| ObjectiveIn this study,we established the model of pressure overload induced heart failure in mice by transverse aortic constriction(TAC)surgery,imitated the occurrence of heart failure induced by hypertension.Postoperation,we gave medication intervention of salvianolic acid B to the mices,comparing with the first-line anti heart failure drug metoprolol succinate sustained-release tablets as control drug.Observed whether it had a heart protective effect in the mices through multiple aspects of cardiac function,anatomy,pathematology and serology.Researched the possible protective mechanism by the method of myocardial cell culture.MethodsAnimal experimentThe C57BL/6 mices were randomly divided into TAC group,MT group and SalB group after TAC,compareing with the SHAM group on sham operation.SHAM group received 0.5ml normal saline by intragastric administration,TAC group received 0.5ml normal saline by intragastric administration,MT group received metoprolol succinate sustained-release tablets suspension 0.5ml by intragastric administration,SalB group received salvianolic acid B 0.5ml solution by intraperitoneal injection.All of the above drugs were given once a day for 14 days,starting from the first day after surgery.After treatment,we obtained AoPg、LVAWd、LVAWs、LVPWd、LVPWs、LVIDd、LVIDs、LVVd、LVVs and HR,then counted EF%、FS%and LVW through mouse echocardiography.Then the mice were anesthetized to death,we got heart,lung,liver and tibia,measured data of BW,HW,LW,LuW,LiW,TL,HW/BW,HW/TL,LW/TL,LuW/BW and LiW/BW.Myocardial tissue stained with HE and Sirius red,we observed the degree of myocardial fibrosis and cardiac myocyte area size,meanwhile we observed the degree of lung congestion,and check the concentration of blood serum BNP.Cell experimentH9c2 cardiomyocytes was divided into blank control group(CON group),isoproterenol group(ISO group),metoprolol group(MT group)and salvianolic acid B group(SalB group).When the cell density in the culture dish up to 70-80%,the cells were cultured in serum free DMEM for 16-24 hours.Then we added 10μmol/L of metoprolol solution in MT group,10μmol/L of salvianolic acid B in SalB group,commensurate DMSO in Con and incubated for 2 hours.Last,added 10μmol/L of isoproterenol in ISO group,MT group and SalB group,incubated for 15 min,then removed the culture dish for cell protein extraction and quantitation.Detected protein expression of p-ERK,ERK,p-AKT,AKT,p-CaMKⅡ,GAPDH,GATA4,and H3 by Western Blot.ResultsAnimal experiment1.Summary of cardiac function results:SHAM group and TAC group comparison:Compared with SHAM group,the figures of AoPg、LVAWd、LVIDs、LVPWd、LVW、LVWc and LVVs were markedly increased,EF and FS markedly decreased in TAC group,with significantly statistical difference(p<0.05 or 0.01).Comparison between four groups:AoPg in TAC group,MT group and SalB group was 19.04-22.15 times of SHAM group.Compared with TAC group,the figures of AoPg、LVAWd、LVIDs、LVPWd、LVW、LVWc and LVVs were markedly decreased,EF and FS markedly increased in MT group and SalB group,with significantly statistical difference(p<0.05 or 0.01).LVAWd in SalB group was more less than in TAC group,with statistical difference(p<0.05).There was no significant difference in LVAWs、LVIDd、LVVd、LVPWs and HR between the four groups(p>0.05).2.Summary of anatomy results:SHAM group and TAC group comparison:Compared with SHAM group,the figures of HW、HW/BW、HW/TL、LW、LW/TL、LuW and LuW/BW were markedly increased in TAC group,with significantly statistical difference(p<0.01).Comparison between four groups:Compared with TAC group,the figures of HW、HW/TL、HW/BW、LW、LW/TL、LuW and LuW/BW were markedly decreased,with significantly statistical difference(p<0.05 or 0.01).In addition,there was a good correlation between echocardiography LW and anatomy LW(R2= 0.6982,p<0.0001).There was no significant difference in TL,LiW and LiW/HW between the four groups.3.Summary of pathology results:SHAM group and TAC group comparison:Compared with SHAM group,myocardial HE staining suggested the myocardium was obviously hypertrophy,myocardial cells significantly increased,the area of myocardial cells significantly increased in TAC group.Sirius red staining indicated a significant increase in myocardial fibrosis in TAC group.The alveolar wall was thickened,the alveolar space was poor opening,the alveolar capillaries and the interstitial blood vessels were dilated and congested,and large erythrocyte deposited in alveolar space in TAC group.Comparison between four groups:Compared with SHAM group,myocardial HE staining suggested the myocardium was hypertrophy,myocardial cells increased,the area of myocardial cells increased in MT group and Sa1B group.Sirius red staining indicated a increase in myocardial fibrosis in MT group and SalB group.The alveolar wall was lightly thickened,the alveolar capillaries and the interstitial blood vessels were lightly dilated and congested,and few erythrocyte deposited in alveolar space in MT group and SalB group.Compared with TAC group,myocardial HE staining suggested that the degree of myocardial hypertrophy was reduced,the extent of myocardial cell hypertrophy decreased,the area of myocardial cells decreased,and the myocardial fibrosis was decreased by Sirius red staining in MT group and SalB group.The alveolar wall was thin,the dilatation and congestion of alveolar capillaries and interstitial blood vessels decreased,and deposition of erythrocyte in alveolar space decreased significantly in MT group and SalB group.4.Summary of serological results:SHAM group and TAC group comparison:Compared with SHAM group,serum BNP concentration significantly increased in TAC group,with significantly statistical difference(p<0.01).Comparison between four groups:The serum BNP concentration in MT group and SalB group was significantly higher than the SHAM group,but was significantly lower than the TAC group,with significantly statistical difference(p<0.01).Cell experiment1.The expression of p-ERK protein:making total ERK(T-ERK)as eference,p-ERK(phosphorylated ERK)expression was inconsistent,p-ERK expression in ISO group was significantly stronger than in CON group without the use of ISO,with statistical difference(p<0.05).The expression of p-ERK in MT group and SalB group was significantly weaker than in ISO group,with significantly statistical difference(p<0.05 or 0.01).2.The expression of p-AKT protein:making total AKT(T-AKT)as eference,p-AKT(phosphorylated AKT)expression was inconsistent,p-AKT expression in ISO group was significantly stronger than in CON group without the use of ISO,with statistical difference(p<0.05).The expression of p-AKT in MT group was significantly weaker than that of ISO group,with statistical difference(p<0.05).The expression of p-AKT in Sa1B group was significantly increased same as in ISO group,significantly stronger than that of Con group and MT group,with statistical difference(p<0.05).3.The expression of GATA4 protein:making Histone H3 as eference,GATA4 expression was inconsistent.The expression of GATA4 in ISO group was significantly increased,while not enhanced in SalB group,the two groups had significantly statistical difference(p<0.01).4.The expression of p-CaMKⅡ protein:making GAPDH as eference,p-CaMK II(phosphorylated CaMKⅡ)expression was inconsistent,p-CaMKⅡ expression in ISO group was significantly stronger than in CON group without the use of ISO,with statistical difference(p<0.05).The expression of p-CaMKⅡ in MT group and SalB group was significantly weaker than in ISO group,with statistical difference(p<0.05 or 0.01).Conclusions1.We proved that the model of pressure overload induced heart failure in mice by TAC was successful through animal experiment.2.Through animal experiment,we proved that salvianolic acid B had heart protective effect of inhibiting cardiac hypertrophy,improving heart function and reducing pulmonary edema.3.Through cell experiment,we found that the inhibition of cardiomyocyte hypertrophy of salvianolic acid B was related to inhibite ERK1/2/GATA4 pathway and CaMKⅡ protein phosphorylation. |
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