The Effect And Mechanism Of Morroniside Inhibiting Angiotensin Ⅱ-induced Cardiac Hypertrophy

Background:Cardiac hypertrophy and fibrosis is one of the common pathological process of realization of the development of cardiovascular disease,persistent lesions may lead to changes in heart structure and ultimately affect the heart function,causing heart failure.Morroniside(mor)is a secoiridoid glycoside from Cornus officinalis.It has been shown that morroniside protects against not only cerebral ischemia/reperfusion injury in rats but also acute myocardial infarction in rats.However,there are no reports about the effects of mononoside on myocardial hypertrophy.Purpose:The aim of this study was to use angiotensin Ⅱ(AngⅡ)to induce myocardial hypertrophy in vitro and in vivo.To investigate whether morroniside can alleviate AngⅡ-induced cardiac hypertrophy and fibrosis and its mechanism.Methods:Male wild type(WT)mice(C57BL/6 background)ranging in age from 10to 12 weeks and weight from 24 to 26 g and H9C2 rat cardiomyocyte cell line were selected for experiments.AngⅡ was injected into mice by subcutaneous implantation of a micropump for 4 weeks to establish a model of cardiac hypertrophy.At the same time,morroniside was given to cardiac hypertrophic mice for 4 weeks by gavage to establish a morroniside treatment group of mice.Hypertrophic cardiomyocyte model was obtained as a model group by AngⅡ stimulation for 48 hours.The cardiomyocytes of morroniside treatment group were established by pretreating the cardiomyocytes with morroniside.Cardiac hypertrophy and fibrosis were assessed by morphology,echocardiographic parameters,hemodynamics,histological analysis,and an analysis of hypertrophy marker expression.Then,western blot was used to detect the expression levels of NF-κB pathway proteins in animal and cell models.Finally,we used NF-κB transcription inhibitors(PDTC)to pre-treat myocardial cells in each group for 1 hour,to detect changes in the size of myocardial cells and the Ca MKⅡ and MAPK pathway proteins.Further clarify the regulatory role of the NF-κB pathway in morroniside on cardiac hypertrophy.Results:The results showed that the model group was smaller than the control group[HW/BW:(4.49±0.1089)mg/g;HW/TL:(7.97±1.043)mg/g;cardiomyocyte surface area[(323.2±18.28)μm~2]The ratio of cardiac mass to body weight(5.716±0.126)mg/g or length of tibia[(11.54±0.6012)mg/g]was significantly increased,and the surface area of cardiomyocytes[(522.3±7.012)μm~2]was also significantly increased,while the HW/BW[(4.716±0.2213)mg/g,HW/TL[(7.939±0.790)mg/g]and myocardial cell surface area[(372.6±19.4)μm~2]in the morroniside treatment group were lower than those in the model group.Statistical significance.Masson and immunohistochemical staining results showed that the fibrosis area of the model group[(4.951±0.674)%]and collagen deposition[collagen I:(5.513±1.046)%;collagen ⅡI:(7.652±2.252)%]Compared with the control group[fibrosis area:(0.783±0.195)%;collagen I:(0.707±0.151)%;collagen ⅡI:(1.019±0.041)%]was significantly increased.However,the fibrosis area[(1.41±0.345)%]and collagen content[collagen I:(1.049±0.231)%;collagen ⅡI:(1.754±0.66)%]in the morroniside treatment group were lower than those in the model group.In addition,the echocardiographic examination of the model group showed that the anterior systolic wall(1.604±0.095)mm,anterior diastolic wall(1.180±0.069)mm,posterior systolic wall thickness(1.809±0.175)mm,and diastolic posterior wall(1.544±0.066)mm is also higher than the control group[anterior wall thickness during systole:1.351±0.138;anterior wall during diastole(0.849±0.043)mm;posterior wall during systole(1.337±0.172)mm;posterior wall during diastole(1.009±0.070)mm],morroniside treatment group[anterior wall thickness during systole:(1.369±0.042)mm;anterior wall during diastole(0.884±0.056)mm;posterior wall thickness during systole(1.549±0.149)mm;after diastole The wall(1.093±0.082)mm]is lower than that of the model group;the ventricular diameter of the model group[diameter during systole:(2.203±0.095)mm;diameter during diastole:(3.096±0.233)mm]and ejection fraction(49.84±2.062)%And shortened fraction(26.88±1.128)%are lower than the control group[diameter during systole(2.367±0.147)mm;diameter during diastole:(3.703±0.24)mm;ejection fraction(63.21±1.693)%;shortened fraction:(36.03±1.055)%]and morroniside treatment group[diameter during systole:(2.347±0.117)mm;diameter during diastole:(3.602±0.177)mm;ejection fraction(59.46±2.384)%;shortened fraction:(32.35±2.063)%],the difference was statistically significant.In in vitro experiments,the cell surface area of the morroniside group[(480.8±24.1)μm2]was also lower than that of the model group[(595.6±43.19)μm2].Moreover,morroniside significantly inhibited the increase of AngⅡ-induced hypertrophy markers,fibrosis markers and inflammatory factor m RNA expression levels.In addition,morroniside can significantly inhibit the nuclear expression of proteins in the NF-κB signaling pathway induced by AngⅡ.In addition,compared with AngⅡ stimulated cells[(598.9±39.47)μm2],the increase in surface area of cells treated with PDTC[(462.3±41.38)μm2]was suppressed,but cells treated with PDTC morroniside[(464.1±28)μm2]compared,the difference was not statistically significant.Conclusions:This study demonstrated in vivo and in vitro experiments that morroniside can attenuate AngⅡ-induced myocardial hypertrophy and fibrosis,respectively.And morroniside may play a role by regulating the NF-κB signaling pathway.