Naringenin Protects Rats against Ang-II Induced Cardiac Hypertrophy and Fibrosis by Downregulating TGF-β1/Smads Signaling Pathways

Background:Naringenin (Nrg), a avone found in several plant foods with various biological properties, has been shown prevention of cardiac remodeling. However, themechanisms underlying this suppression of cardiac remodeling has not been known clearly. Methods: Male Sprague Dawley (SD) rats were AngII infused via osmotic minipumps for 4 weeks and were given Nrg by gavage (100mg/kg/day) at the same time. In vitro experiments used cardiomyocyte and cardiac broblasts(CF) treated with AngII or AngII plus Nrg.Cardiac remodeling was assessed using the echocardiography and histological analysis. And, the effect of Nrg on TGF-β1/Smadssignaling pathway was investigated. Results: Treatmentwith Nrg(100mg/kg/day) decreased the ratio of heart weight to tibia length and hypertrophy markers in rats given AngII infusion. In vitro experiments demonstrated that AngII-induced cardiomyocyte hypertrophy and proliferation of CFs were signicantly inhibited by Nrg administration. Nrg inhibited activation of the TGF-β1/Smad2/3 signaling pathway stimulated by AngII. Conclusions: Nrgsupplementation prevented cardiac remodeling via down-regulating the TGF-β1/Smad2/3 signaling pathway both in cardiomyocyte and CFs, and attenuating cardiac remodeling in AngII-induced rats model. potential role of TGF-β1 signaling pathway in Nrg inhibition of Ang II-induced cardiac remodeling in vitro and in vivo. Our results showed that Nrg markedly improved AngII-induced cardiac hypertrophy and brosis through inhibiting the TGF-β1 signaling pathway. Thus, our result suggest that Nrg might play a protective role in AngII-mediated cardiac remodeling by targeting the TGF-β1 signaling pathway. hypertrophy.


Introduction
Cardiac remodeling is characterized by cardiac hypertrophy and brosis, which has been recognized as a key determinant of clinical outcome in heart disease [1]. Angiotensin II (AngII) is a crucial regulator of cardiac remodeling through inducing cardiomyocyte hypertrophy and proliferation and migration of cardiac broblasts (CFs). Transforming growth factor-β1 (TGF-β1) has been identi ed as a key regulator of extracellular matrix synthesis and degradation, which is believed to partially mediate AngII-induced cardiac remodeling [2]. Naringenin (Nrg) is a avonoid compound found in several plant foods including citrus fruit, tomatoes and gs. Nrg has been identi ed as a potential therapeutic agent as it demonstrates anticancer [3], antiin ammation [4], anti-atherogenic [5] and antimicrobial [6] effect. Previous studies have reported that Nrg ameliorates cardiac hypertrophy induced by high glucose [7] [8] and pressure overload [9]. Although Nrg inhibits TGF-β1 signaling and the subsequent Smad3 phosphorylation for the downstream signal transduction [10], whether Nrg modi es AngII-induced cardiac remodeling through TGF-β1/Smad signaling pathway remains elusive. This study therefore explored the possible prevention by Nrg of cardiac remodeling in vivo, using the AngII-induced rat model, and in vitro on cardiomyocytes and CFs stimulated by AngII plus Nrg.

Animal
Male 8-week-old male Sprague Dawley (SD) rats (150-180 g body weight) were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). All experiments involving rats were approved by the Institutional Animal Care Research Advisory Committee of the National Institute of Biological Science (NIBS) and Animal Care Committee of Zhengzhou University. All rats were kept under a 12-hr light/dark cycle with free access to water and food.

Experimental Design And Treatment Protocol
A rat model of AngII infusion induced cardiac remodeling was established as described previously [11]. In brief, SD rats were quickly anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), then the pre lled osmotic minipumps (Alzet, Model 2002) were implanted into the subcutaneous tissue to deliver AngII (Sigma-Aldrich, A9525) at 400 ng/kg/min for 4 weeks. Rats were randomly assigned to one of the following groups: the sham group (n = 10) received subcutaneous injections of phosphate buffer (PBS) for 4 weeks; the AngII group (n = 10) received subcutaneous injections of AngII (400 ng/kg/min) via osmotic minipumps; the AngII + Nrg group (n = 10) received naringenin by gavage (100mg/kg/day) plus the daily injections of Ang as above.

Echocardiographic Study
Transthoracic echocardiography was used to measure left ventricular (LV) function variables one day before killing. Brie y, rats were placed in a supine position after the induction of general anesthesia. Rats were underwent transthoracic two dimensional guided M-mode echocardiography with a 12L MHz transducer (Sibiscape Co. Ltd.). From the cardiac short axis, the LV anterior wall end-diastolic thickness (LVAWd), the systolic LV anterior wall thickness (LVAWs),the LV internal dimension at end-diastole (LVIDd), the LV internal dimension at end-systole (LVIDs), the LV posterior wall end-diastolic thickness (LVPWd), the LV posterior wall end-systolic thickness (LVPWs) were measured. Echocardiographic measurements were averaged from at least three separate cardiac cycles.

Heart Histological Analysis
The left ventricle were xed in 10% formalin and embedded in para n, and subsequently were sectioned at 4µm and stained with Masson to evaluate the cardiac collagen deposition. To evaluate the size of cardiomyocytes, tissue sections were stained with 1.0 mg/ml Alexa Fluor 488® conjugate of wheat germ agglutinin (WGA) solution (MolecularProbes, Eugene, OR, USA). Ten elds in each region of the heart were selected randomly from four nonconsecutive serial sections, and collagen content was quanti ed by measuring the total blue area per square millimeter using the ImageJ.

Neonatal Rat Ventricular Cardiomyocytes Isolation, Culture And Treatment
Neonatal rat ventricular cardiomyocytes and CFs were obtained from the hearts of 1-2 days old SD rats as described previously [12]. In brief, the ventricles of neonatal rats were harvested after killed by decapitation, and then were cut into ~ 1mm 3 pieces in a dish with cold PBS. 0.125% and 0.05% collagenase type I were used to dissociate cardiomyocytes and broblasts. Cells were cultured in DMEM with 15% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidi ed atmosphere of 5% CO 2 and 95% air at about 37℃. Naringenin were dissolved in dimethyl sulfoxide (DMSO) and diluted with DMEM. Cells were incubated with Nrg (0.1, 1, 10µM) with or without AngII 10uM for 24 h in a 6 well plate.
Cell surface area analysis was performed using confocal microscopy as described previously [13].

Methylthiazolyl Tetrazolium Assay For Cell Viability
Cells were cultured at a density of 4-5 x 10 4 cells per well in 96-well plates for 24 h. The cells were treated with different concentrations of Nrg for 24 h. And then cell viability was determined by the MTT reduction assay. Cells were incubated with MTT solution (5 mg/mL) for 4 h at 37°C. The dark blue formazan crystals that formed in intact cells were solubilized with 150 µL of DMSO, and the absorbance at 490 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA).

Western Blotting
The heart tissues or cells were lysed by RIPA lysis buffer and the protein concentration was detected by using a BCA protein assay kit. Protein (30µg) were separated using 10% SDS-PAGE and then were transferred onto a polyvinylidene di uoride membrane (PVDF). Next, PVDF membranes were blocked with 5% fat-free milk and incubated with primary antibodies overnight at 4℃. Subsequently, the membranes were washed and incubated with secondary antibodies at room temperature. The optical density of the bands was visualized by an ECL system (Pierce). GAPDH was used as an endogenous control. Data was normalized to GAPDH levels.

Rna Isolation And Quantitative Real-time Pcr
Total RNA was extracted from the frozen tissues or cultured cells using Trizol reagent (Invitrogen, USA).
First strand cDNA was synthesized using an RT kit (Invitrogen, USA). qPCR analysis were performed in a MiniOpticon Real-Time PCR Detection System (BioRad Laboratories, USA). Results were expressed as fold differences relative to GAPDH using the 2-ΔΔCT method. All the primers were synthesized by Sangon Biotech (Shanghai, China) and the sequence are listed in Table 1.

Results
Nrg alleviated AngII-induced proliferation and collagen expression of CFs Firstly, we detected the cytotoxicity of Nrg on CFs. The results of methylthiazolyl tetrazolium assay showed that Nrg had no cytotoxic effects on CFs at concentrations less than 200µM (Fig. 1A). Thus, in the following experiments, Nrg concentrations of 200µM were chosen. Then, we examined whether Nrg could inhibit AngII induced proliferation of CFs. CFs were pretreated with Nrg (200µM) following with AngII (0.1µM) for 72h. Our results demonstrated that Nrg could inhibit AngII-induced proliferation of CFs in a concentration-dependent manner (Fig. 1B). Then we examined the effect of Nrg on collagen expression in cardiac broblasts (CFs). Following AngII administration, the brotic markers α-SMA and Col1a1 gene expression were increased in CFs, and Nrg treatment prevented AngII induced CF collagen expression ( Fig. 1C and D

Nrg Ameliorated Angii-induced Cardiac Hypertrophy
Here we analyzed cardiac effects of Nrg treatment in an animal model of AngII-induced cardiac hypertrophy in rats. AngII infusion rats showed a signi cant increase in the ratio of weight/tibia length (HW/TL), and the cell size of cardiomyocytes ( Fig. 3A and D). Examination by echocardiography revealed that the thickness of the left ventricular post wall at the end-diastole (LVPWd) and the end-systole (LVPWs) was higher in AngII infusion rats ( Fig. 3B and C). Compared with AngII group, Nrg treatment markedly ameliorated AngII-induced cardiac hypertrophy, as demonstrated by a signi cantly decrease in HW/TL, cardiomyocyte size, the thickness of the left ventricular post wall at the end-diastole (LVPWd) and the end-systole (LVPWs) (Fig. 3A, B, C and D). Meanwhile, AngII infusion induced increased protein levels of ANP and β-MHC, while their expression was inhibited in Nrg-treated rat (Fig. 3E).

Nrg Attenuated Angii-induced Cardiac Fibrosis
To determine the effect of Nrg on cardiac brosis, heart sections were stained with Masson's staining. Quantitative data revealed increased collagen deposition in AngII-induced rats, while was signi cantly attenuated in Nrg-treated rats (Fig. 4A). As showed in Fig. 4B, AngII infusion induced a signi cant increase in protein levels of α-SMA and Col I, and Nrg treatment reversed cardiac brosis as evidenced by a decreased in collagen deposition and α-SMA and Col I protein level (Fig. 4A and B). Collectively, Nrg treatment can attenuated AngII-induced cardiac hypertrophy and brosis.
Suppression of TGF-β1/Smad2/3 signaling contributes to the anti-hypertrophy effect of Nrg Furthermore, we explored the mechanism underlying the anti-hypertrophy effect of Nrg. As shown in Fig. 5A, the expression of TGF-β1 and phosphorylated Smad2/3 were increased in AngII-induced rat model, which could be attenuated by treatment with Nrg. In cultured cardiomyocytes and cardiac broblasts, AngII induced upregulation of TGF-β1 and phosphorylated Smad2/3 ( Fig. 5B and C), and Nrg treatment inhibited TGF-β1/Smad2/3 signaling as evidenced by attenuating protein expression of TGF-β1 and phosphorylated Smad2/3 ( Fig. 5B and C).

Discussion
Cardiac remodeling is a major driving force in the development and progression of cardiovascular diseases including cardiac hypertrophy, heart failure and myocardial infarction. However, no therapeutic intervention directly targets the brotic response. A better understanding of the mechanisms underlying cardiac remodeling is important for developing more effective diagnostic and therapeutic strategies. In the present study, one of the important ndings is that Nrg treatment markedly improved AngII-induced cardiac hypertrophy and brosis through downregulating TGF-β1 signaling pathway.
AngII, the main effector peptide of renin-angiotensin system (RAS), has been shown to induce TGF-β1 expression and its subsequent signaling and mediates cardiac remodeling [14][15][16]. Pharmacological inhibition of angiotensin converting enzyme and AngII receptor have shown their therapeutic effects for cardiac remodeling [17]. Our previous study also demonstrated that suppressing TGF-β1/Smads signaling pathway inhibits cardiac brosis and improves cardiac function [18]. Nrg, a natural avanone with many pharmacological effects, has been proved to reduce Smad3 phosphorylation and expression in the presence of TGF-β1 [19], and exerted anti-brosis effect [20]. In this work, we therefore attempted to characterize the potential role of TGF-β1 signaling pathway in Nrg inhibition of Ang II-induced cardiac remodeling in vitro and in vivo. Our results showed that Nrg markedly improved AngII-induced cardiac hypertrophy and brosis through inhibiting the TGF-β1 signaling pathway. Thus, our result suggest that Nrg might play a protective role in AngII-mediated cardiac remodeling by targeting the TGF-β1 signaling pathway.
However, it remains obscure what's the inhibition mechanism of Nrg on the AngII-TGF-β1 signaling pathway. One possible explanation is that Nrg can reduced the binding probability of TGF-β1 to its speci c TGF-β1 type II receptor (TβRII). TGF-β1 binding to TβRII is the initial step of TGF-β signaling.
Thus the effect of Nrg on TGF-β ligand-receptor interaction induced inhibition of the receptor dimerization and activation for the signaling complex formation and the subsequent Smad3 phosphorylation for the downstream signal transduction [10].

Conclusions
In present study, we arrived at a conclusion that Nrg targeting TGF-β1/Smad signaling pathway, and promoted cardiac hypertrophy and brosis in AngII-induced rats. Our study supports the notion Nrg has the potential to be developed as a novel inhibitor target for TGF-β signaling, and might be considered as potential prevention strategy for cardiac hypertrophy and brosis.
Our results might help to deepen the understanding of the role and function of TGF-β signaling in cardiac hypertrophy. These ndings offer important insights into fundamental mechanisms underlying functions of Nrg, meanwhile, would provide a potential therapeutic targets for cardiac hypertrophy.

Availability of data and materials
All data generated of analyzed during this study are included in this published article or are available from the corresponding author on reasonable request.
Author's contributions HDL designed the study; XWC and XZ conducted the experiments; HW did sample analysis and data analysis, HDL wrote the manuscript; HDL revised the paper. All authors read and approved the nal manuscript.
Ethics approval and consent to participate This study was approved by the ethics committee of Zhengzhou University.