Galangin ameliorates cardiac remodeling via the MEK1/2–ERK1/2 and PI3K–AKT pathways

Abstract Cardiac remodeling is associated with inflammation and apoptosis. Galangin, as a natural flavonol, has the potent function of regulating inflammation and apoptosis, which are factors related to cardiac remodeling. Beginning 3 days after aortic banding (AB) or Sham surgery, mice were treated with galangin for 4 weeks. Cardiac remodeling was assessed according to echocardiographic parameters, histological analyses, and hypertrophy and fibrosis markers. Our results showed that galangin administration attenuated cardiac hypertrophy, dysfunction, and fibrosis response in AB mice and angiotensin II‐treated H9c2 cells. The inhibitory action of galangin in cardiac remodeling was mediated by MEK1/2–extracellular‐regulated protein kinases 1/2 (ERK1/2)–GATA4 and phosphoinositide 3‐kinase (PI3K)–protein kinase B (AKT)–glycogen synthase kinase 3β (GSK3β) activation. Furthermore, we found that galangin inhibited inflammatory response and apoptosis. Our findings suggest that galangin protects against cardiac remodeling through decreasing inflammatory responses and apoptosis, which are associated with inhibition of the MEK1/2–ERK1/2–GATA4 and PI3K–AKT–GSK3β signals.

essential regulator of the hypertrophic response. ERK1/2 activation via phosphorylation of serine/threonine residues is triggered by MAPK/ERK kinase 1/2 (MEK1/2; Cai et al., 2009). Protein kinase B (AKT) is a serine/threonine kinase involved in the regulation of cardiac growth, proliferation, and migration. Activated-phosphoinositide 3kinase (PI3K) promotes the activation of AKT, directly contributing to the process of cardiac remodeling (J. Li et al., 2017). Overexpression of activated AKT or ERK1/2 contributes to cardiac remodeling, whereas AKT or ERK1/2 knockout mice exhibit inhibition of the intracellular transduction cascades associated with cardiac remodeling.
In recent years, many studies have focused on the beneficial effects of flavonoids for alleviating cardiac remodeling. Flavonoids typically have less toxicity and side effects than chemically synthesized drugs . Galangin (3,5,7-trihydroxyflavone or 3,5,7-trihydroxy-2phenylchromen-4-one) is a natural flavonol, which is abundant in honey and Alpinia officinarum (a plant that has been used as a kind of herbal medicine for multiple ailments in Asia for centuries ).
A series of related studies have demonstrated that galangin possesses several biological activities, including antioxidant (Aloud, Veeramani, Govindasamy, Alsaif, & Al-Numair, 2018), anti-inflammatory and antiapoptosis , anticancer (Y. Wang et al., 2017), and antifibrotic activities (X. Wang et al., 2013). However, the effects of galangin on cardiac remodeling and the potential signaling mechanisms have not yet been elucidated. The aim of this study is to determine whether galangin can attenuate cardiac remodeling induced by pressure overload in vivo and in Ang II cultured-H9c2 cells in vitro, as well as to identify the mechanisms involved in these effects.

| Animals and treatments
Eight-week-old male C57/BL6 mice (23.5-25.5 g) were purchased from the Institute of Laboratory Animal Science, CAMS&PUMC (Beijing, China). The animals were housed in a specific pathogen-free barrier with controlled temperature and humidity. The experimental procedures were approved by the Institutional Guidelines of the Animal Care and Use Committee of Renmin Hospital, which is compliant with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 2011). The diet was based on commonly used diets. Aortic banding (AB) and the corresponding Sham operation were performed in 60 and 30 mice, respectively, after acclimatizing them to the laboratory environment for 1 week as described in previous articles (Ma et al., 2018). The AB operation and data analyses were performed in a blinded fashion for all groups.
Three days after the AB or Sham operation, the animals were treated with the same volume of vehicle (0.5% carboxymethyl cellulose solution) or galangin (5, 25, and 50 mg/kg body weight/day, suspended in 0.5% carboxymethyl cellulose solution) daily for 4 weeks after surgery. At the end of the treatment, the mice were killed by cervical dislocation, and the hearts were dissected and weighed to compare heart weight/body weight (HW/BW, mg/g), lung weight/body weight (LW/BW, mg/g), and heart weight/tibia length (HW/TL, mg/mm) among the six groups.

| Echocardiography
Mice were anesthetized by 1.5% isoflurane. Echocardiography was used to detect cardiac function in each group of mice, which was performed with a MyLab 30CV system (Biosound Esaote, Inc.) with a 10-MHz phased array transducer. Two-dimensional guided M-mode echocardiographic images were obtained at the papillary muscle. End-diastole and end-systole were defined as the phases in which the largest and smallest areas of the LV were obtained, respectively. The LV end-systolic diameter (LVED), LV end-diastolic diameter (LVEDd), and posterior wall thickness (PWT) were measured via LV M-mode tracing with a sweep speed of 50 mm/s. LV-ejection fraction (EF) and fractional shortening (FS) were calculated by the LVEDs and LVEDd values.

| Histological analysis
The arrested hearts were placed in 10% potassium chloride solution immediately to ensure that they were stopped in diastole, and fixed with 10% formalin. The hearts were cut transversely to visualize the LVs and right ventricles and embedded in paraffin. Sections of each heart (5-μm thick) were prepared and stained with hematoxylin and eosin for the evaluation of cross-sectional areas (CSAs) or 0.1% Picro-Sirius red (PSR) for the evaluation of collagen deposition. Subsequently, the slides were visualized by light microscopy (ECLIPSE 80i; Nikon, Japan). Image-Pro Plus version 6.00 (IPP6.0) was used to view the myocardial crosssectional area, to trace a single myocyte (150-200 myocytes in each group), and to measure collagen content.

| Immunofluorescence staining
To identify the H9c2 cells and assess the levels of H9c2 cells hypertrophy, the cells were characterized using immunofluorescence staining for the marker α-actinin (05-384; Millipore). For this experiment, the cells were washed for 3 × 5 min with phosphate-buffered

| Statistical analysis
All values are presented as the means ± standard error of the means and were analyzed by GraphPad Prism 5.0 software (GraphPad Software). One-way analysis of variance followed by post hoc Tukey's test was used for data analysis. Comparisons between two groups were performed by unpaired, two-tailed Student's t tests. A value of p < 0.05 was considered statistically significant.

| Galangin attenuated cardiac hypertrophy and improved cardiac function in vivo
To explore the effects of galangin on cardiac hypertrophy in a murine model, we established an AB-mediated pressure overload model.

| Galangin attenuate cardiac fibrosis in vivo
The extent of cardiac interstitial fibrosis was determined, as indicated by increased-LV collagen volume (evaluated by PSR) and fibrotic markers. After 4 weeks of AB, dramatic cardiac perivascular and interstitial fibrosis were observed in all AB surgery mice, but the extent of fibrosis was markedly reduced in middle-dose (25 mg/kg/day) and high-dose (50 mg/kg/day) galangin-treated mice (Figure 2a

| Galangin attenuate inhibited cardiomyocyte apoptosis
TUNEL assay was performed to assess the Ang II-induced-cell apoptosis in cultured-H9c2 cells. Only 1.255 ± 0.221% and 1.021 ± 0.178% TUNEL-positive nuclei were, respectively, detected in the Con and Con + Gal group cells. A significantly increased percentage of TUNEL-positive nuclei was observed in cells incubated with Ang II (6.240 ± 0.471%; p < 0.001 vs. the control group); however, galangin treatment significantly reduced the percentage of TUNEL-positive cells (4.610 ± 0.376%; p < 0.01 vs. the Ang II-only group; Figure 4a,b). We subsequently investigated the expression of the apoptosis-related protein Bax and Bcl2 in vitro and in vivo. We found that Ang II treatment or AB surgery significantly upregulated proapoptotic protein Bax, and downregulated antiapoptotic protein Bcl2; however, galangin increased Bcl2 expression and decreased Bax expression (Figure 4c-e). These data indicated that galangin protected H9c2 cells from Ang II or AB surgery-induced apoptosis.

| Effects of galangin on PI3K-Akt-GSK3β signaling in vitro and in vivo
To investigate the potential molecular mechanism of galangin on cardiac remodeling, we examined the effects of galangin on the PI3K-Akt-GSK3β signaling pathway. Our data showed that PI3K, Akt, GSK3β were significantly phosphorylated after treatment with Ang II in vitro or 4 weeks after AB surgery in vivo, and middle-dose (25 mg/kg/day) and high-dose (50 mg/kg/day) galangin treatment of mice in vivo and 50 μM galangin in vitro evidently blocked the activation of PI3K, Akt, and GSK3β (Figure 6a-d). The results showed that galangin significantly inhibits cardiac remodeling through inhibition of the PI3K-Akt-GSK3β signaling pathway.

| Effects of galangin on MEK1/2-ERK1/2-GATA4 signaling in vitro and in vivo
We found that the phosphorylated levels of MEK1/2, ERK1/2, and GATA4 were significantly increased in vehicle-treated mice subjected to AB surgery in vivo and postAng II stimulus in vitro. However, the increased phosphorylated levels of MEK1/2, ERK1/2, and GATA4 were blocked in galangin (25, 50 mg/kg/day in vivo or 50 μM in vitro)treated hearts or H9c2 cells (Figure 7a-d). The results showed that galangin significantly inhibits cardiac remodeling through inhibition of the MEK1/2-ERK1/2-GATA4 signaling pathway.

| DISCUSSION
In the present study, we examined the role of galangin in cardiac A. officinarum (a plant that has been used as a kind of herbal medicine for multiple ailments in Asia for centuries; Y. N. Liu et al., 2015).
Galangin has attracted much attention for its potential antiinflammatory, antioxidant, apoptosis regulatory properties (Aloud, Chinnadurai, Govindasamy, Alsaif, & Al-Numair, 2018;Lei et al., 2018;Y. N. Liu et al., 2015). Some studies have found that galangin, as a promising anti-remodeling agent in asthma, may involve in the TGF-β1-ROS-MAPK pathway (Y. N. Liu et al., 2015). Meanwhile, galangin can inhibit CCl4-mediated liver fibrosis in rats, its mechanism may be related to scavenging oxygen-free radicals, reducing lipid peroxidation, and inhibiting the activation and proliferation of hepatic stellate cells (X. Wang et al., 2013). However, the effects of galangin on cardiac remodeling and the potential signaling mechanisms have not yet been elucidated.  et al., 2006;J. Li et al., 2017). Our team also demonstrated that inhibiting AKT/GSK3β attenuated pressure overload-induced myocardial fibrosis and blocked cardiac fibroblasts activation and transformation in vitro (Yan et al., 2011). We observed that the phosphorylation levels of PI3K, AKT, and GSK3β are increased in the remodeling heart caused by pressure overload and in H9c2 cells under Ang II-stimulation and that these effects are inhibited by galangin.
ERK1/2 (extracellular-regulated protein kinases 1/2) is a class of serine/threonine protein kinases, which is a signal transduction protein that transmits mitogen signals. It is normally located in the cytoplasm and translocates to the nucleus when activated by phosphorylation, thus regulating the activity of transcription factors and producing corresponding cellular effects (Linke et al., 2014;Thei, Rocha-Ferreira, Peebles, Raivich, & Hristova, 2018). As an important member of the MAPK signaling pathway, ERK1/2 can directly modify a series of transcription factors that promote cardiac gene expression and ultimately lead to cardiac hypertrophy (Ma et al., 2016). The activation of the ERK1/2 is triggered by MAPK1/2 (MEK1/2) via phosphorylation of serine/ threonine residues (Zong et al., 2013). Because ERK1/2 can be activated by a variety of stimuli, leading to cardiac remodeling, it may be an ideal target for improving cardiac remodeling (Zhong et al., 2015). GATA4 (GATA-Binding Factor 4) is a member of the GATA family of zinc-finger transcription factors (Arceci, King, Simon, Orkin, & Wilson, 1993  IκBα, NF-κB p65 is activated from inactivated state and transferred from cytoplasm to nucleus, binding to the corresponding inflammation-related genes, initiating inflammatory cytokine transcription, and inducing inflammation (Tanaka & Iino, 2016). In our study, we showed that both AB surgery in vivo and Ang II stimulus in vitro increases the phosphorylation of IκBα□ and phosphorylation of NF-κB p65. However, galangin administration reduces the heart expressions of phosphor-IκBα□ and phospho-NF-κB p65 in vivo and in vitro.
Cardiomyocytes apoptosis increases when the heart is subjected to pressure overload or when cardiomyocytes are stimulated by Ang II (Yang et al., 2012;Yu, Hu, Li, Wang, & Chen, 2018). Galangin has been shown to modulate cell apoptosis (Tomar et al., 2017). In our experiments, we found that both AB surgery and Ang II-stimulation increased the apoptosis ratio of H9c2 cells, increased the expression of apoptotic protein Bax, and decreased the expression of antiapoptotic protein Bcl2. However, galangin increased Bcl2 expression and decreased Bax expression in vivo and in vitro, as well as decreased the apoptosis ratio of H9c2 cells in vitro.
Myocardial fibrosis is another important pathological feature of pressure overload cardiac remodeling, which is characterized by the accumulation of collagen, increased extracellular matrix (ECM) deposition, impairs diastolic relaxation, and causes cardiac dysfunction, which is characterized by increased accumulation of collagen and deposition of ECM, decreased diastolic function and cardiac dysfunction. We observed the galangin reduced the cardiac fibrosis and inhibited collagen synthesis in vivo. To further discuss the molecular mechanism, we evaluated the role of galangin in TGF-β/Smad signaling F I G U R E 7 Effects of galangin on the MEK1/2-ERK1/2-GATA4 signaling pathway. (a, b) Representative western blots for total and phosphorylation of MEK1/2, ERK1/2, and GATA4. (c, d) Quantitative results of western blot (n = 5 per experimental group) # p < 0.05 versus the Sham group or the control group; *p< 0.05 or **p < 0.01, versus AB or Ang II group. AB: aortic banding; Ang II: angiotensin II; ERK1/2: extracellular-regulated protein kinases 1/2 (a key role in the transcription of profibrotic genes). We have found that mice with galangin treatment attenuate the expression of TGF-β1 and p-smad2/t-smad2 induced by pressure overload.
Taking the results of our study together, we showed the first evidence that galangin protects against cardiac hypertrophy, inflammation, apoptosis, and fibrosis was induced by pressure overload. Galangin also suppressed the activation of PI3K-Akt-GSK3β and MEK1/2-ERK1/2-GATA4 signaling in vitro and in vivo.
Therefore, galangin may offer a promising effective approach for preventing cardiac remodeling.