Introduction

Molecular Medicine Reports

1791-2997 1791-3004

D.A. Spandidos

10.3892/mmr.2022.12822

MMR-26-04-12822

Articles

Puerarin attenuates isoproterenol-induced myocardial hypertrophy via inhibition of the Wnt/β-catenin signaling pathway

Wang

Xiaoying

1 2 * He

Kai

1 2 * Ma

Linlin

2 Wu

Lan

2 Yang

Yan

3 Li

Yanfei

1 2

1Graduate School, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China 2College of Medical Technology, Shanghai University of Medicine and Health Sciences, Shanghai 201318, P.R. China 3Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China

Correspondence to: Professor Yan Yang, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Building 12, 1000 Jinqi Road, Fengxian, Shanghai 201106, P.R. China, E-mail: yangyan@saas.sh.cn Professor Yanfei Li, College of Medical Technology, Shanghai University of Medicine and Health Sciences, 279 Zhouzhu Road, Pudong New Area, Shanghai 201318, P.R. China, E-mail: liyf@sumhs.edu.cn *

Contributed equally

10 2022

09 08 2022

26 4

306

23032022 20072022

2022

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Myocardial hypertrophy (MH) is an independent risk factor for cardiovascular disease, which in turn lead to arrhythmia or heart failure. Therefore, attention must be paid to formulation of therapeutic strategies for MH. Puerarin is a key bioactive ingredient isolated from Pueraria genera of plants that is beneficial for the treatment of MH. However, its molecular mechanism of action has not been fully determined. In the present study, 40 µM puerarin was demonstrated to be a safe dose for human AC16 cells using Cell Counting Kit-8 assay. The protective effects of puerarin against MH were demonstrated in AC16 cells stimulated with isoproterenol (ISO). These effects were characterized by a significant decrease in surface area of cells (assessed using fluorescence staining) and mRNA and protein expression levels of MH-associated biomarkers, including atrial and brain natriuretic peptide, assessed using reverse transcription-quantitative PCR and western blotting, as well as β-myosin heavy chain mRNA expression levels. Mechanistically, western blotting demonstrated that puerarin inhibited activation of the Wnt signaling pathway. Puerarin also significantly decreased phosphorylation of p65; this was mediated via crosstalk between the Wnt and NF-κB signaling pathways. An inhibitor (Dickkopf-1) and activator (IM-12) of the Wnt signaling pathway were used to demonstrate that puerarin-mediated effects alleviated ISO-induced MH via the Wnt signaling pathway. The results of the present study demonstrated that puerarin pre-treatment may be a potential therapeutic strategy for preventing ISO-induced MH and managing MH in the future.

puerarin isoproterenol cardiac hypertrophy Wnt/β-catenin signaling pathway phosphorylation of NF-κB p65

Shanghai Special Project of Biomedical Science and Technology

21S11901700

The present study was supported by Shanghai Special Project of Biomedical Science and Technology (grant no. 21S11901700).

Introduction

Cardiovascular disease (CVD) is the leading cause of human mortality worldwide accounted for 32.9% of all deaths with a steadily reaching of the number of CVD deaths from 12.1 million in 1990 to 18.6 million in 2019 (1), and places a notable burden on human health (2,3). At present, management of patients with CVD is challenging due to the high mortality, disability rates and rising health care costs (4). According to the World Health Organization, three-quarters of CVD deaths could be prevented through early detection and lifestyle interventions (5). As a result, CVD has attracted the attention of research communities and healthcare professionals to ensure that measures, including developing the care of cardiovascular disease in the ambulatory setting, improving approaches to the prevention of cardiovascular disease, expanding digital health interventions and universal health coverage (6), have been taken. Pharmaceutical developments and technological improvements are used in the Western world to decrease CVD-associated mortality, alongside interventions to control risk factors for CVD (7). Among risk factors for CVD, pathological myocardial hypertrophy (MH) caused by long-term overload is an independent risk factor for adverse cardiovascular events (8). Therefore, reversing MH may minimize the risk of cardiovascular events.

MH refers to an adaptive and compensatory change in heart structure that maintains cardiac output during adverse stimulation (9). The hypertrophic process is categorized as physiological or pathological. Physiological hypertrophy is key to preservation of normal heart structure and function; it is a tightly regulated but reversible phenomenon, such as the hypertrophic process that occurs in the heart following continuous exercise (10). This is a compliant change (an adaptive response to stimuli) in the heart and causes negligible effects in the body. However, pathological hypertrophy is associated with fibrosis, increased proinflammatory cytokine production and cellular dysfunction (11). This type of maladaptive cardiac hypertrophy, typically caused by chronically high, continuous hemodynamic pressure, is irreversible and leads to heart failure in severe cases (12).

Angiotensin-converting enzyme inhibitors (13,14), angiotensin II receptor blockers (15,16), calcium channel antagonists, β-receptor blockers and diuretics (17,18) are currently the primary treatments for MH. They have all been reported to improve the clinical symptoms of patients to a certain extent; however, the single-target therapeutic efficacy of these agents remains unsatisfactory as none of them significantly decrease mortality rate (19). Therefore, MH needs to be addressed in the CVD research. Traditional Chinese medicine (TCM) has been reported to improve the symptoms and quality of life of patients with heart failure (20). It has been reported that TCM confers therapeutic benefits using natural, biologically active ingredients that have multiple targets (21). Furthermore, agents such as Radixastragali (Huangqi) and Ginseng (Renshen) (22,23) have gained attention due to their stable therapeutic effects by targeting multiple targets underlying the development of MH. Astragaloside IV(AS-IV) which is the main component of Radixastragali, improves cardiac function and ameliorate MH, which is mediated by attenuating inflammatory reaction (24) and inactivating TANK-binding kinase 1 (TBK1)/PI3K/AKT signaling pathway (25). The pharmacological properties of ginseng are mainly attributed to ginsenosides which plays a robust antihypertrophic role via inhibition of NHE-1-dependent calcineurin activation (26) and the suppression of MCT-activated calcineurin and ERK signaling pathways (27).

Based on clinical experience and systematic theory, advances have been achieved in the research of TCM formulas, extracts and compounds (28). For example, research and development of ‘compound Danshen dropping pill’ has contributed to treatment of coronary heart disease and angina pectoris (29). Numerous compounds isolated from TCM products have important properties, such as the antimalarial effects of artemisinin (30) and the anti-inflammatory properties of licorice extract, which contains 3 triterpenes and 13 flavonoids (31). Furthermore, the curative effects of novel TCM compounds that exert specific pharmacological effects may be enhanced by elucidating their mechanism individually whilst also characterizing their target profile. Ultimately, treatment strategies for MH may be developed using the multi-faceted profiles of TCM compounds combined with metabolomics and traditional pharmacological methods (32).

Puerarin (PU) is an isoflavone chemical component (33) found within the Pueraria genera of plants (34). PU has been reported to exert numerous therapeutic effects, including anti-cancer, vasodilatory and heart protective effects (35). Furthermore, it has been previously reported that PU ameliorates pressure overload-induced MH in ovariectomized rats via activation of the peroxisome proliferator-activated receptor-α/peroxisome proliferator-activated receptor γ-1 signaling pathway and regulation of energy metabolism remodeling (36). However, the multi-target characteristics of TCM monomers suggest that PU has multiple therapeutic targets (37). Numerous signaling pathways are associated with the pathophysiology of MH, including the 5′AMP-activated protein kinase/mTOR (38), PI3K/AKT/mTOR (39) and insulin-like growth factor 1/PI3K/AKT (40) signaling pathways. Therefore, the exploration of the mechanism of PU on MH to achieve the effect of precise treatment is urgent. Numerous studies have reported that the Wnt signaling pathway, which is activated in early embryonic development to promote cardiac development and silenced in adulthood to maintain normal cardiac function, serves an important role in CVD (41,42). As experimental data on the effects of PU on the Wnt signaling pathway in the context of MH remain incomplete, the present study was preformed to address this.

Materials and methods <sec> <title>Chemicals and reagents

PU (purity, 99.00%) was purchased from TargetMol Chemicals, Inc. Isoproterenol hydrochloride (ISO), Dickkopf-1 (DKK1) and IM-12 were purchased from MedChemExpress. Cell Counting Kit-8 (CCK-8) assay was purchased from Dojindo Molecular Technologies, Inc. PVDF membranes, RIPA buffer, Actin-Tracker Green (microfilament green fluorescent probe), DAPI Staining Solution and BCA Protein Assay kit were purchased from Beyotime Institute of Biotechnology. TransScript^® Green Two-Step qRT-PCR SuperMix kit was purchased from TransGen Biotech Co., Ltd. Antibodies against low-density lipoprotein receptor-related protein 6 (LRP6; cat. no. 3395), phosphorylated (p-)p65 (cat. no. 3033), NF-κB p65 (cat. no. C22B4), Tubulin (cat. no. 2128S), β-catenin (cat. no. 8480), Wnt5a/b (cat. no. 2530), c-Myc (cat. no. 5605), glycogen synthase kinase-3β (GSK3β; cat. no. 12456) and p-GSK3β (cat. no. 9322) were purchased from Cell Signaling Technology, Inc. Atrial natriuretic peptide (ANP; cat. no. ab189921) and brain natriuretic peptide (BNP, cat. no. ab236101) were purchased from Abcam. GAPDH (cat. no. BM1623) and the secondary antibodies including HRP Conjugated AffiniPure Goat Anti-Rabbit IgG (cat. No. BA1054) and HRP Conjugated AffiniPure Mouse Anti-Human IgG (cat. No. BM2002) was from Boster.

Cells

The human cardiomyocyte AC16 cell line (MingzhouBio Inc., cat. no. MZ-4038) was used. Cells were cultured in culture flasks with DMEM (BasalMedia Inc.) at 37°C under 5% CO₂ in a humidified atmosphere. When cells reached the logarithmic growth phase, they were evenly distributed into six-well plates. Experimental treatments were performed after 24 h.

In vitro cardiac hypertrophy model and drug treatment

Cells were randomly divided as follows: i) Control, cells treated with DMEM; ii) ISO, cells treated with 10 µM ISO for 24 h; iii) PU, cells treated with 40 µM PU for 6 h followed by 10 µM ISO for 24 h; iv) DKK1, cells treated with 780 nM DKK1 for 6 h followed by 10 µM ISO for 24 h; v) IM12, cells treated with 30 nM IM-12 for 24 h; vi) PU + IM12, cells treated with 40 µM PU for 6 h followed by 30 nM IM-12 for 24 h and vi) PU + ISO + IM12, cells treated with 40 µM PU for 6 h followed by 10 µM ISO for 24 h and 30 nM IM-12 for 6 h. A total of 1.5×10⁵ cells were plated in culture flasks with DMEM at 37°C under 5% CO₂ in a humidified atmosphere. The cells were washed three times with PBS before addition of new drugs to avoid mixing of different drug components.

CCK-8 assay

PU was dissolved in DMSO and DMEM to produce a range of concentrations (5, 10, 20, 40 and 80 µM) for subsequent use. The final DMSO content was 0.2%. Cells were evenly dispersed into six groups containing different concentrations of PU (0, 5, 10, 20, 40 and 80 µM) and inoculated into a 96-well plate (~5,000 cells/well). PU incubation was performed for 6 h at 37°C under 5% CO₂ before 100 µl CCK-8 reagent was added, followed by incubation for 2 h at 37°C under 5% CO₂ in a humidified atmosphere. The 96-well plate was assessed using a PT-3502PC microplate reader (Beijing Potenov Technology Co. Ltd.) to quantify the absorption value of each well at 450 nm. A line chart was constructed plotting optical density (OD) values against drug concentrations to assess the effect of PU on cell viability.

Cytoskeletal staining

The surface area of AC16 cardiomyocytes in the different treatment groups was assessed using cytoskeletal staining. Control, ISO and PU), cells were washed with PBS twice and fixed using 3.7% formaldehyde solution at room temperature for 10 min in the dark according to the manufacturer's protocol for the Actin-Tracker Green. PBS containing 0.1% Triton X-100 was added to permeabilize the fixed cells for 10 min at room temperature. The cells were stained with Actin-Tracker Green working solution diluted with PBS containing 1–5% BSA and 0.1% Triton X-100 at a ratio of 1:50 for 30 min at room temperature. Following rinsing with PBS, 5 mg/ml DAPI (sufficient to cover each section) was added to each well for 3–5 min at room temperature before imaging using a fluorescence microscope (Olympus Corporation). The magnification was 400×. A total of five non-overlapping fields located at the upper and lower left and right and center of each well was selected. The cells in each field were numbered and sampled by a random number generator. A total of four cells was randomly extracted from each field. Finally, the size of cells was quantified using ImageJ2 (version 1.53; National Institutes of Health). The mean area of the selected cells was determined for each well and normalized to the control.

Reverse transcription-quantitative (RT-q)PCR

RNA was extracted from AC16 cells (Control, ISO and PU) using TRIzol^® (Thermo Fisher Scientific, Inc.). The concentration of RNA was detected using a NanoDrop^® spectrophotometer (Thermo Fisher Scientific, Inc.). Complementary DNA synthesis, using incubation at 42°C for 15 min and heating at 85°C for 5 sec, was performed according to the manufacturer's protocols of the TransScript^® Green Two-Step qRT-PCR SuperMix kit. The primer sequences were as follows: ANP forward (F), 5′-CAACGCAGACCTGATGGATTT-3′ and reverse (R), 5′-AGCCCCCGCTTCTTCATTC-3′; BNP F, 5′-TGGAAACGTCCGGGTTACAG-3′ and R, 5′-CTGATCCGGTCCATCTTCCT-3′; β-myosin heavy chain (β-MHC) F, 5′-TCACCAACAACCCCTACGATT-3′ and R, 5′-CTCCTCAGCGTCATCAATGGA-3′ and GAPDH F, 5′-CTGGGCTACACTGAGCACC-3′ and R, 5′-AAGTGGTCGTTGAGGGCAATG-3′. The reaction systems of each primer sequence were added into a 96-well fluorescent qPCR plate and CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: 94°C initial denaturation for 30 sec, followed by 40 cycles of 5 sec at 94°C and 30 sec at 60°C. The 2^−ΔΔCq method (43) was used to assess mRNA expression levels.

Western blotting

The cells in each group (Control, ISO, PU, DKK1, IM12, PU + IM12, PU + ISO + IM12) were digested with 0.25% trypsin for 25 sec at 37°C under 5% CO₂ in a humidified atmosphere, washed with PBS three times and lysed with RIPA lysis buffer containing protease and phosphate inhibitors at 4°C for 30 min. The supernatant was collected following centrifugation (12,000 × g at 4°C for 10 min), before protein concentration was quantified via BCA assay. An equal amount of protein (15 µg protein/lane) was separated by 10% SDS-PAGE and transferred onto PVDF membranes. PVDF membranes were blocked with 5% skimmed milk powder solution at room temperature for 1 h before being incubated at 4°C overnight with specific primary antibodies. The antibodies were as follows: LRP6 (1:1,000), p-p65 (1:1,000), p65 (1:1,000), Tubulin (1:10,000), β-catenin (1:1,000), Wnt5a/b (1:1,000), GAPDH (1:5,000), c-Myc (1:1,000), GSK3β (1:1,000), p-GSK3β (1:1,000), ANP (1:1,000) and BNP (1:1,000). After washing with TBST (0.1% Tween-20), membranes were incubated with anti-rabbit (1:5,000) and anti-mouse secondary antibodies (1:5,000) at room temperature for 1 h. The membranes were rinsed again and treated with BeyoECL Moon (cat. no. P0018FS, Beyotime Institute of Biotechnology) before bands were visualized using the Tanon-4600 Chemiluminescence Imaging System. Finally, blots were analyzed using ImageJ2 (version 1.53; National Institutes of Health) to semi-quantify the protein expression levels.

Statistical analysis

All statistical analysis was performed using GraphPad Prism (version, 9.1.1; GraphPad Software, Inc.). Comparisons between groups were performed using one-way ANOVA with Dunnett's (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7) or Tukey's post hoc test (Fig. 8). Data are presented as the mean ± SEM of ≥3 independent experimental repeats. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>PU is not toxic to cardiomyocytes

CCK-8 assay OD values were used to assess the effects of PU on AC16 cell viability. The results demonstrated no significant difference in cell viability between treatment groups (Fig. 1). These data demonstrated that the highest concentration of PU (80 µM) had little effect on cell viability after 6 h.

PU decreases ANP, BNP and β-MHC transcription

The mRNA expression levels of MH markers ANP, BNP and β-MHC were quantified using RT-qPCR. The results demonstrated significant increases in mRNA expression levels of ANP (P<0.05; Fig. 2A), BNP (P<0.01; Fig. 2B) and β-MHC (P<0.01; Fig. 2C) in the ISO group compared with the control. By contrast, treatment with PU significantly decreased mRNA expression levels of ANP (P<0.05), BNP (P<0.01) and β-MHC (P<0.05) compared with the ISO group. These results suggested that the ISO-induced MH model was successfully established and the effect of ISO was reversed by PU.

PU decreases protein expression levels of ANP and BNP

To assess the effect of PU on ISO-induced MH, western blotting was performed to determine protein expression levels of ANP and BNP. Following 24 h induction with 10 µM ISO, protein expression levels of ANP and BNP in the AC16 cardiomyocytes were significantly higher compared with those in the control (P<0.01; Fig. 3). However, expression levels of ANP and BNP protein in AC16 cardiomyocytes pretreated with PU were significantly lower compared with those in the ISO group (P<0.01). These results demonstrated that cells pretreated with PU were resistant to ISO-induced MH.

ISO-treated AC16 cell surface area decreases significantly following PU pretreatment

Changes in AC16 myocardial cell area were observed using fluorescence microscopy. The surface area of AC16 cells in the ISO group significantly increased compared with those in the control (P<0.01; Fig. 4A, B and D). However, PU pretreatment significantly decreased surface area of AC16 cells compared with those in the ISO group (P<0.05; Fig. 4C). These results suggested that MH was prevented by PU pretreatment.

PU inhibits the Wnt/β-catenin signaling pathway

To determine the potential anti-MH mechanism of PU, core components in the Wnt/β-catenin and NF-κB signaling pathways were assessed using western blotting. The protein expression levels of LRP6, β-catenin, Wnt-5a/b and c-Myc and phosphorylation levels of p65 and GSK3β, significantly increased in the ISO group compared with the control group (P<0.01; Fig. 5A-F and H-K). However, the expression of GSK3β was significantly decreased by ISO compared with the control group (P<0.01; Fig. 5G). There was a significant decrease in the expression of LRP6 (P<0.05), β-catenin (P<0.01), Wnt-5a/b (P<0.01) and c-Myc (P<0.01) as well as phosphorylation levels of p65 (P<0.01) and GSK3β (P<0.01) in the AC16 cells pretreated with PU compared with the ISO group. Furthermore, expression of GSK3β (P<0.01) was significantly increased by PU pretreatment compared with the ISO group. These results suggested that the protective effects of PU on MH may be mediated by Wnt and NF-κB signaling pathway activity.

PU serves as an inhibitor of the Wnt signaling pathway by antagonizing its activator

A Wnt inhibitor and activator were used to verify if PU regulated the Wnt signaling pathway. DKK1 is an inhibitor of the Wnt signaling pathway that has been reported to regulate embryonic development (44). Wnt signaling transduction is blocked by DKK1 binding, which induces LRP6 internalization (45).

After cells were pretreated with DKK1, protein expression levels of components in the Wnt signaling pathway, such as LRP6, β-catenin, Wnt5a/b and c-Myc, were significantly suppressed compared with ISO group (P<0.01; Fig. 6A-G); this effect was similar to the aforementioned effect mediated by PU. Furthermore, expression of ANP, a cardiac hypertrophy marker, was significantly decreased by DKK1 pretreatment (P<0.01; Fig. 6G), which suggested that inhibition of the Wnt signaling pathway may be beneficial for the treatment of MH.

IM-12 enhances Wnt signaling, primarily by inhibiting GSK-3β (46). In the present study, IM-12 significantly decreased GSK3β expression compared with the control (P<0.01; Fig. 7G). The protein expression levels of LRP6, β-catenin, Wnt-5a/b and c-Myc were significantly increased by IM-12 compared with the control (P<0.01; Fig. 7A-F). However, protein expression levels of LRP6 (P<0.05), β-catenin (P<0.01), Wnt-5a/b (P<0.01) and c-Myc (P<0.01) were significantly decreased by PU pretreatment. These results suggested that PU suppressed IM-12-induced activation of the Wnt/β-catenin signaling pathway.

The effect of IM-12 on expression of markers in the Wnt signaling pathway and ISO-induced MH in the presence of PU pretreatment were assessed (Fig. 8). Following treatment with IM-12, PU-induced inhibition of Wnt signaling pathway protein expression was partially reversed. With the exception of protein expression of GSK3β, which was increased by PU but significantly reversed by IM-12 compared with the PU + ISO group, expression levels of all proteins in the PU + ISO + IM12 group were significantly increased compared with PU group (P<0.01). Moreover, the inhibitory effects of PU on the MH marker ANP was partially but significantly reversed by IM-12 treatment compared with the PU + ISO group (P<0.01). These results demonstrated that the protective effects of PU on MH may be mediated by inhibition of the Wnt signaling pathway.

Discussion

Despite therapeutic advances, there is a lack of effective therapeutic treatment strategies for MH. Pathological cardiac hypertrophy is induced by various factors, including pressure (47) and volume overload (48) and pharmaceutical induction, such as induction using angiotensin II (49) and ISO (50). ISO is a β-receptor agonist that causes acute myocardial contraction, ischemia, hypoxia and cause damage to myocardial cells due to production of a large number of peroxynitrite and oxygen free radicals in rats (51). Prabhu et al (52) reported that activities of myocardial mitochondrial cathepsin-D and β-glucuronidase are significantly decreased by high-dose ISO and that ISO decreases stability of the myocardial mitochondrial membrane. ISO is widely used to induce MH (50,53). There is evidence demonstrating that ISO stimulation leads to MH (53). ISO activates MAPK in cardiomyocytes and phosphorylates Raf/MEK/ERK signaling pathway components (54). The increase of p-ERK1/2 protein expression levels is a downstream effect of ISO-induced protein kinase C ε activation, which leads to MH (55). A previous study reported that chronic infusion of ISO results in compensatory cardiac hypertrophy associated with protein kinase A activity and N-terminal cleaved histone deacetylase production (56). The mechanism of ISO-induced myocardial injury may be associated with oxidative stress, calcium overload, inflammation and lipid peroxidation (57). It has been confirmed that in ISO-induced MH, ISO treatment markedly increases reactive oxygen species levels in cardiomyocytes (58), activates the NF-κB signaling pathway in AC16 cells and induces an inflammatory response (59). Further studies are required to determine the mechanism underlying ISO-induced MH. Based on a previous study (60), 10 µM ISO was chosen as the induction concentration for the present study.

In failing human hearts, there is a significant increase in both mRNA and protein expression of ANP, BNP and β-MHC (61); whereas expression of these three genes is normally stable in normal cardiomyocytes (62,63). A previous study reported that PU decreases mRNA expression levels of MH markers ANP and BNP in a dose-dependent manner, with 40 µM being the optimal concentration (64). Combined with the results from the present CCK-8 assay, 40 µM was selected as the concentration to induce MH for subsequent experimentation in the present study. It has been reported that the early stage of MH is characterized by the increase of the surface area of cardiomyocytes (65). Changes in cell surface area (quantified and analyzed using immunofluorescence) directly reflect progression of cardiac hypertrophy; this method is used to assess MH. In a previous study, H9c2 cells were stained with crystal violet and surface area was observed as a measure of MH to confirm that the surface area of CD38 knockdown cells was not enlarged (66). Another study used wheat germ agglutinin staining to evaluate the size of cardiomyocytes from heart sections (67). A previous study used surface area of cells stained with gentian violet to assess MH (68). Studies have also aimed to use cell surface area to assess the role of miR-339-5p in cardiomyocyte hypertrophy (69) and determine the therapeutic potential of pyrroloquinoline quinone for ISO-induced cardiac hypertrophy (59). In the present study, pretreatment with PU was demonstrated to exert protective effects against ISO-induced MH, demonstrated by decreased area of cardiomyocytes and expression of markers of cardiac hypertrophy. Therefore, PU may be a promising clinical agent for the prevention and treatment of MH, although its mechanism remains to be fully elucidated.

The signal transduction mechanism underlying MH is complex. Inflammation (70) and oxidative stress (71) have both been previously demonstrated to cause alterations leading to MH and heart failure. A previous systematic study of Wnt on a genome-wide level reported an association between MH and the Wnt expression profile (72). The Wnt signaling pathway has also been reported to be associated with cardiac disease; excessive stimulation of Wnt signaling is detrimental to cardiovascular pathology (42). PU has been reported to demonstrate anti-inflammatory and antioxidant activities (73). However, the potential effects of PU on MH and the Wnt signaling pathway in cardiomyocytes remain poorly understood. Therefore, the present study aimed to explore if PU demonstrate effects against MH in an in vitro model, with specific focus on the Wnt/β-catenin signaling pathway.

The Wnt signal transduction pathway is divided into classical and non-classical branches (74). The present study verified the expression of associated proteins and demonstrated that PU inhibited ISO-induced MH by blocking the classical branch of the Wnt/β-catenin signaling pathway. Wnt signaling is typically activated by the membrane receptor complex consisting of frizzled (Fzd), LRP5/6 and disheveled (Dvl) (75). Following activation of the Wnt receptor, Fzd binds to Wnt protein and LRP5/6 to form a receptor complex (76). Dvl is recruited by the receptor complex and activated via phosphorylation before binding to the β-catenin degradation complex, which separates β-catenin from the degradation complex to promote stabilization and aggregation before β-catenin translocates into the nucleus (77). β-catenin is a core component of the Wnt/β-catenin signaling pathway and is reported to be upregulated in cardiomyocytes from patients with acute myocardial infarction and heart tissue of spontaneously hypertensive rats (78). However, GSK3β mediates both β-catenin-dependent and -independent cascades (79). A previous study reported that GSK3β exerts negative regulatory effects on the Wnt signaling pathway (80). In the nucleus, β-catenin binds to T cell factor/lymphoid enhancer factor (Tcf/Lef) to activate transcription of Wnt target genes (81). c-Myc and Tcf/Lef are downstream targets of the Wnt signaling pathway; a previous study reported that activity of these targets is inhibited by low expression of β-catenin (82).

In the present study, western blotting was used to assess expression of core proteins in the Wnt signaling pathway; expression levels of these proteins in the ISO group was significantly increased but significantly reversed by PU pretreatment. However, GSK3β protein expression levels exhibited an opposite trend, which is likely due to its negative regulatory mechanism of Wnt; following activation of Wnt signaling, activity of GSK3β is hampered (83). A previous study reported that decreased expression of GSK3β in cardiac fibroblasts that led to adverse ventricular remodeling (84). Therefore, inhibition of GSK3β may increase the activity of Wnt signaling, thereby leading to cardiac hypertrophy. PU induces expression of GSK3β, as demonstrated in the present study, and serves a protective role. Furthermore, numerous studies have demonstrated that the Wnt/β-catenin and NF-κB signaling pathways exhibit crosstalk and this interaction is enhanced by the inflammatory response (85,86). In the present study, it was demonstrated that, in the ISO group, the protein expression of Wnt5a/b and β-catenin and the phosphorylation levels of p65 were significantly increased compared with the control but significantly decreased by pretreatment with PU. These observations are in accordance with those in previous studies which suggested that there may be synergy between the two signaling pathways during the development of MH (87,88). However, more evidence is needed to support the synergistic effect.

To verify whether inhibition of the Wnt signaling pathway ameliorated ISO-induced MH, DKK1, an inhibitor of the Wnt signaling pathway, was used. DKK1 is a member of the DKK family that is reported to antagonize the Wnt/β-catenin signaling pathway by decreasing β-catenin and increasing OCT4 expression (89). The results of the present study demonstrated that the DKK1 group exhibited trends in the expression of protein in the Wnt signaling pathway that were comparable to those follow PU pretreatment, in that PU exerted inhibitory effects on Wnt signaling comparable with those mediated by the known Wnt inhibitor. IM-12, an activator of the Wnt signaling pathway, has been reported to increase expression of β-catenin and downstream proteins while suppressing expression of GSK-3β (46). The present study assessed whether IM-12 reversed the inhibitory effects of PU on ISO-induced MH, thus confirming whether PU exerted its effects via inhibition of the Wnt signaling pathway. The present study demonstrated that PU inhibited expression of Wnt signaling pathway proteins induced by IM-12 and increased expression of GSK-3β. Furthermore, IM-12, the specific activator of the Wnt signaling pathway, was demonstrated to eliminate the protective effects of PU on MH. In conclusion, PU served a cardioprotective role partially via inhibition of the Wnt signaling pathway.

However, in the present study only core proteins in the Wnt signaling pathway were assessed; therefore the detailed underlying molecular mechanism in the upstream regulation of Wnt/β-catenin signaling during MH was not fully elucidated. Since Wnt signaling induces nuclear localization of β-catenin (90), it is important to determine the localization and expression of β-catenin. These limitations of the present study require addressing in future. In future studies, the inhibitor-like mechanism of PU and its effects on other disease caused by aberrant activation of the Wnt signaling pathway, including colon cancer, hepatocellular carcinoma and pancreatic, lung and ovarian cancer (91), should be explored.

In summary, the present study demonstrated that pretreatment of AC16 cells with PU attenuated ISO-induced MH. The underlying mechanism was associated with inhibition of protein expression in the Wnt/β-catenin signaling pathway and NF-κB activity. These results suggested that PU may be a potential agent for treating MH. Based on the importance of the Wnt signaling pathway for initiation, maintenance, progression and relapse of MH, PU may serve an inhibitor-like role for the treatment of cardiovascular and associated disease caused by hyperactivation of the Wnt signaling pathway. Furthermore, the present study may provide a novel direction for development and use of agents for MH treatment.

Acknowledgements

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XW performed experiments, analyzed data and wrote the manuscript. KH performed experiments and data analysis. LM designed the study, analyzed data and revised the manuscript. LW performed data analysis. YY participated in discussions and analyzed data. YL designed the study and obtained funding. XW, KH, LM and YL confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for participation

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Roth

Mensah

Johnson

Addolorato

Ammirati

Baddour

Barengo

Beaton

Benjamin

Benziger

Global burden of cardiovascular diseases and risk factors, 1990–2019: Update from the GBD 2019 study

J Am Coll Cardiol76298230212020

10.1016/j.jacc.2020.11.010

33309175

Cho

Moon

Ryu

A study on clinical and healthcare recommending service based on cardiovascula disease pattern analysis

Int J Biosci Biotechnol82872942016

Nalban

Sangaraju

Alavala

Mir

Jerald

Sistla

Arbutin attenuates isoproterenol-induced cardiac hypertrophy by inhibiting TLR-4/NF-κB pathway in mice

Cardiovasc Toxicol202352482020

10.1007/s12012-019-09548-3

31485892

Roth

Mensah

Fuster

The global burden of cardiovascular diseases and risks: A compass for global action

J Am Coll Cardiol76298029812020

10.1016/j.jacc.2020.11.021

33309174

Kim

Lee

Won

Kim

Yang

Kim

Lee

Machine learning-based diagnosis and risk factor analysis of cardiocerebrovascular disease based on KNHANES

Sci Rep1222502022

10.1038/s41598-022-06333-1

35145205

Leong

Joseph

McKee

Anand

Teo

Schwalm

Yusuf

Reducing the global burden of cardiovascular disease, part 2: Prevention and treatment of cardiovascular disease

Circ Res1216957102017

10.1161/CIRCRESAHA.117.311849

28860319

Van Camp

Cardiovascular disease prevention

Acta Clin Belg694074112014

10.1179/2295333714Y.0000000069

25176558

Zhao

Jia

Ren

Xiao

Jin

Lin

Difluoromethylornithine attenuates isoproterenol-induced cardiac hypertrophy by regulating apoptosis, autophagy and the mitochondria-associated membranes pathway

Exp Ther Med228702021

10.3892/etm.2021.10302

34194548

Gallo

Vitacolonna

Bonzano

Comoglio

Crepaldi

ERK: A key player in the pathophysiology of cardiac hypertrophy

Int J Mol Sci2021642019

10.3390/ijms20092164

31052420

Ellison

Waring

Vicinanza

Torella

Physiological cardiac remodelling in response to endurance exercise training: Cellular and molecular mechanisms

Heart985102012

10.1136/heartjnl-2011-300639

21880653

Selvetella

Hirsch

Notte

Tarone

Lembo

Adaptive and maladaptive hypertrophic pathways: Points of convergence and divergence

Cardiovasc Res633733802004

10.1016/j.cardiores.2004.04.031

15276462

Shimizu

Minamino

Physiological and pathological cardiac hypertrophy

J Mol Cell Cardiol972452622016

10.1016/j.yjmcc.2016.06.001

27262674

Kurosawa

Kojima

Kato

Ohashi

Minami

Narita

Protective action of angiotensin converting enzyme inhibitors on cardiac hypertrophy in the aortic-banded rat

Jpn Heart J406456541999

10.1536/jhj.40.645

10888384

A Romero

Mathew

Wasinski

Reed

Brody

Dawood

Twiner

McNaughton

Fridman

Flack

Angiotensin-converting enzyme inhibitors increase anti-fibrotic biomarkers in African Americans with left ventricular hypertrophy

J Clin Hypertens (Greenwich)23100810162021

10.1111/jch.14206

33694311

Liu

Shen

Wang

Liu

Zheng

Luo

EndophilinA2 protects against angiotensin II-induced cardiac hypertrophy by inhibiting angiotensin II type 1 receptor trafficking in neonatal rat cardiomyocytes

J Cell Biochem119829083032018

10.1002/jcb.26862

29923351

Walsh-Wilkinson

Drolet

Le Houillier

Roy

ÈM

Arsenault

Couet

Sex differences in the response to angiotensin II receptor blockade in a rat model of eccentric cardiac hypertrophy

PeerJ7e74612019

10.7717/peerj.7461

31404429

Chang

Tsai

Sung

Chen

Pandya

Maeda

Tsai

Diuretics prevent thiazolidinedione-induced cardiac hypertrophy without compromising insulin-sensitizing effects in mice

Am J Pathol1844424532014

10.1016/j.ajpath.2013.10.020

24287404

Okura

Miyoshi

Irita

Enomoto

Jotoku

Nagao

Watanabe

Matsuokan

Ashihara

Higaki

Comparison of the effect of combination therapy with an angiotensin II receptor blocker and either a low-dose diuretic or calcium channel blocker on cardiac hypertrophy in patients with hypertension

Clin Exp Hypertens355635692013

10.3109/10641963.2013.764892

23402476

Zhang

Wang

Loss of LRRC25 accelerates pathological cardiac hypertrophy through promoting fibrosis and inflammation regulated by TGF-β1

Biochem Biophys Res Commun5061371442018

10.1016/j.bbrc.2018.09.065

30340835

Zang

Wan

Zhang

Huang

Liu

Zhang

An updated role of astragaloside IV in heart failure

Biomed Pharmacother1261100122020

10.1016/j.biopha.2020.110012

32213428

Kang

Liu

Research progress in prevention and cure of fibrosis by traditional Chinese medicine

Mod Appl Sci21271322008

10.5539/mas.v2n5p127

Yang

Chen

Sun

Research progress on mechanism of action of Radix Astragalus in the treatment of heart failure

Chin J Integr Med182352402012

10.1007/s11655-012-1022-1

22466951

Karmazyn

Gan

Treatment of the cardiac hypertrophic response and heart failure with ginseng, ginsenosides, and ginseng-related products

Can J Physiol Pharmacol95117011762017

10.1139/cjpp-2017-0092

28505464

Yang

Wang

Zhang

Yang

Zhang

Astragaloside IV attenuates inflammatory cytokines by inhibiting TLR4/NF-кB signaling pathway in isoproterenol-induced myocardial hypertrophy

J Ethnopharmacol150106210702013

10.1016/j.jep.2013.10.017

24432369

Liu

Wang

Astragaloside IV protects against the pathological cardiac hypertrophy in mice

Biomed Pharmacother97146814782018

10.1016/j.biopha.2017.09.092

29793309

Guo

Gan

Haist

Rajapurohitam

Zeidan

Faruq

Karmazyn

Ginseng inhibits cardiomyocyte hypertrophy and heart failure via NHE-1 inhibition and attenuation of calcineurin activation

Circ Heart Fail479882011

10.1161/CIRCHEARTFAILURE.110.957969

20971938

Qin

Gong

Wei

Huang

Total ginsenosides inhibit the right ventricular hypertrophy induced by monocrotaline in rats

Biol Pharm Bull31153015352008

10.1248/bpb.31.1530

18670084

Dai

Zhou

Liu

Chen

Peng

Xiong

Traditional Chinese medicine formulas, extracts, and compounds promote angiogenesis

Biomed Pharmacother1321108552020

10.1016/j.biopha.2020.110855

33059257

Luo

Chen

Systematic review of compound danshen dropping pill: A chinese patent medicine for acute myocardial infarction

Evid Based Complement Alternat Med20138080762013

10.1155/2013/808076

23843882

Artemisinin-A gift from traditional Chinese medicine to the world (nobel lecture)

Angew Chem Int Ed Engl5510210102262016

10.1002/anie.201601967

27488942

Yang

Yuan

Zhou

Liu

The anti-inflammatory activity of licorice, a widely used Chinese herb

Pharm Biol555182017

10.1080/13880209.2016.1225775

27650551

Duan

Bian

Zhai

Shang

Lin

Zhao

Yin

Wen

Metabonomic strategy for the evaluation of Chinese medicine Salvia miltiorrhiza and Dalbergia odorifera interfering with myocardial ischemia/reperfusion injury in rats

Rejuvenation Res202632772017

10.1089/rej.2016.1884

28093038

Wang

Zhang

Wang

Gao

Dai

A comprehensive review on Pueraria: Insights on its chemistry and medicinal value

Biomed Pharmacother1311107342020

10.1016/j.biopha.2020.110734

32942158

Hou

Huang

Cai

Yuan

Liu

Zhao

Qiu

Cheng

Puerarin ameliorated pressure overload-induced cardiac hypertrophy in ovariectomized rats through activation of the PPARα/PGC-1 pathway

Acta Pharmacol Sin4255672021

10.1038/s41401-020-0401-y

32504066

Yuan

Shi

Jia

Shi

Zhang

Shou

Zhu

Use of network pharmacology to explore the mechanism of Gegen (Puerariae lobatae Radix) in the treatment of type 2 diabetes mellitus associated with hyperlipidemia

Evid Based Complement Alternat Med202166334022021

10.1155/2021/6633402

33953784

Zhou

Zhang

Peng

Puerarin: A review of pharmacological effects

Phytother Res289619752014

10.1002/ptr.5083

24339367

Liu

Zhang

Cai

Wang

Zhou

Yang

Liu

A diet formula of Puerariae radix, Lycium barbarum, Crataegus pinnatifida, and Polygonati rhizoma alleviates insulin resistance and hepatic steatosis in CD-1 mice and HepG2 cells

Food Funct5103810492014

10.1039/C3FO60524H

24626737

Liu

Huang

Zhang

Liu

Luo

Chen

Puerarin prevents cardiac hypertrophy induced by pressure overload through activation of autophagy

Biochem Biophys Res Commun4649089152015

10.1016/j.bbrc.2015.07.065

26188094

Gao

Guo

Zhao

Chen

Zhang

Yan

Liao

Chi

Carboxypeptidase A4 promotes cardiomyocyte hypertrophy through activating PI3K-AKT-mTOR signaling

Biosci Rep40BSR202006692020

10.1042/BSR20200669

32347291

Foulquier

Daskalopoulos

Lluri

Hermans

KCM

Deb

Blankesteijn

WNT signaling in cardiac and vascular disease

Pharmacol Rev70681412018

10.1124/pr.117.013896

29247129

Weeks

Bernardo

Ooi

JYY

Patterson

McMullen

The IGF1-PI3K-Akt signaling pathway in mediating exercise-induced cardiac hypertrophy and protection

Adv Exp Med Biol10001872102017

10.1007/978-981-10-4304-8_12

29098623

Fan

Qiu

Shu

Hagenmueller

Riffel

Meryer

Zhang

Hardt

Wang

Recombinant frizzled1 protein attenuated cardiac hypertrophy after myocardial infarction via the canonical Wnt signaling pathway

Oncotarget9306930802018

10.18632/oncotarget.23149

29423029

Livak

Schmittgen

Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method

Methods254024082001

10.1006/meth.2001.1262

11846609

Lieven

Knobloch

Rüther

The regulation of Dkk1 expression during embryonic development

Dev Biol3402562682010

10.1016/j.ydbio.2010.01.037

20144607

King

Liu

Bijur

Dkk1 stabilizes Wnt co-receptor LRP6: Implication for Wnt ligand-induced LRP6 down-regulation

PLoS One5e110142010

10.1371/journal.pone.0011014

20543981

Wang

Duan

Liu

Sui

Huang

Wen

IM-12 activates the Wnt-β-catenin signaling pathway and attenuates rtPA-induced hemorrhagic transformation in rats after acute ischemic stroke

Biochem Cell Biol977027082019

10.1139/bcb-2018-0384

31770017

Cheng

Shen

Chen

Liu

Lin

Chen

Qingda granule attenuates angiotensin II-induced cardiac hypertrophy and apoptosis and modulates the PI3K/AKT pathway

Biomed Pharmacother1331110222021

10.1016/j.biopha.2020.111022

33378940

Guo

Gong

Kesteven

Iismaa

Chan

Holman

Pinto

Pironet

The Ca²⁺-activated cation channel TRPM4 is a positive regulator of pressure overload-induced cardiac hypertrophy

Elife10e665822021

10.7554/eLife.66582

34190686

Schnelle

Chong

Zoccarato

Elkenani

Sawyer

Hasenfuss

Ludwig

Shah

In vivo [U-¹³C]glucose labeling to assess heart metabolism in murine models of pressure and volume overload

Am J Physiol Heart Circ Physiol319H422H4312020

10.1152/ajpheart.00219.2020

32648823

Song

Chen

Shen

Zhang

Distinct actions of intermittent and sustained β-adrenoceptor stimulation on cardiac remodeling

Sci China Life Sci544935012011

10.1007/s11427-011-4183-9

21706409

Ribeiro

Buttros

Oshima

Bergamaschi

Campos

Ascorbic acid prevents acute myocardial infarction induced by isoproterenol in rats: Role of inducible nitric oxide synthase production

J Mol Histol40991052009

10.1007/s10735-009-9218-1

19466570

Prabhu

Narayan

Devi

Mechanism of protective action of mangiferin on suppression of inflammatory response and lysosomal instability in rat model of myocardial infarction

Phytother Res237567602009

10.1002/ptr.2549

19172585

Wang

Chen

Zhao

Tao

YTHDF2 alleviates cardiac hypertrophy via regulating Myh7 mRNA decoy

Cell Biosci111322021

10.1186/s13578-021-00649-7

34266473

Zhang

Kimura

Murao

Obata

Matsuyoshi

Takaki

Effects of angiotensin type I receptor blockade on the cardiac Raf/MEK/ERK cascade activated via adrenergic receptors

J Pharmacol Sci1132242332010

10.1254/jphs.09336FP

20562518

Cai

Liu

Guo

β-Adrenergic stimulation activates protein kinase Cε and induces extracellular signal-regulated kinase phosphorylation and cardiomyocyte hypertrophy

Mol Med Rep11437343802015

10.3892/mmr.2015.3316

25672459

Werhahn

Kreusser

Hagenmüller

Beckendorf

Diemert

Hoffmann

Schultz

Backs

Dewenter

Adaptive versus maladaptive cardiac remodelling in response to sustained β-adrenergic stimulation in a new ‘ISO on/off model’

PLoS One16e02489332021

10.1371/journal.pone.0248933

34138844

Garg

Khanna

Exploration of pharmacological interventions to prevent isoproterenol-induced myocardial infarction in experimental models

Ther Adv Cardiovasc Dis81551692014

10.1177/1753944714531638

24817146

Liu

Ding

Chang

Zheng

Zhang

Wang

Baicalein attenuates cardiac hypertrophy in mice via suppressing oxidative stress and activating autophagy in cardiomyocytes

Acta Pharmacol Sin427017142021

10.1038/s41401-020-0496-1

32796955

Wen

Shen

Zhou

Zhao

Dai

Jin

Pyrroloquinoline quinone attenuates isoproterenol hydrochloride-induced cardiac hypertrophy in AC16 cells by inhibiting the NF-κB signaling pathway

Int J Mol Med458738852020

31922230

Zhao

Jiang

Chen

Zhang

Zhu

Yao

Dissection of mechanisms of Chinese medicinal formula Si-Miao-Yong-an decoction protects against cardiac hypertrophy and fibrosis in isoprenaline-induced heart failure

J Ethnopharmacol2481120502020

10.1016/j.jep.2019.112050

31265887

Zhang

Wang

Lin

Liu

Yuan

Lin

GDF11 attenuated ANG II-induced hypertrophic cardiomyopathy and expression of ANP, BNP and beta-MHC through down-regulating CCL11 in mice

Curr Mol Med186616712018

10.2174/1566524019666190204112753

30714521

Cameron

Rademaker

Ellmers

Espiner

Nicholls

Richards

Atrial (ANP) and brain natriuretic peptide (BNP) expression after myocardial infarction in sheep: ANP is synthesized by fibroblasts infiltrating the infarct

Endocrinology141469046972000

10.1210/endo.141.12.7847

11108284

Edwards

Cardiac MHC gene expression: More complexity and a step forward

Am J Physiol Heart Circ Physiol294H14H152008

10.1152/ajpheart.01297.2007

17993592

Yuan

Zong

Zhou

Bian

Deng

Dai

Gan

Yang

Tang

Puerarin attenuates pressure overload-induced cardiac hypertrophy

J Cardiol6373812014

10.1016/j.jjcc.2013.06.008

23906530

Yeh

Tsai

Cheng

Pai

Chen

Lai

Huang

Padma

Huang

Mechanism of Taiwan Mingjian Oolong tea to inhibit isoproterenol-induced hypertrophy and apoptosis in cardiomyoblasts

Am J Chin Med4477862016

10.1142/S0192415X16500051

26916915

Guan

Hong

Zhao

Liu

Xiao

Chen

Deng

Wang

CD38 promotes angiotensin II-induced cardiac hypertrophy

J Cell Mol Med21149215022017

10.1111/jcmm.13076

28296029

Jiang

Cao

Zhang

Jiang

Tian

Feng

Dou

Gorospe

Zheng

HuR regulates phospholamban expression in isoproterenol-induced cardiac remodelling

Cardiovasc Res1169449552020

10.1093/cvr/cvz205

31373621

Huo

Shi

Yan

Luo

Guo

Lin

Zhang

Alleviation of inflammation and oxidative stress in pressure overload-induced cardiac remodeling and heart failure via IL-6/STAT3 inhibition by raloxifene

Oxid Med Cell Longev202166990542021

10.1155/2021/6699054

33824698

Zhang

Yuan

Liu

MiRNA-339-5p promotes isoproterenol-induced cardiomyocyte hypertrophy by targeting VCP to activate the mTOR signaling

Cell Biol Int462882992022

10.1002/cbin.11731

34854520

Han

Shi

Zheng

Shi

Jiang

Han

DL-3-n-butylphthalide attenuates myocardial hypertrophy by targeting gasdermin D and inhibiting gasdermin D mediated inflammation

Front Pharmacol126881402021

10.3389/fphar.2021.688140

34168567

Shah

Bhullar

Elimban

Dhalla

Oxidative stress as a mechanism for functional alterations in cardiac hypertrophy and heart failure

Antioxidants (Basel)109312021

10.3390/antiox10060931

34201261

Gai

Wang

Tian

Gong

Zhao

Whole genome level analysis of the Wnt and DIX gene families in mice and their coordination relationship in regulating cardiac hypertrophy

Front Genet126089362021

10.3389/fgene.2021.608936

34168671

Qin

Zhang

Wang

Puerarin suppresses Na+-K+-ATPase-mediated systemic inflammation and CD36 expression, and alleviates cardiac lipotoxicity in vitro and in vivo

J Cardiovasc Pharmacol684654722016

10.1097/FJC.0000000000000431

27606935

Moon

Kohn

De Ferrari

Kaykas

WNT and beta-catenin signalling: Diseases and therapies

Nat Rev Genet56917012004

10.1038/nrg1427

15372092

Agostino

Pohl

SÖ

The structural biology of canonical Wnt signalling

Biochem Soc Trans48176517802020

10.1042/BST20200243

32725184

Hua

Yang

Zhu

Wang

Gan

Oligomerization of Frizzled and LRP5/6 protein initiates intracellular signaling for the canonical WNT/β-catenin pathway

J Biol Chem29319710197242018

10.1074/jbc.RA118.004434

30361437

Gao

Chen

Dishevelled: The hub of Wnt signaling

Cell Signal227177272010

10.1016/j.cellsig.2009.11.021

20006983

Zeng

Wang

Guo

Liu

Wang

Feng

Zhang

Liao

Zhao

Small molecule induces mitochondrial fusion for neuroprotection via targeting CK2 without affecting its conventional kinase activity

Signal Transduct Target Ther6712021

10.1038/s41392-021-00533-3

33602894

Ríos

Godoy

Inestrosa

Wnt3a ligand facilitates autophagy in hippocampal neurons by modulating a novel GSK-3β-AMPK axis

Cell Commun Signal16152018

10.1186/s12964-018-0227-0

29642895

Barker

Morin

Clevers

The Yin-Yang of TCF/beta-catenin signaling

Adv Cancer Res771242000

10.1016/S0065-230X(08)60783-6

10549354

Piazza

Manni

Tubi

Montini

Pavan

Colpo

Gnoato

Cabrelle

Adami

Zambello

Glycogen synthase kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

BMC Cancer105262010

10.1186/1471-2407-10-526

20920357

Guo

Gupte

Umbarkar

Singh

Sui

Force

Lal

Entanglement of GSK-3β, β-catenin and TGF-β1 signaling network to regulate myocardial fibrosis

J Mol Cell Cardiol1101091202017

10.1016/j.yjmcc.2017.07.011

28756206

Guan

Wei

Shi

Zhao

Pan

Han

Hou

Yang

Crosstalk between Wnt/β-catenin signaling and NF-κB signaling contributes to apical periodontitis

Int Immunopharmacol981078432021

10.1016/j.intimp.2021.107843

34153668

Jia

Yang

Liu

Tan

Ooi

Chi

Filion

Figeys

Wang

β-Catenin and NF-κB co-activation triggered by TLR3 stimulation facilitates stem cell-like phenotypes in breast cancer

Cell Death Differ222983102015

10.1038/cdd.2014.145

25257174

Shang

Hua

The regulation of β-catenin activity and function in cancer: Therapeutic opportunities

Oncotarget833972339892017

10.18632/oncotarget.15687

28430641

Gitau

Zhao

Guo

Liang

Qian

Wang

Acetyl salicylic acid attenuates cardiac hypertrophy through Wnt signaling

Front Med94444562015

10.1007/s11684-015-0421-z

26626190

Olsen

Dimaano

Fritz-Hansen

Sogaard

Chakir

Eskesen

Steenbergen

Kass

Abraham

Hypertrophy signaling pathways in experimental chronic aortic regurgitation

J Cardiovasc Transl Res68528602013

10.1007/s12265-013-9503-y

23888404

Liu

Shentu

Wang

Zhang

Sun

Wang

Inhibition of NF-κB and Wnt/β-catenin/GSK3β signaling pathways ameliorates cardiomyocyte hypertrophy and fibrosis in streptozotocin (STZ)-induced type 1 diabetic rats

Curr Med Sci4035472020

10.1007/s11596-020-2144-x

32166663

Fang

Tang

Qiao

Zhang

Wang

Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

Mol Med Rep137207302016

10.3892/mmr.2015.4586

26648540

Kim

Song

Lee

Kim

Kwon

Kim

Jeong

Lee

PARsylated transcription factor EB (TFEB) regulates the expression of a subset of Wnt target genes by forming a complex with β-catenin-TCF/LEF1

Cell Death Differ28255525702021

10.1038/s41418-021-00770-7

33753903

Zhang

Guo

Wang

Geng

Han

The protective effect of kaempferol on heart via the regulation of Nrf2, NF-κβ, and PI3K/Akt/GSK-3β signaling pathways in isoproterenol-induced heart failure in diabetic rats

Drug Dev Res802943092019

10.1002/ddr.21495

30864233

Figure 1.

Effect of PU (5–80 µM) on myocardial AC16 cell viability. Association between PU concentration and cell viability. PU, puerarin.

Figure 2.

Effect of PU on transcription of ANP, BNP and β-MHC. mRNA expression levels of (A) ANP, (B) BNP and (C) β-MHC in ISO- and PU + ISO-treated AC16 cells were semi-quantified using reverse transcription-quantitative PCR. *P<0.05 and **P<0.01. CN, control; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain; ISO, isoproterenol; PU, puerarin.

Figure 3.

Effect of PU on ANP and BNP protein expression levels. (A) Representative western blotting images of ANP and BNP protein expression. (B) ANP and (C) BNP protein expression levels were semi-quantified using ImageJ. **P<0.01. CN, control; ISO, isoproterenol; PU, puerarin; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide.

Figure 4.

Effect of PU on surface area of AC16 cells. Fluorescent images of (A) control and cells treated with (B) ISO or (C) PU and ISO. (D) Cell surface area was quantified using ImageJ software. Magnification, 400×. *P<0.05 and **P<0.01. CN, control; ISO, isoproterenol; PU, puerarin.

Figure 5.

Effect of PU on expression of components in the Wnt/β-catenin and NF-κB signaling pathways. (A) Representative western blotting images of LRP6, c-Myc, p-GSK3β and GSK3β. (B) Representative western blotting images of p-GSK3β and GSK3β. (C) Representative western blotting images of β-catenin, Wnt5a/b, p-p65 and p65. (D) LRP6, (E) c-Myc, (F) p-GSK3β, (G) GSK3β, (H) β-catenin, (I) Wnt5a/b and (J) p-p65 protein expression levels were semi-quantified. (K) Ratio of p-p65/p-65. The blots were analyzed using ImageJ. *P<0.05 and **P<0.01. CN, control; ISO, isoproterenol; PU, puerarin; LRP6, low-density lipoprotein receptor-related protein 6; p-, phosphorylated; GSK3β, glycogen synthase kinase 3β.

Figure 6.

Effect of DKK1 on expression of protein in the signaling Wnt pathway. (A) Representative western blotting images of LRP6. (B) Representative western blotting images of β-catenin, Wnt5a/b, c-Myc and ANP. (C) LRP6, (D) β-catenin, (E) c-Myc, (F) Wnt5a/b and (G) ANP protein expression levels were semi-quantified. The blots were analyzed using ImageJ. **P<0.01. DKK1, Dickkopf-1; CN, control; ISO, isoproterenol; LRP6, low-density lipoprotein receptor-related protein 6; ANP, atrial natriuretic peptide.

Figure 7.

Effect of IM-12 and PU on expression of proteins in the Wnt signaling pathway. (A) Representative western blotting images of LRP6, β-catenin and Wnt5a/b expression. (B) Representative western blotting images of c-Myc and GSK3β expression. (C) LRP6, (D) β-catenin, (E) Wnt5a/b, (F) c-Myc and (G) GSK3β protein expression levels were semi-quantified. Blots were analyzed using ImageJ. *P<0.05 and **P<0.01. CN, control; PU, puerarin; LRP6, low-density lipoprotein receptor-related protein 6; GSK3β, glycogen synthase kinase 3β.

Figure 8.

Effect of ISO, PU and IM-12 on expression of proteins in the Wnt signaling pathway. (A) Representative western blotting images of LRP6, GSK3β and β-catenin expression. (B) Representative western blotting images of c-Myc, Wnt5a/b and ANP expression. (C) LRP6, (D) β-catenin, (E) GSK3β, (F) c-Myc, (G) Wnt5a/b and (H) ANP protein expression levels were semi-quantified. The blots were analyzed using ImageJ. *P<0.05 and **P<0.01. CN, control; ISO, isoproterenol; PU, puerarin; LRP6, low-density lipoprotein receptor-related protein 6; GSK3β, glycogen synthase kinase 3β; ANP, atrial natriuretic peptide; ns, not significant.