Kaempferol Attenuates Cardiac Hypertrophy via Regulation of ASK1/MAPK Signaling Pathway and Oxidative Stress

• Abstract
• Introduction
• Results
• Discussion
• Material and Methods
• Animals and aorta banding surgery protocol
• Cardiac function with echocardiography
• Histological analyses
• Western blot
• Quantitative real-time polymerase chain reaction
• Reactive oxygen species, glutathione, oxidized glutathione, and superoxide dismutase assays
• Cell culture and immunofluorescence staining of H9c2 cardiomyocytes
• Reactive oxygen species determination of H9c2 cardiomyocytes
• Statistical analysis
• References

Abstract

Kaempferol has been demonstrated to provide benefits for the treatment of atherosclerosis, coronary heart disease, hyperlipidemia, and diabetes through its antioxidant and anti-inflammatory properties. However, its role in cardiac hypertrophy remains to be elucidated. The aim of our study was to investigate the effects of kaempferol on cardiac hypertrophy and the underlying mechanism. Mice subjected to aorta banding were treated with or without kaempferol (100 mg/kg/d, p. o.) for 6 weeks. Echocardiography was performed to evaluate cardiac function. Mice hearts were collected for pathological observation and molecular mechanism investigation. H9c2 cardiomyocytes were stimulated with or without phenylephrine for in vitro study. Kaempferol significantly attenuated cardiac hypertrophy induced by aorta banding as evidenced by decreased cardiomyocyte areas and interstitial fibrosis, accompanied with improved cardiac functions and decreased apoptosis. The ASK1/MAPK signaling pathways (JNK1/2 and p38) were markedly activated in the aorta banding mouse heart but inhibited by kaempferol treatment. In in vitro experiments, kaempferol also inhibited the activity of ASK1/JNK1/2/p38 signaling pathway and the enlargement of H9c2 cardiomyocytes. Furthermore, our study revealed that kaempferol could protect the mouse heart and H9c2 cells from pathological oxidative stress. Our investigation indicated that treatment with kaempferol protects against cardiac hypertrophy, and its cardioprotection may be partially explained by the inhibition of the ASK1/MAPK signaling pathway and the regulation of oxidative stress.

Introduction

HF remains a rising global epidemic problem across the world with more than 37.7 million patients [1]. Despite recent advances of therapy strategies for heart failure, the current 1-year mortality rate after diagnosis of symptomatic HF remains as high as 25 to 40 % [2]. Given the extremely low 5-year survival rate, the severity of symptomatic HF has been considered as comparative as malignant tumors [1], [2]. Cardiac hypertrophy, with alterations in size, geometry, shape, composition, and, finally, dysfunction of the heart, is a common pathophysiological process and a hallmark of varies cardiovascular diseases [3]. Mounting evidences from epidemiological, preclinical, and clinical studies have highlighted the maladaptive features of cardiac hypertrophy. Sustained pathological cardiac hypertrophy progressively leads to HF and effective suppression of it is essential for retarding the development to HF [3]. Thus, new strategies aiming at cardiac hypertrophy are of great importance in order to illuminate the future of HF patients.

MAPK signaling pathways are overactivated in pathological cardiac hypertrophy [4]. The intricate role of MAPKs has been indicated in different scenarios of cardiac hypertrophy [4]. Once the MAPK signaling cascades have been activated, the MAPK members (p38, JNK, and ERK) could phosphorylate a variety of downstream signaling targets, which initiate the process of cardiac hypertrophy [4]. Therefore, drugs targeting MAPK cascades may be useful candidates for the treatment of cardiac hypertrophy [5].

Kp, one of the most common dietary flavonoids, is a polyphenol antioxidant which could be isolated from a vary of fruits and vegetables including tea, broccoli, apples, strawberries, and beans [6]. Substantial studies have demonstrated that Kp possesses multiple beneficial functions including antioxidative, antidiabetes, and antitumor. During 12 years of follow-up, people who have a high intake of Kp have a relative low morbidity and mortality of CHD [7]. In an isolated rat heart model of I/R, Kp could improve cardiac function and inhibit cardiomyocyte apoptosis [8]. In a recently published research article, Kp prevented the rat heart from I/R injury and improved cardiac performance. Kp exerts its protection function by suppressing the p38/JNK/TNF- α/NF-kBp65 pathway, but increasing the phosphorylation of ERK1/2 [9]. Taken together, Kp may be an effective drug for heart disease through regulating MAPK signaling pathways.

However, it remains to be investigated whether Kp can prevent the heart from pressure overload-induced cardiac hypertrophy. In this study, we used an AB mouse model in vivo and PE-treated H9c2 in vitro to examine the effectiveness of Kp in the treatment of cardiac hypertrophy.

Results

In order to determine the effect of Kp on cardiac hypertrophy, AB surgery was conducted by descending aorta ligation in male C57BL/6 mice. Kp (dissolved in saline) or saline were administrated by intragastric administration on AB and CON mice, respectively, 3 days after surgery. At the end of 6 weeks of Kp treatment, systolic and diastolic functions of mice subjected to pressure overload were improved compared to the AB group ([Fig. 1]), while there was no significant difference between the CON and Kp groups.

The inhibitive effect of Kp on cardiac hypertrophy was also evidenced by the decreased ratios of HW/BW, HW/TL ([Table 1]), and the cross-sectional area of cardiomyocyte ([Fig. 2 A–C]). Additionally, RT-PCR was used to further examine the change of hypertrophic indicators and the result was in accordance with echocardiography and morphology findings. Specifically, an obvious decrease of ANP, BNP ([Fig. 2 D, E]), α-MHC, and β-MHC (Fig. S1 A, B, Supporting Information) was observed in the Kp + AB group compared to the AB group.

The Masson trichrome stain was used to analyze the effect of Kp treatment on cardiac fibrosis. As shown in [Fig. 3 A, B], a marked increase of interstitial fibrosis was observed in AB mice compared with that of the control group and this was significantly attenuated with Kp treatment. The fibrosis associated markers, including fibronectin, CTGF, and collagen I and III, were obviously upregulated but blocked by Kp treatment ([Fig. 3 C]). The TGF-β/Smad signaling pathway plays key roles in the development of cardiac fibrosis. Western blotting analysis revealed that Kp treatment blocked the activation TGF-β, Smad3, and Smad1/5 in mice that suffered from pressure overload ([Fig. 4 A, B]). Additionally, cardiac fibrosis of normal mice with or without Kp administration did not differ significantly.

To explore the underlying mechanism, we examined the total and phosphorylated protein change of the MAPK pathway. Pressure overload induced a high activation of the MAPK pathway, and Kp treatment, interestingly, only inhibited the phosphorylation of JNK1/2 and P38, without affecting ERK phosphorylation ([Fig. 5]). Therefore, Kp treatment was considered to modulate a common upstream of JNK1/2 and P38. Through Western blot analysis, we found Kp treatment downregulated the phosphorylation level of ASK1, a mediator for both JNK1/2 and P38.

Immunochemistry exams were conducted to evaluate the expression of 4-HNE in heart tissue. A significant increase was observed in mice that suffered from pressure overload and this was largely abolished by Kp treatment ([Fig. 6 A, B]). Moreover, oxidative stress-related assay kits were used to determine the change of the GSH/GSSG ratio and SOD in the myocardium. In the AB group, the GSH/GSSG ratio and SOD level were markedly decreased when compared to the control group, indicating a situation of exaggerated oxidative stress induced by pressure overload ([Fig. 6 C, D]). The downregulation of these antioxidative enzymes was significantly cancelled by Kp treatment ([Fig. 6 C, D]). These provide further evidence of cardioprotective effects of Kp through regulating oxidative stress.

To further validate the efficacy of Kp treatment on cardiac hypertrophy, H9c2 cardiomyocytes were treated with PE for 48 h. As shown in [Fig. 7 A, B], Kp treatment clearly prevented the increase of cell surface area of H9c2 cardiomyocytes caused by PE. Coinciding with in vivo studies, the ASK1/MAPK signaling pathway activated by PE was inhibited by treatment with Kp in vitro ([Fig. 7 C, D]). We also tested the reactive oxygen species (ROS) level during in vitro experiments. Firstly, through observing fluorescence intensity directly under a microscope, we found that after treatment with PE (50 µM) for 3 h the fluorescence intensity enhanced significantly, while treatment with Kp markedly decreased the fluorescence intensity (Fig. S1 C, Supporting Information). Then, in order to quantify the ROS level, we collected the H9c2 cells for performing flow cytometry and the results ([Fig. 8 A, B]) showed PE significantly stimulated the accumulation of ROS in H9c2 cells but Kp efficiently blocked the production of ROS. Finally, we also determined the change of the GSH/GSSG ratio and SOD ([Fig. 8 C, D]) in the H9c2 cell lysis, which once again demonstrated that Kp can effectively antagonize the oxidative stress induced by treatment with PE in vitro.

ASK1 and MAPK (JNK1/2 and p38) regulated the downstream apoptosis and inflammation. To provide evidence to the inhibitive function of Kp on the ASK1/MAPK signaling pathway, we detected the downstream of this signaling pathway such as Bcl2, Bax, and NFκB/P65. Apoptotic-related proteins, including Bcl2 and BAX, of in vitro and in vivo experiments were analyzed by Western Blot. As shown in Figs. S2 and S3, Supporting Information, Bcl2, an antiapoptotic protein, was downregulated under pressure overload or PE stimuli, and Kp treatment effectively prohibited this change. Meanwhile, Kp treatment blocked the increase of BAX, a proapoptosis protein induced by AB or PE. The phosphorylation of P65 increased by AB or PE was also inhibited by the treatment of Kp (Figs. S2 and S3, Supporting Information).

Discussion

Our investigation demonstrated that Kp alleviates cardiac hypertrophy both in vivo and in vitro. The underlying mechanism might be related to the inhibition of inflammation, apoptosis, and interstitial fibrosis through blunting the ASK1/MAPK signaling pathway and oxidative stress. In in vitro experiments, we further confirmed that Kp treatment showed cardiomyocytes protective effects by attenuating oxidative stress and modulating the ASK1/MAPK signaling pathway in PE-treated H9c2 cells.

Several published studies have showed that Kp could be a potent candidate for cardiovascular disease therapies [10]. In a recent study, Suchal et al. [9] reported that Kp treatment protected the heart from I/R injury by suppressing the JNK/P38 signaling pathway and activating the ERK signaling pathway. It has been demonstrated that hyperactivity of MPAKs (JNK, P38, ERK) signaling pathways play critical roles in the development of cardiac hypertrophy that resulted in transforming to malignant heart failure [4], [5]. The data from our study showed that Kp administration can inhibit the activation of the JNK1/2 and p38 signaling pathways but has no significant impact on the ERK1/2 signaling pathway, which is different from the results of a previous investigation [9]. This divergent between the two studies might be partly explained by the different hemodynamic, cellular, and molecular changes between acute ischemia injury [11] and chronic pressure overload [3]. Besides, Kp might target specific substrates answering to different stimuli. Our experiment also implied that Kp might target a common upstream target of p38 and JNK1/2. Previous published data have showed that ASK1 is a major upstream regulator of p38 and JNK1/2 [10], [12], [13]. In this study, we firstly showed that Kp could effectively blunt the phosphorylation of ASK1. So, it was obviously that Kp protects the mouse heart from hypertrophy partly through the regulation of the ASK1/MAPKs (JNK/P38) signaling pathway.

Overactivation of ASK1 in cardiomyocytes aggravated apoptosis, resulting in maladaptive left ventricle remodeling induced by pressure overload [14]. Mice with a global deficiency of AKS1 showed markedly increased aldosterone-induced inflammation as evidenced by inhibition of macrophage infiltration and monocyte chemotactic protein 1 expression [15]. ASK1 could also mediate inflammation by inhibiting the activity of NF-kB. Chronic inhibition of ASK1 with a highly selective ASK1 inhibitor or genetic method [16] ameliorated the enlarged left ventricle with marked mitigation of cardiomyocyte apoptosis and fibrosis. In this study, we confirmed that Kp effectively blunted the ASK1/JNK/P38 signaling pathway, directly contributing to the downregulated expression of apoptosis and inflammation-related proteins.

Flavonoids have been considered owing potency to antagonize oxidative stress both in vitro and in vivo as the most active constituent [17]. Kp is one of the most common natural flavonoids and also possesses potent antioxidative stress properties [6]. In this study, we confirmed that Kp still exerted a strong antioxidative function under hypertrophic stimuli. Oxidative stress has long been demonstrated to play an important role in cardiac hypertrophy induced by pressure overload and neurohumor factors [18], [19]. Oxidative stress can also contribute to the activation of ASK1 by separating thioredoxin from ASK1 and its later overphosphorylation, followed by increased activity of downstream signaling pathways [20], [21]. However, results from studies regarding the effects of classical antioxidants such as vitamin C, vitamin E, or folic acid in combination with vitamin E have been frustrating since they did not present an ideal therapeutical effect [22]. The natural flavonoids, especially the ones isolated from dietary vegetables and fruits, such as Kp, resveratrol, and curcumin, may be better candidates in future basic and clinical investigations.

Cardiac fibrosis, an integral feature of cardiac hypertrophy, is characterized by the accumulation of collagen I/III and other extracellular matrix constituents [23]. ASK1 deficiency significantly attenuated interstitial fibrosis, perivascular fibrosis, and the enhancement of TGF-β and collagen deposition through the inhibition of inflammation [15]. TGF-β1/Smad is a well-established signaling pathway related to cardiac fibrosis [24]. The cross talk between the MAPK and TGF-β1/Smad has been demonstrated in several previous studies [25], [26]. Considering the potential inhibitory effects of Kp on the ASK1/MAPK (JNK1/2 and p38) signaling pathway and oxidative stress in vitro and in vivo mentioned above, it is not surprising that Kp can significantly decrease the interstitial fibrosis during the process of cardiac hypertrophy.

Finally, flavonoids contain multiple phenolic hydroxyls and easily undergo metabolism catalysis by hepatic and intestinal metabolic enzymes, resulting in low bioavailabilities [27]. But we think that it is obligatory and interesting to detect the function and underlying mechanism through oral administration because Kp is taken orally from our daily food. In this study, we indicated that oral administration of Kp seemed to be as effective as other manners, such as intraperitoneal administration or intravenous adminstration. Recently, Zheng L et al. [28] presented that kaempferol by oral administration was mostly biotransformed to kaempferol-3-glucuronide (K-3-G), kaempferol-7-glucuronide (K-7-G), and kaempferol-7-sulfate in plasma, and K-3-G represented the major metabolite. K-3-G has been shown to be the predominant form in plasma after ingestion of Kp [29]. So, it is likely that the inhibition effect of cardiac remodeling observed in this study may be derived from metabolites of Kp (especially K-3-G) or by an unknown indirect effect. Our investigation showed Kp treatment in vitro had a similar effect on the regulation of ASK1/MAPK signaling pathways and oxidative stress with its metabolite in vivo. It seems that the addition of glucuronide in Kp might not contribute to the different bioactivity of Kp. Kp is a natural flavonoid present in vegetables and fruits such as tea and wine [30]. Intake of these foods has been demonstrated to reduce cardiovascular disease [30], [31]. We thought that an investigation of the administration manner of Kp was very interesting and essential. In follow-up experiments, it may also be very interesting to investigate the sole metabolite bioactivity in vivo.

Material and Methods

Animals and aorta banding surgery protocol

Male C57BL/6 mice (age: 6–8 weeks; body weight: 23.5–27.5 g) were purchased from the animal center of Zhongnan Hospital of Wuhan University and maintained in the animal room at 22 °C with a 12-hour light/12-hour dark cycle. With approval from the Animal Care and Use Committee of Zhongnan Hospital of Wuhan University (Approval number: 2 016 026, date: 2016–05–08), all the procedures were performed in accordance with the Guideline for the Care of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). After a 1-week adaption to the environment, mice were randomly distributed into the CON or AB group, followed by 6 weeks of treatment with Kp (> 98 % purity; Shanghai Winherb Medical Science) or normal saline through intragastric administration at a dose of 100 mg/kg body weight/day (100 mg/kg/d), respectively. Previous studies gave Kp at a dose of 20–25 mg through intraperitoneal administration (i. p.) [9]. We prescribed Kp in this study by oral administration and the dose is 4 times higher than that of i. p. or intravenous administration. We also considered it to be safe at the dose of 100 mg/kg body weight/day for mice [32], [33]. Thus, the mice were allocated into four groups: CON (n = 10), Kp (n = 10), AB (n = 16), AB + Kp (n = 16).

A cardiac hypertrophy model was established by AB [34]. Concisely, mice were anesthetized with pentobarbital sodium (> 98 % purity; Shanghai Winherb Medical Science) through intraperitoneal injection, and a horizontal skin incision was made at the 2–3 intercostal space. Following the isolation of the descending aorta, a 24-gage (mice weight: 23.5 to 25 g, n = 20) needle was placed next to the aorta. Subsequently, a 7–0 silk suture was tied to band the aorta and the needle was quickly removed after ligation. Similar procedures without AB were performed with the mice from the CON group.

Cardiac function with echocardiography

Echocardiography measurements were performed in all experimental mice at baseline and the end of the study. In detail, mice were conscious and cardiac function was measured with a high-resolution Vevo 2100 System equipped with a 30 MHz MS400 linear array transducer (VisualSonics, Inc.). B-mode and M-mode images were obtained in the parasternal short axis view at the mid-papillary muscle level. The LVID; s, LVID; d, and wall thickness were measured from three to five consecutive cardiac cycles in M-mode images. According to the protocol from the manufacturer, LV Vol; d and LV Vol; s were measured digitally. Subsequently, the EF was calculated as [(LV Vol; d – LV Vol; s)/LV Vol; d] × 100 %. The FS was calculated as [(LVID; d – LVID; s)/LVID; d] × 100 %.

Histological analyses

At the end of our study, mice were sacrificed by cervical dislocation. Hearts were harvested and arrested in diastole with 10 % KCl for a good morphological analysis. After being fixed with 10 % formalin, hearts were embedded in paraffin and cut transversely in sections of 4–5 mm. To identify cardiomyocyte CSA, HE staining was applied and followed by tracing the outline of a single myocyte with Image Pro 6.0 (Media Cybernetics). In each group, independent hearts (n = 5) were performed with the abovementioned procedures and at least 40 myocytes of each independent heart were randomly selected and traced. As for collagen deposition, tissue sections were stained with Masson-trichrome staining. Collagen fibers were presented as blue, and pictures were obtained and analyzed with Image Pro 6.0. For each section, five independent fields were obtained and measured under a 20× objective lens. Collagen contents were quantified and averaged for all sections.

The heart paraffin slides from the different groups indicated above were incubated with 4-HNE (Alpha Diagnostic International) antibody at the concentration of 1 : 400 dilution for 12 h. After washing with PBS the next day, sections were incubated with horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. Finally, immunohistochemical staining for 4-HNE was accomplished by treatment with peroxidase substrate DAB and counterstaining with hematoxylin. The 4-HNE was presented in brown color and Image Pro 6.0 was used to calculate the results.

Western blot

Protein samples of each group were obtained by homogenizing left ventricle tissue in the RIPA lysis buffer. A bicinchoninic assay kit (Thermo Fisher Scientific) was applied for quantification of protein concentration. Protein samples of 50 µg were loaded and separated in SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore). After blocking with 5 % nonfat milk or bovine serum albumin (for detecting phosphorylated protein), the membranes were incubated overnight at 4 °C with primary antibodies for p-Smad1/5, p-Smad3, p-JNK1/2, JNK1/2, p-ERK1/2, ERK1/2, p-P38, P38, p-ASK1, ASK1, GAPDH, Bcl-2, BAX, P65, and p-P65 from Cell Signaling Technology and TGF-β from ABCAM at a concentration of 1 : 1000. Following washing three times with TBST (Tris buffered saline with Tween-20), membranes were incubated with the appropriate secondary antibody (all from Santa Cruz Biotechnology) at room temperature. Target proteins were obtained and visualized with an ECL (enhanced chemiluminescence) kit.

Quantitative real-time polymerase chain reaction

TRIzol reagent (Introvitrogen) was used to extract the total RNA from LV tissue and real-time polymerase chain reaction (PCR) was completed with the Transcriptor first Strand cDNA Synthesis Kit (Roche). For each sample, PCR amplifications of 20 µL were completed with a LightCycler 480 SYBR Green Master Mix (Roche Diagnostics GmbH) under specialized cycle conditions [95 °C/30 s + 40 × (95 °C/5 s + 60 °C/30 s) + 95 °C/5 s + 60 °C/1 min + 95 °C/5 s]. For hypertrophic evaluation, the mRNA level of ANP, BNP, α-MHC, and β-MHC was measured. The levels of fibronectin, CTGF, and collagens I and III were investigated in order to assess the cardiac fibrosis. The primers used in this study were shown in the table (Table S1, Supporting Information)

Reactive oxygen species, glutathione, oxidized glutathione, and superoxide dismutase assays

The GSH and GSSG Assay Kits, the Reactive Oxygen Species Assay Kit and the Total Superoxide Dismutase Assay Kit with WST-8 activity assay kits were purchased from Beyotime Co. The experiments were performed in cardiac tissue and H9c2 cardiomyocytes according to the manufacturerʼs instructions.

Cell culture and immunofluorescence staining of H9c2 cardiomyocytes

H9c2 cardiomyocytes (Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM (GIBCO) supplemented with 10 % (v/v) fetal bovine serum (GIBCO), penicillin (100 U/mL), and streptomycin (100 mg/mL) (GIBCO) in a humidified CO₂ incubator (NUAIRE) with 5 % CO₂ at 37 °C. Cells at exponential growth were dissociated with 0.25 % trypsin (GIBCO), seeded in a six-well plate (1 × 10⁶ cells/well) or twenty-four-well plate (3 × 10³ cells/well) and incubated for 24 h. Then cells were starved with serum-free DMEM for another 12 h before different treatments for the different assays, followed by Kp (25 µM) for 2 h before treatment with or without PE (50 µM). Protein was extracted from cells cultured in a six-well plate for Western blotting and determination of GSH, GSSG, and SOD.

For CSA determination of the in vitro experiment, after discarding culture medium, cells were washed three times with PBS and fixed with 4 % paraformaldehyde. 0.2 % Triton was used for membrane rupture and 8 % serum for blocking. Following incubation with 20 µL actin antibody overnight, goat anti-mouse secondary antibody was added before DAPI staining. Five random fields each containing five cells (four slides for each group) were traced and photographed. Finally, CSA from 100 cells in each group was calculated and averaged. Image-Pro Plus was applied to conduct analysis.

Reactive oxygen species determination of H9c2 cardiomyocytes

For determining ROS, H9c2 cells were seeded in six-well plates and treated with Kp (25 µM) for 2 h before treatment with or without PE (50 µM) for another 3 h. Then cells were collected and washed three times with PBS. After removing PBS, the collected cells were incubated with 10 µM of DCFH-DA for 30 min at 37 °C in the dark. The fluorescence density was analyzed by a flow cytometer (BD). Moreover, cells were seeded in 24-well plates. Instead of collecting the H9c2 cells described above, the DCFH-DA (10 µM) was directly added into the 24-well plates and incubation was performed for 30 min at 37 °C in dark. Then the fluorescence was observed directly under a microscope (Olympus).

Statistical analysis

All values are presented as the mean ± SEM. Data was analyzed with one-way ANOVA followed by a least significance difference (LSD) test. SPSS 13.0 (SPSS, Inc.) was used for data analysis and the result was considered statistically significant only when the p value was < 0.05.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81270304 and 81420108004).

^* Hong Feng and Jianlei Cao contributed equally to this work.

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• 31 Geleijnse JM, Launer LJ, Van der Kuip DA, Hofman A, Witteman JC. Inverse association of tea and flavonoid intakes with incident myocardial infarction: the Rotterdam Study. Am J Clin Nutr 2002; 75: 880-886
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• 32 Al-Numair KS, Veeramani C, Alsaif MA, Chandramohan G. Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats. Pharm Biol 2015; 53: 1372-1378
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• 33 Hoang MH, Jia Y, Mok B, Jun HJ, Hwang KY, Lee SJ. Kaempferol ameliorates symptoms of metabolic syndrome by regulating activities of liver x receptor-beta. J Nutr Biochem 2015; 26: 868-875
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• 34 Marano G, Ferrari AU. Surgical animal model of ventricular hypertrophy. Methods Mol Med 2007; 139: 95-104
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Correspondence

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  PubMedSearch in Google Scholar

• Supplementary Material