Zingerone attenuates aortic banding‐induced cardiac remodelling via activating the eNOS/Nrf2 pathway

Abstract Cardiac remodelling refers to a series of changes in the size, shape, wall thickness and tissue structure of the ventricle because of myocardial injury or increased pressure load. Studies have shown that cardiac remodelling plays a significant role in the development of heart failure. Zingerone, a monomer component extracted from ginger, has been proven to possess various properties including antioxidant, anti‐inflammatory, anticancer and antidiabetic properties. As oxidative stress and inflammation contribute to acute and chronic myocardial injury, we explored the role of zingerone in cardiac remodelling. Mice were subjected to aortic banding (AB) or sham surgery and then received intragastric administration of zingerone or saline for 25 days. In vitro, neonatal rat cardiomyocytes (NRCMs) were treated with zingerone (50 and 250 μmol/L) when challenged with phenylephrine (PE). We observed that zingerone effectively suppressed cardiac hypertrophy, fibrosis, oxidative stress and inflammation. Mechanistically, Zingerone enhanced the nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2)/antioxidant response element (ARE) activation via increasing the phosphorylation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Additionally, we used Nrf2‐knockout (KO) and eNOS‐KO mice and found that Nrf2 or eNOS deficiency counteracts these cardioprotective effects of zingerone in vivo. Together, we concluded that zingerone may be a potent treatment for cardiac remodelling that suppresses oxidative stress via the eNOS/Nrf2 pathway.

autophagy alterations, endoplasmic reticulum (ER) stress and metabolism impairment). 2 However, our understanding of the key processes responsible for the transition to HF remains incomplete and far less is known about how cardiac remodelling is suppressed.
Oxidative stress refers to the excessive production of reactive oxygen species (ROS) when cells are exposed to harmful stimuli. ROS can directly react with membrane lipids, proteins and nucleic acids to cause apoptosis and necrosis. 3,4 Additionally, ROS can act as signalling molecules that trigger the production of pro-inflammatory cytokines. 5 Accumulating evidence has suggested that oxidative stress is an important factor that exacerbates cardiac remodelling. 6 Inhibition of oxidative stress could be of significant therapeutic value in the treatment of cardiac remodelling and HF.
Zingerone is a nontoxic and inexpensive compound extracted from a common seasoning in Chinese food, dried ginger, with varied pharmacological activities including antioxidant, anti-inflammatory, anticancer and antidiabetic activities. 7 Numerous studies have found that zingerone is a potent antioxidant. It protects DNA against stannous chloride-induced ROS oxidative damage, 8 prevents oxidative stress in intestine smooth muscles 9 and reduces mitochondrial injury and lipid peroxidation. 10 Furthermore, zingerone has higher antioxidant activity than ascorbic acid 8 and exerts a scavenging effect against peroxynitrite formed from the reaction of superoxide and nitric oxide. 11 Recently, accumulating evidence has suggested that zingerone protects the heart from myocardial infarction injury via its antioxidant and free radical scavenging properties. [12][13][14] Another study revealed that pretreatment with zingerone relieves hyperlipidaemia and cardiac hypertrophy in isoproterenol-induced myocardial infarcted rats. 15 However, it is not clear whether zingerone exerts protective effects in AB-induced cardiac remodelling.
Therefore, this investigation was designed to clarify the role of zingerone in cardiac remodelling induced by AB surgery.

| Animals and animal models
Male C57/B6J mice were obtained from the Beijing HFK Bioscience CO LTD. eNOS-knockout (KO) (Stock no. 002684) and Nrf2-KO (Stock no. 017009) mice were obtained from the Jackson Laboratory. Mice were subject to AB or sham operation as described in the previous study. 16 Briefly, the thoracic cavity was opened from the second and third ribs on the left side of the mouse, and the thoracic aorta was separated.
Then, a 27-gauge (bodyweight of 24-25 g) or 26-gauge (bodyweight of 26-27 g) needle was placed in the direction parallel to the blood vessel, the blood vessel was ligated with the needle, and the needle was quickly pulled out the of the vessel. Once the blood vessel formed approximately 70% stenosis, the thoracic cavity was closed. In the sham operation group, only a line was hung, but the vessel was not ligated, the rest of the steps were the same as those performed in the surgery group. The male C57/B6 mice were randomly grouped into sham or an AB surgery group with or without zingerone treatment. In the mechanism research section, the Nrf2-KO and eNOS-KO mice were used and were also grouped as before. Four weeks post-AB or sham procedure, the hearts and lungs of the mice were harvested and weighed.

| Dosage information
Zingerone was obtained from Winherb Medical Science Co Ltd and was dissolved in sterile saline. Zingerone was intragastrically administered to mice 3 days after AB surgery and repeatedly for another 25 days at a dose of 10 or 20 mg/kg/d according to the previous studies. 17,18

| Echocardiography and haemodynamics
Echocardiography and haemodynamics were performed according to our previous studies. 19,20 Briefly, a MyLab 30CV ultrasound Instruments). The data of end-systolic pressure (ESP), end-diastolic pressure (EDP), minimal rate of pressure decay (dp/dt min) and maximal rate of pressure development (dp/dt max) were recorded and processed by PVAN software.

| Quantitative real-time RT-PCR
Total RNA of heart tissues and cells was collected with TRIzol (Invitrogen) and reverse transcribed into cDNA with oligo-dT primers and reverse transcriptase. Light Cycler 480 SYBR Green 1 Master Mix (Roche) was used for RT-PCR analysis. The transcript quantities were normalized to the amount of GAPDH gene expression.

| Western blot
Western blots were performed according to our previous studies. 19,20 A Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology) was used for the protein extraction.

| Cell culture and treatment
Neonatal rat cardiomyocytes were isolated and cultured as our previous study. 21

| ROS detection in NRCMs
Dichlorofluorescein diacetate (DCFH-DA) was diluted 1:1000 with serum-free medium to a final concentration of 10 μmol/L. The cell culture medium was removed, and the appropriate volume of diluted DCFH-DA was added. One well of a six-well plate received 1 mL of the diluted DCFH-DA. The plate was incubated for 30 minutes at 37°C in a cell incubator. The cells were washed three times with serum-free cell culture medium to remove DCFH-DA that did not enter the cells, and immunofluorescence was observed by a laser confocal microscope.

| NO production and SOD activity detection
A Total Nitric Oxide Assay Kit was used to measure NO production (Beyotime Institute of Biotechnology). NO itself is extremely unstable and is rapidly metabolized to nitrate and nitrite in cells, and nitrate reductase is used to reduce nitrate to nitrite; then, the nitrite is detected by the classical Griess reagent to indirectly determine total nitric oxide. The activity of superoxide dismutase (SOD) was detected by a Total Superoxide Dismutase Assay Kit with WST-8 (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

| Cell viability assay
The cells were incubated in a 96-well plate. After the cells were attached, different concentrations of zingerone were added to each well and incubated for 24 hours at 37°C. Then, MTT (5 mg/mL) was added and incubated for 4 hours. The culture solution was discarded, 100 µL of DMSO was added to each well, and the absorbance value was measured after 10-minutes incubation.

| Immunofluorescence
The cells were incubated in a 24-well plate; after the cells were attached, the medium was discarded, 4% paraformaldehyde was added to fix the cells, and 0.1% Triton X-100 was added to permeabilize them. Primary α-actinin (Abcam) and Nrf2 (Abcam) antibodies were applied at 4°C overnight; goat anti-rabbit (LI-COR) was used as the secondary antibody and was added to the cells and incubated for 60 minutes at 37°C. Immunofluorescence was observed by a laser confocal microscope.

| Data and statistical analysis
The data are expressed as the means ± SD and were analysed by SPSS software, version 23.0. One-way ANOVA followed by Tukey's post hoc test was used for group comparisons. Two-group comparisons were performed by Student's unpaired t test. The value of P < .05 was defined as significantly different.

| Zingerone suppressed PE-induced NRCMs hypertrophy
To investigate the effects of zingerone on cardiac hypertrophy, we treated NRCMs with a hypertrophic agonist, PE (50 μmol/L), and assessed the effect of zingerone on NRCM hypertrophy. First, we determined different zingerone dose-responses in NRCMs. As shown in Figure

| Zingerone prevented cardiac hypertrophy and fibrosis induced by AB surgery
To explore the effects of zingerone on cardiac remodelling, all mice were subjected to AB surgery or a sham operation with or  Figure 2H-I, the degree of perivascular and interstitial fibrosis was remarkably reduced in zingerone-treated mice subjected to AB. Additionally, the mRNA levels of fibrosis markers, transforming growth factor β1 (TGF-β1), connective tissue growth factor (CTGF), and collagen I and collagen III were dramatically decreased in zingerone-treated mice subjected to AB ( Figure 2J). These results indicated that zingerone abated the cardiac hypertrophy and fibrosis induced by AB surgery.

| Zingerone relieved pressure overload-induced heart dysfunction in mice
As shown in Table S1 in Appendix S1, vehicle-treated mice after AB surgery exhibited aggravated cardiac function, with reduced LVEF, LVFS and dilated left ventricular diameter (increased LVEDd, LVESd) and wall hypertrophy (increased LVPWd).
However, the cardiac dysfunction was alleviated dramatically in zingerone-treated mice after AB, which was assessed by the elevated LVEF and LVFS and reduced LV diameter and wall thickness. Additionally, haemodynamic parameters also displayed better systolic and diastolic LV functions in zingerone-treated mice after AB, as assessed by ESP, EDP, dp/dt min and dp/dt max. Accordingly, zingerone administration could alleviate heart dysfunction in mice after AB surgery.

| Zingerone attenuated oxidative stress in vitro
In vitro, we detected the cellular ROS generation in each group, and we found that zingerone treatment dramatically inhibited

| Zingerone enhanced Nrf2/ARE activation in vivo and in vitro
Nrf2 is a defensive pathway involved in oxidative and chemical stress. We next explored whether zingerone affected the activation of Nrf2. We found decreased protein expression levels of nucleus-Nrf2 (N-Nrf2), heme oxygenase-1 (HO-1) and SOD and increased protein expression levels of Kelch-like ECH-associated protein

| The effects of Nrf2 in zingerone-mediated cardioprotection in vivo and in vitro
To further evaluate the necessity of the Nrf2 pathway on the anti-remodelling effects of zingerone, NRCMs were treated with si Nrf2 to knock-down Nrf2 ( Figure S2A-B in Appendix S1). Interestingly, zingerone-mediated cardioprotective effects (anti-hypertrophy and antioxidative stress) were counteracted by Nrf2 knock-down ( Figure 6A-D). Furthermore, the transcription levels of the hypertrophic markers and NADPH oxidase such   Figure S2E in Appendix S1). In vivo, we used Nrf 2-KO mice and found that the HW/BW, LW/BW, HW/TL and LW/TL ratios increased in both the zingerone-treated and vehicle-treated mice after AB surgery ( Figure 6E). Additionally, the heart function (Table S2 in Appendix S1), cardiomyocyte area

| Zingerone improved the eNOS activation and NO production in vivo and in vitro
It is well-established that the transcriptional regulation of those antioxidant genes by Nrf2 signalling is mainly through NO. 22 The biosynthesis of NO by NOS enzymes is well defined. Additionally, increased phosphorylation at Ser1177/9 leads to eNOS activation and to elevated NO production. 23 To further evaluate the mechanism underlying how zingerone affects the Nrf2 pathway, we next investigated the eNOS-NO axis. We found that the eNOS expression levels were unchanged, but p-eNOS (Ser1177) expression levels were reduced in mouse heart after AB surgery in vivo and in NRCMs with PE

| Zingerone-mediated cardioprotection depends on the eNOS/Nrf2 pathway in vitro
To further evaluate the effects of eNOS on zingerone-mediated cardioprotection, we treated the NRCMs with si eNOS ( Figure   S2C-D in Appendix S1). As expected, zingerone-mediated cardioprotective effects (anti-hypertrophy and antioxidative stress) in NRCMs were effectively abolished by silencing eNOS (Figure 7G-J, Figure S2F in Appendix S1). Moreover, the protein expression of N-Nrf2 was significantly decreased after PE + si RNA treatment and dramatically increased with PE + si RNA + zingerone treatment; however, after eNOS knock-down, the N-Nrf2 protein expression was significantly down-regulated ( Figure 7K-L). Additionally, Nrf2 silencing did not influence the expression of eNOS, p-eNOS (S1177) and NO production in NRCMs ( Figure S4A-C in Appendix S1).

| eNOS deficiency counteracted the cardioprotection effects of Zingerone in vivo
In vivo, we further used eNOS-KO mice to investigate the effect of eNOS on zingerone-mediated cardioprotection. Consistent with the results in vitro, the cardioprotection of zingerone treatment after AB surgery was effectively abolished by eNOS deletion, as demonstrated by the same increase in the HW/BW, LW/BW, HW/TL and LW/TL ratios between zingerone-treated and vehicle-treated mice after AB surgery ( Figure 8A). In addition, the heart function (Table S3 in Appendix S1), cardiomyocyte area ( Figure 8B-C), LV collagen volume ( Figure 8D-E), 4-HNE production ( Figure 8F-G) and transcription level of hypertrophic, fibrotic and oxidative stress markers showed no significant differences between the zingeroneand vehicle-treated mice after AB ( Figure S3C-E in Appendix S1).
Additionally, zingerone treatment did not enhance Nrf2 activation because of the same decreased N-Nrf2 expression in zingeronetreated and vehicle-treated mice after the AB surgery ( Figure 8H-I).
These data suggested that eNOS ablation completely eliminated the cardioprotection of zingerone treatment. And that zingerone-mediated cardioprotection depends on the eNOS/Nrf2 pathway.

| D ISCUSS I ON
Ginger is one of the most common spices originating in South-East Asia. Zingerone is present in significant amounts (9.25%) in ginger, and cooking or drying also converts gingerol (another component in ginger) into zingerone by a retro-aldol reaction. Zingerone is known to have potent pharmacological activities, such as antioxidant and anti-inflammatory properties. 7 Numerous studies have suggested that zingerone protects against cardiac oxidative stress and inflammation damage in the streptozotocin-induced diabetic mice, 24 isoproterenol-induced myocardial infarction rats, 13,14 chemo/radiotherapy-mediated cardiac damage, among others. 25 These data suggest a potential cardioprotection of zingerone in heart diseases, especially in cardiac remodelling. Our research further explored its effect in pressure overload-induced cardiac remodelling and found that zingerone significantly inhibited pathological cardiac hypertrophy, fibrosis, heart dysfunction, inflammation and oxidative stress induced by hypertrophic stimuli. Furthermore, we also revealed that the mechanism by which zingerone inhibits oxidative stress following hypertrophic stress is through the eNOS-NO-Nrf2 signalling pathway.
Oxidative stress is generated when ROS overwhelm the antioxidant enzymes. To combat the harm caused by oxidative stress, cells defend themselves with enzymes such as SOD, catalase and glutathione peroxidase. 26 There is overwhelming evidence indicating that increased oxidative stress and reduced activity of antioxidants are closely related to the development and propagation of cardiac hypertrophy and HF in animal models and humans. 5,6 Nrf2 is a redoxsensitive transcription factor that normally resides in the cytoplasm bound to keap1. Upon exposure to pro-oxidative or electrophilic stimuli, cysteine residues of keap1 are oxidized or covalently modified, and Nrf2 is released to the nucleus. By binding to ARE consensus sequences, Nrf2 initiates many antioxidant genes that preserve cellular homeostasis. 27,28 In the mechanism of zingerone-mediated cardioprotection against pathological cardiac remodelling, we found F I G U R E 5 Zingerone enhanced the Nrf2 nuclear translocation in vivo and in vitro. A, Immunofluorescence staining of Nrf 2 was used to detect the Nrf2 nuclear translocation on myocardial sections (n = 6 samples). B, Immunofluorescence staining of Nrf 2 was used to detect the Nrf2 nuclear translocation on NRCMs (n = 6 samples, 50 + cells per group) a dramatic inhibition of oxidative stress by zingerone, and the ef- at the Ser1177 site in response to various stimuli. 30,31 In our study, p-eNOS (Ser1177) expression dramatically increased with zingerone treatment after AB surgery in vivo and after PE challenge in vitro, thus leading to increased production of NO.
Accumulating evidence indicates the importance of NO in heart diseases with a spectrum of related pathologies, which include hypertension, 32 CVD 33 and atherogenesis. 34 It is well known that NO is Based on the available information, it is apparent that zingerone promotes eNOS activation, increasing NO production and targeting Nrf2-associated antioxidant gene transcription in cardiac remodelling. Therefore, zingerone may be considered of therapeutic interest in the prevention and treatment of cardiac remodelling and HF.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data will be made available after been required upon request from the corresponding author.