with H&E(×400 magnification,×20 magnification).x400:NS+TAC mice were bigger than NS+SHAM mice(P<0.001),LBM+SHAM mice(P<0.001)and LBM+TAC mice(P<0.01).x20:TAC mice:ventricular wall thickened much,heart chamber volume became smaller,showed concentric type thicken,NS+TAC group was more obvious.The SHAM mice were normal.TAC mice:stromal hyperplasia,disorganized with cell lysis,the LBM+TAC group showed better.5.Sirius stainingSirius red(red staining,×40 magnifications,x20 magnifications).×40:Mean cardiac fibrosis formed much more in the TAC mice than SHAM mice,the LBM+TAC group were better than NS+TAC group,the same to the 20 magnifications.6.MortalitySHAM mice were 100%survival.Survival rate was significantly higher in the NS+TAC mice(94.118%)than the LBM+TAC mice(83.3%).LBM+TAC mice displayed a significantly improved survival compared to NS+TAC mice.Experiment 31.Phosphorylation of AKT:AKT phosphorylations were enhanced in NS+TAC group,compared with other groups(P<0.001),the other three groups were no significant difference(P>0.05).2.Phosphorylation of ERK1/2:ERK1/2 phosphorylations of threonines at 202th and 204th sites were enhanced in NS+TAC group,compared with other groups(P<0.05),the other three groups were no significant difference(P>0.05).3.The expression of GATA4:GATA4 protein were enhanced in NS+TAC group,compared with other groups(P<0.001),the other three groups were no significant difference(P>0.05).4.The expression of CAN:CAN protein were enhanced in NS+TAC group,compared with other groups(P<0.001),LBM+TAC mice were up than LBM+SHAM mice(P<0.05),the other two groups were no significant difference(P>0.05).5.The expression of NFATc3:NFATc3 protein were enhanced in NS+TAC group,compared with other groups(P<0.001),LBM+TAC mice were up than LBM+SHAM mice(P<0.05),the other two groups were no significant difference(P>0.05).Conclusions1.The experiments constructed cardiac hypertrophy model by using transverse aortic constriction(TAC)surgery to impose pressure overload on mouse truly.2.At the end of experiments,quality of Life in LBM+TAC group were greatly better than NS+TAC group.Mortality,heart function and anatomical data improved in LBM+TAC group compared with NS+TAC group.Then histological examination stained with HE and sirus were superior in LBM+TAC group.3.LBM effected cardiac hypertrophy through mitogen-activated protein kinase 1/2/GATA4 signaling pathway,AKT/GATA4 signaling pathway and CAN-NFATc3 signaling pathway.