Orange Flavonoid Hesperetin Protects Against Cardiac Remodeling Induced By Pressure Overload In Mice

Background:Cardiac remodeling, which is caused by long-term cardiac overload caused by hypertension and other diseases, is a key factor of heart failure. In the initial stages, cardiac remodeling has compensatory function, but myocardial fibrosis may lead to cardiac dysfunction, which is characterized by cardiac hypertrophy, fibrosis, oxidative stress and apoptosis of myocardial cells. It has been reported that Akt, PKC and other signaling pathways were involved in various processes of cardiac remodeling. There are a variety of drugs in clinical treatment of heart failure in the processes of cardiac remodeling. Hesperetin, which belongs to citrus flavonoids, is mainly expressed in flavonoids. It has several pharmacological properties, such as anti-inflammatory, anti-tumor and so on. Hesperetin have some antioxidant and anti-apoptotic function, however, the role of hesperetin in cardiac remodeling remains elusive. So we study the effect of hesperetin on the process of cardiac remodeling, including myocardial hypertrophy, fibrosis, oxidative stress and cardiac myocyte apoptosis, by aortic banding (AB).Methods: Wild-type C57BL/6mice (8-10weeks old male) were randomly were randomly assigned to four groups: vehicle-sham (n=10), hesperetin-sham (n=9), vehicle-AB (n=10) and hesperetin-AB (n=10). Aortic banding (AB) was performed as described previously. Then,1week after AB or surgery, the animals were treated with30mg/kg/day of hesperetin or vehicle for7weeks. We mainly study cardiac remodeling process and molecular mechanisms underlying the case through the following indicators:A. cardiac hypertrophy:1. detected different groups of mice heart rate and IVSd, LVDd, LVPWd, IVSs, LVDs, LVPWs by echocardiography;2. monitored mice in each group LVEF, LVFS, ESP and dP/dT max by hemodynamic apparatus;3. investigated mice heart weight (HW), lung weight (LW), heart weight ratio (HW/BW) and heart weight rib diameter ratio (HW/TL) by anatomical studies;4. studied cardiac volume and cardiac myocyte cross-sectional area by using WGA staining and HE staining;5. studied mRNA expression of ANP, BNP, a-myosin heavy chain (a-MHC) and other proliferative marker by using real-time quantitative PCR6. investigated the protein expression of mice heart PKC-Akt and MAPK signaling pathways signaling molecules by western blot studiesB. myocardial fibrosis:1. studied myocardial fibrosis mice in each group situations using PSR staining;2. detected the expression of cardiac fibrosis media by real-time quantitative PCR3. detected expression of Smad proteins cascade related protein using western blotC. Oxidative StressDetected mRNA expression mouse myocardium oxidase subunit by real-time quantitative PCRD. Apoptosis1.detected myocyte apoptosis using TUNEL assay2.detected the expression of Smad proteins cascade and cleavaged PARP, BAX, BCL-2and other apoptosis-related protein by western blot;Results:Part I:cardiac hypertrophy1. Aorta banding significantly increased heart rate. The heart rate of vehicle-sham group is518±13.34, while heart rate increased to522.38±11.90in vehicle-AB group. There is no significant difference between hesperetin-AB group and vehicle-sham group;2. LVEF, LVFS significantly reduced in vehicle-AB mice;3. HE sections showed that heart volume and cross-sectional area of myocardial cells increased in vehicle-AB group, and hespertin can significantly reduce increasement of the volume and cardiac myocyte cross-sectional area, which is caused by AB surgery;4. Real-time quantitative PCR indicated that myocardial proliferation markers,such as ANP mRNA, was significantly increased after AB surgery, hesperetin can significantly reduce cardiac markers proliferation;5. Western blot results showed that in mice with chronic pressure overload induced the activation of myocardial PKCa/β Ⅱ-AKT and JNK signaling pathway, and hesperetin can reduce the activation of these two signaling pathways. Part II:Myocardial fibrosis1. PSR staining showed that AB surgery-induced left ventricular pressure overload increased areas of fibrosis, collagen volume and content. Hesperetin significantly reduce this phenomenon;2. Real-time quantitative PCR results showed that chronic pressure overload increased mRNA expression of myocardial tissue fibrosis media such as TGF-β1. Hespertin can significantly reduce the expression of these fibrosis medium;3. Western blot results showed that pressure overload significantly increased phosphorylation of Smad2and Smad3expression. While compared with the AB, hesperetin significantly reduced phosphorylation levels of Smad2and Smad3.Part III:First,Oxidative stressReal-time quantitative PCR results showed that chronic pressure overload increased mRNA expression of NADPH oxidase subunit, reduced mRNA expression of SOD1and SOD2, whereas hesperetin can reduce the mRNA expression of NADPH oxidase subunit increased mRNA expression of SOD1and SOD2.Second, Apoptosis1. TUNEL results showed that chronic pressure overload increased myocardial apoptosis and hespertin can reduce cardiac myocyte apoptosis;2. Western blot experiments showed that chronic pressure overload significant increased protein expression of cleavaged PARP, BAX, BCL-2, BCL-XL, Bak and cleaved caspase-3, and hesperetin can inhibit the protein expression of cleavaged PARP, Bax/Bcl-2, Bak and cleaved caspase-3, etc.Conclusions:We found that orange flavonoid hesperetin protected against cardiac hypertrophy, fibrosis, apoptosis and dysfunction induced by aortic banding. These findings possible exploit a potential therapeutic drug to cardiac remodeling and heart failure.