Astragaloside IV Prevents Myocardial Hypertrophy Induced By Mechanical Stress By Activating Autophagy And Reducing Inflammation

Objective The aim of the present study was to investigate the effects of astragaloside IV(As-IV)on myocardial hypertrophy by ligating and narrowing the abdominal aorta of Sprague-Dawley(SD)rats and flexcell FX-5000 tension system to stretch primary cardiomyocytes as a model.Methods Healthy male Sprague-Dawley rats weighing 200-250 g.Twelve hours before surgery,fasting but not water and 20% urethane(1g/kg)were used to fix the anesthetized rat on the operating table,and the abdominal aorta above the left renal artery was exposed.Cause partial narrowing of the abdominal aorta(abdominal aorta construction,AAC).The surgery treatment rats was divided into four groups: aortic banding group(AB),40 and 80 mg/kg/d As-IV(intragastric administration)and 1 mg/kg/d rapamycin(rapa)(intraperitoneal injection).The sham-operated group had a similar operation,except for the application of a silver clip.The rats received intraperitoneal injection of gentamicin for a week after the surgery.Surviving rats were randomly divided into groups one week after surgery(n = 10),Astragaloside group was administered orally for 8 weeks,Rapamycin is given intraperitoneally for 8weeks,AAC,Sham and Rapa groups were given the same volume of water,AAC group,Sham group and As-IV group were injected intraperitoneally with the same volume of normal saline.After dosing Rats were anaesthetized with20% urethane(1g/kg),maintained in a supine position,and skin preparation (shaving)was performed,after which the Philips IE33 echocardiographic system was used to perform two-dimensional(2D)-guided M-mode echocardiography(Philips Medical Systems Nederland BV).The following parameters were measured: left ventricular ejection fraction(LVEF),left ventricular posterior wall end-diastolic thickness(LVPWd),left ventricular posterior wall end-systolic thickness(LVPWs),interventricular septum end diastolic thickness(IVSd),and interventricular septum end systolic thickness(IVSs).Weigh rat weight,heart and left ventricle weight,The heart weight index was calculated(heart weight/body weight ratio,HWI = HW/BW),and left ventricular weight index(left ventricular weight/body weight ratio,LVWI =LVW/BW)was also calculated.HE staining was used to observe the arrangement;TNF-α and IL-18 levels in serum was measured by ELISA;detection of ROS in myocardial tissue;the expression of ANP,BNP,LC3 II,p62,NLRP3,and IL-1β in myocardial tissue was detected by Western blot;determination of NLRP3 expression in tissues by immunohistochemistry.SPF grade SD rats aged 1-3 days were sucked from the ventricle to the middle and lower 2/3 ventricles.The D-Hanks solution was used to wash away the blood and cut the tissue into a pulpy form.Then use 0.4% collagenase and0.05% trypsin = 2: 1 for digestion and obtain the ventricular muscle cells by differential centrifugation.Rat primary cardiomyocytes were isolated and cultured in DMEM with 10% fetal bovine serum(Hy Clone,USA).Then,primary cardiomyocytes were placed into Bio Flex Plate-Collagen Type.After 48 hours of culture,serum-free DMEM was added,and 100 μM As-IV or 100 n M rapamycin was administered in the corresponding treatment group.The isolated primary cardiomyocytes were stretched for 24 hours by a Flexcell FX-5000 Tension System(1 HZ,20% elongation).The control group was not stretched,and the inhibitor group was administered 3-MA without stretching.The model group was not administered under the same conditions;the autophagy inhibitor 3-MA group was only administered without extension under the same conditions;the blank group was not administered without extension under the same conditions.The cells were divided into 5 groups:Control group;Stretch group;As-IV group,Rapa group,3-MA group.After the procedure,each group of cells was collected for subsequent experiments.The cell size was measured by phalloidin-tetramethyl in vitro;determination of Reactive Oxygen Species(ROS)in Cardiomyocytes by DHE staining;the expression of ANP,BNP,LC3 II,p62,NLRP3,and IL-1β in cardiomyocytes was detected by Western blot;TNF-α and IL-18 levels in cell supernatant was measured by ELISA.Results(1)In vitro experiment,Model group compared with Sham operation group,(1)Echocardiography showed that the cardiac hypertrophy indexes LVPWd,LVPWs,IVSd,and IVSs in rats significantly increased in the AAC group,and the whole heart weight index and left ventricular weight index increased.The cardiac myocytes of AB rats were hypertrophied,and the myocardial fibers were disordered.(2)The levels of TNF-α and 1L-18 in serum were increased.(3)The expression of ANP and BNP in myocardial tissue was significantly increased.(4)The expression of autophagy-related protein LC3 II was significantly down-regulated in myocardial tissues,and the expression of P62 was significantly up-regulated.(5)expression of NLRP3 and1L-1β in myocardial tissues was significantly increased.(6)The ROS production in the myocardial muscle was significantly increased.(7)The brown-yellow positive expression of NLRP3 in myocardial tissue of AAC rats was significantly increased.AAC + As-IV(40,80)group,AAC + Rapa group compared with AAC group,(1)Echocardiography showed that the cardiac hypertrophy indexes LVPWd,LVPWs,IVSd,and IVSs in rats were significantly reduced in the administration group and the positive control group,the whole heart weight index and left ventricular weight index were reduced,and AAC rats with hypertrophy and large myocardial tissue cells have improved.(2)The levels of TNF-α and 1L-18 in serum were reduced.(3)The expression of ANP and BNP in myocardial tissue was significantly reduced.(4)The expression of autophagy-related protein LC3 II was significantly up-regulated in myocardial tissues,and the expression of p62 was significantly down-regulated.(5)expression of NLRP3 and 1L-1β in myocardial tissues was significantly reduced.(6)The ROS production in the myocardial muscle was significantly reduced.(7)The brown-yellow positive expression of NLRP3 in myocardial tissue of rats in each treatment group was reduced to varying degrees.(2)In vitro experiment,compared with the Control group,(1)Myocardial cells enlarged in the Stretch and 3-MA groups.(2)The ROS production in the Stretch and 3-MA groups were increased intracellular.(3)The expression of ANP and BNP in cardiomyocytes were increased in the Stretch and 3-MA groups.(4)The expression of inflammation was significantly up-regulated in the Stretch and 3-MA groups.(5)Autophagy was inhibited in both the Stretch and 3-MA groups.Compared with Stretch group,(1)As-IV and Rapamycin(Rapa)have protective effects on hypertrophic cardiomyocytes.(2)As-IV and Rapamycin can reduce ROS content in cells.(3)As-IV and rapamycin can reduce the expression of inflammation.(4)As-IV and Rapamycin can up-regulate LC3 II and down-regulate p62 to activate autophagy.Conclusion(1)In the abdominal aorta construction model,As-IV can improve myocardial hypertrophy.abdominal aorta construction(2)In the abdominal aorta construction model,As-IV activates autophagy and reduces cardiac inflammation.(3)As-IV has a protective effect on abdominal aortic constriction rats,which may be related to astragaloside IV activation of autophagy,reduction of inflammatory infiltration and oxidative stress.