Effect And Mechanism Of Guanxinning Tablet On Myocardial Hypertrophy Model In Vivo And In Vitro

Myocardial hypertrophy is a compensatory response to maintain cardiac output under various physiological and pathological stimuli.However,long-term cardiac hypertrophy often leads to heart decompensation,heart failure and sudden death.It is also an important cause of incidence rate and mortality worldwide.The main drugs now for the treatment of cardiac hypertrophy are β-blockers and renin angiotensin aldosterone system inhibitors,which have many problems and defects,such as side effects,the best dose uncontrolled,and the individual difference of curative effect.Traditional Chinese medicine has the characteristics of various components,mild efficacy,small side effects,and treating both the symptoms and the root causes.It can regulate and treat the body from multi-dimensional and multi-level.Guanxinning tablet(GXN)is an oral preparation extracted from Salvia miltiorrhiza and Ligusticum chuanxiong.Studies have shown that GXN has vasodilator and antithrombotic effects,and is used in the treatment of coronary heart disease.However,its effect on anti-cardiac hypertrophy is unknown.At present,the research and development of anti myocardial hypertrophy are in experimental stage,and GXN is a Chinese medicine preparation which has been used in clinical practice.The purpose of this project is to promote the clinical application of GXN in the treatment of myocardial hypertrophy by studying the pharmacodynamic effect and mechanism of GXN against myocardial hypertrophy,which can effectively shorten the research and development time and save the research and development cost.Compared with other research and development stages of traditional Chinese medicine,it has great advantages,and it is also of great significance to promote the modernization of traditional Chinese medicine.The effect of GXN on myocardial hypertrophy was evaluated in spontaneously hypertensive rats.1.the results of echocardiography showed that SHR rats had obvious pathological characteristics of myocardial hypertrophy from 15 weeks old,while the correlation indexes of ventricular wall thickness(IVSd,IVSs,LVPWs and LVPWd)of SHR rats in GXN group were significantly lower than those in the model control group(P<0.05,P<0.01),and the effect of GXN high dose group was significantly better than that of amlodipine group.2.The results of heart weight and coefficient of SHR rats showed that after drug intervention,the heart weight and coefficient of each drug group decreased in varying degrees,and the heart coefficient of GXN high-dose group decreased significantly(P<0.05),which was also better than that of amlodipine positive group.3.Compared with the model control group,the area of myocardial cells by WGA staining decreased significantly after GXN treatment(P<0.01);Masson staining showed that compared with the model group,the score of myocardial fibrosis decreased after GXN treatment,and GXN high-dose group decreased the same extent as the positive control group(P<0.05).RT-PCR was used to detect mRNA expression of ANP,BNP and RCANl in SHR.The results showed that compared with the model group,mRNA levels of ANP,BNP and RCAN1 in GXN group was significantly decreased(P<0.05,P<0.01),and the results were close to or even better than those in positive group.In order to evaluate the anti hypertrophic effect of GXN in vitro,the primary rat cardiomyocytes were isolated and induced by isoproterenol(Iso).1.MTT assay was used to detect the effect of GXN on the proliferation of cardiomyocytes stimulated by 10 μg/mL Iso.The results showed that GXN had no significant effect on the proliferation of cardiomyocytes induced by Iso in the concentration range of 50μg/mL～1200 μg/mL(P>0.05),indicating that GXN had high safety on cardiomyocytes.2.Cells were seeded on 12 well plates followed by different treatments:control group,model group(isoproterenol Iso,10 μmol/L),GXN low dose group(GXN 200μg/mL)and GXN high dose group(GXN 400 μg/mL).12 hours of GXN were pre-treated before Iso was added.After 48 hours of culture,the cells were observed under confocal microscope and photographed and the area of single cell was measured by image analysis system.Our results showed that compared with the model group,the cross-sectional area of myocardial cells in GXN high-dose group was significantly decreased(P<0.01).3.Flow cytometry showed that GXN could inhibit cardiomyocyte apoptosis.4.Detection in the mRNA levels of Bcl2 and Bax by RT-PCR showed that after 400 μg/mL GXN pretreatment,the mRNA expression of Bcl2 gene increased significantly(P<0.01),and the mRNA expression of Bax gene decreased significantly(P<0.01).5.protein expression of MEK,ERK1/2,p-ERK1/2,Bcl2 and Bax were examined by western blot.The results showed that the phosphorylation expressions of MEK and ERK1/2 were significantly increased in the model group(P<0.01),and the phosphorylation expressions of these two proteins were significantly decreased after GXN pretreatment(P<0.05).Consistent with the results of RT-PCR,expression of Bcl2 protein increased significantly(P<0.05)and Bax protein decreased significantly(P<0.05)after GXN pretreatment.This paper confirmed the applicability of SHR rats as an animal model of myocardial hypertrophy,and reported the anti myocardial hypertrophy effect of GXN for the first time in vivo and in vitro by combining with Iso induced primary cardiomyocytes,which provided theoretical basis for GXN as an anti cardiac mast drug.Our preliminary study shows that the mechanism of GXN anti myocardial hypertrophy is related to the inhibition of abnormal activation of ERK signaling pathway and the inhibition of apoptosis of cardiomyocytes.