Effect And Mechanism Of Qili Qiangxin Capsule On Ventricular Remodeling In Mice After Relieving High Pressure Loa

Purpose:As the population ages,the morbidity of heart failure is on the rise,while the rate of re-hospitalization and mortality remains high.Aortic stenosis is the most common type of heart valve lesion in clinical and can induce left ventricular remodeling due to chronic pressure overload,resulting in the development of heart failure.At present,the main means of clinical treatment is surgical treatment for aortic stenosis,especially in recent years transcatheter aortic valve replacement(TAVR)surgery has become a promise for minimally invasive surgery for severe aortic stenosis.However,left ventricular reverse remodeling following successful TAVR is incomplete and is characterized by persistent cardiac fibrosis,oxidative stress,inflammation,and apoptosis,leading to a poor prognosis.The treatment philosophy of Qiliqiangxin Capsule is common treatment and separated elimination of qi,blood and water,which has advantages of holistic treatment,multi-target effect,individualized treatment based on syndrome differentiation and slight side effect.In previous studies on a background of standard treatment,Qiliqiangxin Capsules further reduced the levels of N terminal-pro brain natriuretic peptide(NT-pro BNP)as well as improve patient quality of life in chronic heart failure patients.Meanwhile Qiliqiangxin capsule delays the progression of myocardial hypertrophy,ventricular remodeling and cardiac dysfunction due to pressure overload in mice.However,the basic mechanism of Qiliqiangxin Capsules effectiveness is poorly understood,and little is known about its protective effects against cardiac remodeling and improve cardiac dysfunction after successful TAVR.Therefore,in the current study we established a mice model through pressure overloading and subsequent unloading to closely mimic TAVR surgery.To observe the effects of Qiliqiangxin Capsules on ventricular remodeling and cardiac function after release of pressure overload and to explore the possible mechanism of Qiliqiangxin Capsule on ventricular remodeling caused by aortic stenosis.Material and method:1.Forty 8-week-old C57BL/6J mice were randomly divided into control group(n=8)and model group(n=32).Transverse aortic constriction(TAC)surgery was performed in model mice to establish pressure overload-induced cardiac hypertrophy model simulates the pathophysiological process of aortic stenosis.After 8 weeks of feeding in the model group,cardiac function and cardiac structure were evaluated by echocardiography,which confirmed that TAC modeling was successful.Then,the model group was divided into consistent pressure overload group(aortic banding,AB group,n=8),AB combined with Qiliqiangxin capsule treatment group(ABQ,n=8),aortic debanding group(aortic debanding,DB group,n=8)and DB combined with Qiliqiangxin Capsule treatment group(DBQ group,n=8).Mice in DB and DBQ groups underwent a second surgery to separate the aorta from the silk thread needle and relieve the pressure overload.The other mice,including those in the control,AB and ABQ groups,also underwent a second open thoracotomy surgery as controls.Finally,ABQ and DBQ groups were given 1g/kg/d by intragastric administration every morning,once a day,for 4 weeks.Other groups were given the same volume of distilled water.After 4weeks,the cardiac function and ventricular structure of mice in each group were evaluated by small animal ultrasound.Ultrasonic indicators were collected.Such as,ejection fraction(LVEF%)of the left ventricle,left ventricular fractional shortening(FS%),left ventricular internal diameter at end-systole(LVIDS),left ventricular internal diameter at end-diastole(LVIDD),left ventricular mass(LVmass)and other indicators.After body weight(BW)was measured,mice were sacrificed,heart weight(HW)was weighed,and tibia length(TL)was measured.Calculate the ratio of heart weight to body weight(HW/BW)and the ratio of heart weight to tibial length(HW/TL).Pathological sections of mice heart tissue were performed.HE staining was used to observe the gross cardiac morphology of the heart,WGA staining was used to observe the cardiomyocyte size,and myocardial fibrosis were detected by MASSON staining.Real-time PCR were used to detect the m RNA expression related to myocardial hypertrophy indicators include brain natriuretic peptide(BNP)and myosin heavy chain-7(MYH7).2.The expression of 4-hydroxynonenal(4-HNE)in mice myocardial tissue was determined by immunohistochemistry staining.Real-time PCR was used to detect oxidative stress indexes in myocardial tissue of mice in each group: nuclear factor erythrocyte 2 related factor 2(Nr F2),heme oxygenase-1(Heme Oxygenase-1,HO-1)and superoxide dismutase(SOD2)m RNA expression levels.The protein expressions of Nr F2,HO-1,Catalase,cyclooxygenase 2(COX2)and SOD2 in myocardial tissue of mice in each group were detected by Western blot.Results:1.The heart failure model of mice with pressure overload was established by TAC surgery,and then the pressure overload caused by aortic coarctation was removed by surgery.The animal model was used to simulate the mice undergoing TAVR surgery.2.In terms of cardiac function,LVEF and FS values of AB group were significantly decreased compared with control group.At the same time,LVEF and FS values of DB group were decreased compared with control group.Compared with AB and ABQ groups,LVEF and FS values in DB group were significantly increased.Compared with AB group,ABQ group and DB group,the LVEF and FS values of DBQ group were significantly improved.3.In terms of the cardiac structure of mice,the levels of LVIDD,LVIDS and LVMass in group AB were significantly increased compared with the control group.Compared with control group,LVIDD,LVIDS and LVMass values of DB group were higher than those of control group.Compared with AB and ABQ groups,LVIDD,LVIDS and LVMass values of mice in DB group were significantly decreased.Compared with AB group,ABQ group and DB group,DBQ group showed significant improvement in LVIDD,LVIDS and LVMass levels.4.In terms of cardiac hypertrophy in mice,the general morphology of heart in AB group was significantly larger than that in the control group.After the second surgery and/or administration of Qiliqiangxin Capsule,the cardiac hypertrophy in DB group and DBQ group could be improved compared with that in AB group.In terms of HW/BW ratio,compared with the control group,the HW/BW ratio of mice in AB and ABQ groups was significantly increased,while the HW/BW ratio of mice in DB and DBQ groups was significantly decreased compared with AB and ABQ groups.The ratio of HW/TL in AB group was significantly higher than that in DBQ and control groups.Meanwhile,the ratio of HW/TL in ABQ group was significantly higher than that in control group.RT-PCR was used to detect the m RNA level of BNP and MYH7 in myocardial tissue of mice in each group.According to whether the mice relieved the pressure overload,compared with the control group,the m RNA levels of BNP and MYH7 in group AB were significantly increased,while the m RNA levels of BNP and MYH7 in group DB were decreased compared with those in group AB,but still higher than those in the control group.After 4 weeks treatment with Qiliqiangxin Capsule,m RNA levels of BNP and MYH7 in ABQ group were significantly reduced compared with AB group.After the mice were relieved of pressure overload and further treated with Qiliqiangxin Capsule for 4 weeks,m RNA levels of BNP and MYH7 in DBQ group were reduced compared with DB group,but there was no statistical significance.5.The general morphology of the mice heart was observed by HE staining.The results showed that the gross picture of the heart of mice in group AB were significantly larger than those in the control group.After Qiliqiangxin Capsule or release of pressure overload,myocardial hypertrophy induced by pressure overload was alleviated in ABQ and DB groups,while myocardial hypertrophy induced by pressure overload was further significantly alleviated in DBQ group.The cross-sectional area of left ventricular cardiomyocytes was evaluated by WGA staining.Compared with the control group,the cross sectional area of myocardial cells in AB group,ABQ group and DB group increased,and there was no statistical difference between the cross sectional area of myocardial cells in DBQ group mice and the control group.After the mice with pressure overload were treated with Qiliqiangxin Capsule for 4 weeks,the cross sectional area of myocardial cells in ABQ group decreased compared with that in AB group.Compared with DB group,the cross sectional area of cardiomyocytes in DBQ group was significantly reduced after treatment with Qiliqiangxin Capsule for 4 weeks after pressure overload relief.6.Masson staining was used to evaluate myocardial fibrosis in mice.The results showed that compared with the control group,the area of myocardial fibrosis in AB group,ABQ group and DB group increased,and there was no statistical difference in the area of myocardial fibrosis in DBQ group mice compared with the control group.After the mice with pressure overload were treated with Qiliqiangxin Capsule for 4 weeks,compared with AB group,the area of myocardial fibrosis in ABQ group was decreased.After relieving the pressure overload of the mice and further giving Qiliqiangxin capsule for 4 weeks,the area of myocardial fibrosis in DBQ group was significantly lower than that in DB group.7.The expression of 4-HNE in myocardial tissue of mice in each group was detected by immunohistochemical staining to evaluate the oxidative stress in myocardial tissue.The results showed that,compared with the control group,the expression of 4-HNE in group AB was significantly increased.Compared with AB group,the expression of 4-HNE in DB group was significantly decreased,but still more than that in control group.The expression of4-HNE was decreased in ABQ group compared with AB group.The expression of 4-HNE in DBQ group was further reduced than that in DB group.8.Real-time PCR and Western blot were used to detect the m RNA and protein expressions related to indexes of oxidative stress.The results showed that compared with the control group,the m RNA levels of Nr F2,HO-1 and SOD2 in AB group were significantly decreased,while the m RNA levels of Nr F2,HO-1 and SOD2 in DB group were increased compared with AB group,but they were still lower than those in the control group.Compared with AB group,the m RNA levels of Nr F2,HO-1 and SOD2 in ABQ group were significantly increased in AB group.Compared with DB group,m RNA levels of Nr F2,HO-1 and SOD2 in DBQ group were significantly increased.Compared with the control group,the protein levels of Nr F2,HO-1,SOD2 and Catalase in the myocardial tissue of mice in AB group were significantly decreased,while those in DB group were increased compared with those in AB group,but still lower than those in the control group.In addition,compared with the control group,the expression of COX2 protein in AB group increased significantly,and the expression of COX2 protein in DB group decreased compared with AB group,but still higher than the control group.Compared with DB group,the protein expression levels of Nr F2,HO-1,SOD2 and Catalase in DBQ group were significantly increased,while the protein expression levels of COX2 were significantly decreased.Conclusion:TAC surgery was performed in C57BL/6J mice to establish pressure overload-induced cardiac hypertrophy model,The cardiac hypertrophy model was used to relieve the pressure overload after a second surgery,which safely and effectively simulated the TAVR surgery process of aortic valve stenosis and the pathological process of disease development.Qiliqiangxin capsule further reduces myocardial injury,improves cardiac fibrosis,reduces oxidative stress injury,reverse ventricular remodeling and improves cardiac function for mice after release of pressure overload.The underlying mechanism may be related to Nr F2/HO-1pathway.