| BackgroundCardiac hypertrophy is a common pathophysiological pathway that variuos cardiovascular diseases develop into heart failure.The characteristics of cardiac hypertrophy include increase of protein synthesis which lead to cardiomyocytes hypertrophy,activation of fibroblasts which lead to fibrosis of myocardial interstitium,endothelial cells injury which lead to of myocardium ischemia,infiltration of inflammatory cells which lead to myocardium inflammatory cascade reaction.In the early stages of cardiovascular diseases,compensatory cardiac hypertrophy can increase cardiac output.However,persistent cardiac hypertrophy will gradually develop into decompensation and eventually lead to irreversible heart failure.However,there is no specific drug for the treatment of cardiac hypertrophy.The few anti-hypertrophic drugs for heart failure including angiotensin converting enzyme inhibitors,beta-blockers,aldosterone receptor antagonists and angiotensin receptor neprilysin inhibitors.However,these drugs have limited effects,thus the incidence and mortality of heart failure increasing year by year.Therefore,it is of great importance to identify novel pharmacological agents for the prevention and treatment of cardiac hypertrophy.Sanggenon C is a flavonoid,mainly extracted from mulberry bark.Till now,the biological and pharmacological effects of Sanggenon C have been raraly reported.Previous studies have shown Sanggenon C is able to inhibit the growth of tumor cells.In addition,it has been reported that Sanggenon C inhibited protein tyrosine phosphatase 1B and decreased blood pressure.Recently,researchers found that Sanggenon C has anti-oxidant and anti-inflammatory effects.Sanggenon C can inhibit TNF-?-induced cell adhesion and VCAM-1 expression through regulation of the NF-?B pathway.Sanggenon C was able to suppress the protein expression of ICAM-1 at post-translational levels.Since Sanggenon C possesses antioxidant and anti-inflammatory activities,which play a key role in cardiac hypertrophy,it may serve as a potential antihypertrophic agent.In this study,we use the aortic banding surgery to induced cardiac hypertrophy mice model in vivo,and phenylephrine(PE)stimulated H9C2 cells to induce cardiomyocyte hypertrophy model in vitro.The aim of this study is to explore the protective effect of Sanggenon C on cardiac hypertrophy and its specific mechanism.Clarifying the role of Sanggenon C in cardiac hypertrophy will provide new targets and strategies for the prevention and treatment of cardiac hypertrophy.Method1.Cardiac hypertrophy model was induced by aortic banding in mice.The experimental animals were divided into four groups: sham-operated group(control group n=15,intraperitoneal injection of saline containing 1% Tween 80,0.1ml/100g),model group(n=15,one week after aortic banding,intraperitoneal injection of saline containing 1% Tween-80,0.1ml/100g);low-dose Sanggenon C treatment group(n=15,one week after aortic banding,the mice were subjected to intraperitoneally injected with Sanggenon C,10mg/kg/d,diluted with saline containing 1% Tween-80 for 3 weeks)and the high-dose Sanggenon C treatment group(n=15,1 week after aortic banding,the mice were subjected intraperitoneal injection of Sanggenon C,20mg/kg/d,diluted with saline containing 1% Tween-80 for 3 weeks).Four weeks after operation,echocardiographic analysis and hemodynamic measurements were performed to assess cardiac function in mice.In addition,the degree of myocardial hypertrophy and fibrosis was assessed by pathological staining and molecular analysis in each group.Real-time quantitative reverse transcription-polymerase chain reaction was used to detect the expression of hypertrophic markers.2.Cultured H9c2 cardiomyocytes were randomly divided into four groups: control group,phenylephrine(PE)stimulated group,1 ?mol/L Sanggenon C treatment group and 10 ?mol/L Sangenone C treatment group.Cardiomyocytes hypertrophy was induced by PE stimulation,then cells were incubated with different concentrations of Sanggenon C.MTT test was used to detect cell viability in each group.?-actin immunofluorescence was used to detect the area of cardiomyocytes.RT-PCR was used to detect the expression of hypertrophic markers.Weatern blot was used to detect the the expression of signal pathway related proteins in heart tissue of mice in each group.Then H9c2 cardiomyocytes were divided into four groups: control group,PE stimulated group,10?mol/L Sanggenon C treatment group and CaN overexpression+10?mol/L Sanggenon C treatment group.?-actin immunofluorescence and reverse transcription-polymerase chain reaction were used to evaluation the hypertrophy of cardiomyocytes.Result1.Echocardiography result showed that the diameter of left ventricle in model group was significantly larger than that in control group,as evidenced by icreased left ventricle diastolic and systolic diameters in model group compared with that in control group(P < 0.05).The diastolic and systolic function of left ventricle in model group was significantly lower than that in control group as evidenced by decreased left ventricular ejection fraction and fractional shortening in model group compared with that in control group(P < 0.05).End-diastolic pressure was higher than that of the control group.Cardiac output was lower than that of the control group,and the maximum rate of left ventricular pressure development and decay was lower than that of the control group.In the LD-Sanggenon C and HD-Sanggenon C treated group,the left ventricular end-diastolic diameter and left ventricular end-systolic diameter of the mice were lower than the model group(P<0.05);left ventricular ejection fraction and fractional shortening were significantly higher than those in the model group(P<0.05);but in the LD-Sanggenon C treated group,the end-diastolic pressure was lower than the model group(P<0.05),the cardiac output and the maximum rate of left ventricular pressure development and decay have no difference from model group(P>0.05).However,in the HD-Sanggenon C treated group,the end-diastolic pressure,cardiac output and the maximum rate of left ventricular pressure development and decay were all improved significantly.The results of heart sampling showed that the heart mass,cross-sectional area of myocardial cells,heart weight/body weight,heart weight/tibia length,lung weight/tibia length ratio in model group were significantly higher than those in control group(P < 0.05).The cross-sectional area of myocardial cells,heart weight/body weight,heart weight/tibia length,lung weight/tibia length ratio in Sanggenon C treatment treatment group(including low-dose group and high-dose group)were significantly lower than those in model group(P < 0.05).RT-PCR analysis showed that the transcription levels of atrial natriuretic peptide(ANP),B-type natriuretic peptide(BNP),beta-myosin heavy chain(?-MHC)in mice hearts in model group were significantly higher than those in control group;while the transcription levels of ANP,BNP and?-MHC Sanggenon C treatment treatment group(including low-dose group and high-dose group)were significantly lower than those in model group(P < 0.05).PSR staining showed that the perivascular and interstitial fibrosis in the model group was significantly higher than that in the control group(P < 0.05).And the perivascular and interstitial fibrosis in Sanggenon C treatment treatment group(including low-dose group and high-dose group)was significantly lower than that in the model group(P < 0.05).RT-PCR analysis showed that the mRNA expression levels of collagen I,collagen III,fibronectin and connective tissue growth factor in the model group were significantly higher than those in the control group(P < 0.05).The mRNA expression level of those factors in Sanggenon C treatment treatment group(including low-dose group and high-dose group)was significantly lower than that of the model group(P < 0.05).2.H9c2 cells were treated with different concentrations of Sanggenon C(1,2,4,8,10 ?mol/L)for 48 hours.MTT results showed that there was no difference in cardiomyocytes viability among different concentrantions of Sanggenon C treatment group and control group(P > 0.05).The results of a-actin immunofluorescence staining showed that the surface area of cardiomyocytes in PE stimulation group was significantly higher than that in control group(P < 0.05).The area of myocardial cells in 1 and 10 ?mol/L Sanggenon C group was significantly smaller than that in PE stimulation group(P < 0.05).RT-PCR results showed that the transcription levels of ANP,BNP and beta-MHC in PE stimulation group were significantly higher than those in control group,while the transcription levels of cardiac hypertrophic marker in 1 and 10 ?mol/L Sanggenon C treatment group were significantly lower than those in PE stimulation group(P < 0.05).Western blot results showed that the expression of calcineurin and the nuclear expression of nuclear factor-activated T cell protein 2(NFAT2)were significantly higher in the model group than in the control group(P < 0.05),whereas the phosphorylation and the total expression of NFAT2 was significantly lower in the model group than in the control group(P < 0.05).The expression of calcineurin and nuclear expression of NFAT2 in the heart of Sanggenon C treatment group(low-dose group and high-dose group)mice were significantly lower than those in the model group(P < 0.05).The phosphorylated and total NFAT2 in the heart of Sanggenon C treatment group(low-dose group and high-dose group)mice were significantly higher than those in the model group(P < 0.05).NFAT2 immunofluorescence staining showed that the nuclear translocation of NFAT2 in PE stimulation group was significantly higher than that in control group,while the nuclear translocation of NFAT2 in H9C2 cells of 1 and 10 ?mol/L Sanggenon C treatment group was significantly lower than that of PE stimulation group.The results of a-actin immunofluorescence staining showed there is no significant difference between CaN overexpression+10 ?mol/L Sanggenon C treatment group and PE stimulation group of the surface area of cardiomyocytes(P>0.05).RT-PCR results showed that there is no significant difference between CaN overexpression+10 ?mol/L Sanggenon C treatment group and PE stimulation group of the hypertrophic markers(P>0.05).Conclusion1.Sanggenon C improves the impaired cardiac function in mice following aortic banding.2.Sanggenon C protects against cardiac hypertrophy and fibrosis induced by aortic banding in mice.3.Sanggenon C attenuates phenylephrine-induced cardiomyocyte hypertrophy.4.Sanggenon C protects cardiac hypertrophy through the regulation of calcinuerin/NFAT2 signaling pathway. |
PDF Full Text Request