Thymoquinone ameliorates pressure overload‐induced cardiac hypertrophy by activating the AMPK signalling pathway

Abstract Prolonged pathological myocardial hypertrophy leads to end‐stage heart failure. Thymoquinone (TQ), a bioactive component extracted from Nigella sativa seeds, is extensively used in ethnomedicine to treat a broad spectrum of disorders. However, it remains unclear whether TQ protects the heart from pathological hypertrophy. This study was conducted to examine the potential utility of TQ for treatment of pathological cardiac hypertrophy and if so, to elucidate the underlying mechanisms. Male C57BL/6J mice underwent either transverse aortic constriction (TAC) or sham operation, followed by TQ treatment for six consecutive weeks. In vitro experiments consisted of neonatal rat cardiomyocytes (NRCMs) that were exposed to phenylephrine (PE) stimulation to induce cardiomyocyte hypertrophy. In this study, we observed that systemic administration of TQ preserved cardiac contractile function, and alleviated cardiac hypertrophy, fibrosis and oxidative stress in TAC‐challenged mice. The in vitro experiments showed that TQ treatment attenuated the PE‐induced hypertrophic response in NRCMs. Mechanistical experiments showed that supplementation of TQ induced reactivation of the AMP‐activated protein kinase (AMPK) with concomitant inhibition of ERK 1/2, p38 and JNK1/2 MAPK cascades. Furthermore, we demonstrated that compound C, an AMPK inhibitor, abolished the protective effects of TQ in in vivo and in vitro experiments. Altogether, our study disclosed that TQ provides protection against myocardial hypertrophy in an AMPK‐dependent manner and identified it as a promising agent for the treatment of myocardial hypertrophy.

fully meet the clinical needs. Therefore, the identification of novel protective agents is of great interest for improving preventive and therapeutic strategies.
AMP-activated protein kinase (AMPK) is a kinase that plays a crucial role in cell growth regulation and mitochondrial function during metabolic stress. 6 Accumulating evidence has suggested AMPK as an inhibitor of cardiac hypertrophy due to its inhibition on protein synthesis and reactive oxygen species (ROS) production. [7][8][9][10] Under physiological conditions, intracellular ROS are produced and cleared in an equilibrium state. When cardiomyocytes are exposed to pathogenic stimuli, ROS accumulate and stimulate intracellular signalling proteins with a critical role, such as mitogen-activated protein kinase (MAPK). MAPK transduces signals to transcription factors and reactivates fetal gene expression. [11][12][13] Thymoquinone (TQ, Figure S1) is a bioactive natural product mainly derived from Nigella sativa seeds (black cumin). It has been widely used in ethnomedicine to treat disorders including diabetes, cancer, rheumatism and neurological diseases. 14 With regard to the cardiovascular system, several population and rodent studies have shown that TQ provides protection against doxorubicin-induced cardiotoxicity, 15 allows for substantial recovery of cardiac function after ischemia/reperfusion injury, 16 and exhibits a blood pressurelowering effect. 17 The primary mechanism underlying these effects may be its antioxidant activity, mediated by its ROS scavenger property and the preservation of endogenous antioxidants. 18,19 Although previous studies have explored the effect of Nigella sativa (the origin plant of which TQ is derived) on physiological cardiac hypertrophy, 20,21 it has not been yet elucidated whether this plant or TQ has a therapeutic effect on pathological cardiac hypertrophy.
In this study, we tested the hypothesis that TQ may be a promising biological therapy to slow the progression of pathological cardiac hypertrophy. We also investigated the potential underlying mechanisms using a selective inhibitor of AMPK in in vivo and in vitro experiments.

| Animals and treatment
All experimental animal procedures were approved by the Tab First, the mice were randomly assigned to one of the following four groups after 1 week of adaptation: sham operation + corn oil (sham + vehicle), sham operation + TQ (sham + TQ), transverse aortic constriction (TAC) operation + corn oil (TAC + vehicle) and TAC operation + TQ treatment (TAC + TQ). The TAC-induced pressure overload model was established according to that described in a previous study. 22 Mice in the sham group underwent a similar procedure without constricting the aorta. Two days after the operation, TQ (50 mg/kg, dissolved in corn oil) or the same volumes of corn oil were orally administrated once daily for six consecutive weeks.
Then, experiments were performed to examine the mechanism of action of TQ. Animals were randomly assigned to one of the following four groups: sham + vehicle, TAC + vehicle, TAC + TQ and TAC + TQ + CpC. TQ was administered at the same daily dose as previously described. The AMPK inhibitor CpC was administered intraperitoneally at 20 mg/kg/day. At 6 weeks, mice underwent echocardiography and were sacrificed for the other experiments.
Subsequently, transthoracic echocardiography was performed by a skilled blinded technologist using a GE Vivid E95 ultrasound system (General Electric Company) equipped with an 18-MHz probe. Data

| Histological analysis
The ratios of heart weight/body weight (HW/BW) and HW/tibia length (HW/TL) were calculated to assess the severity of LV hypertrophy. The hearts were fixed in 4% paraformaldehyde immediately after isolation. After 16 h, the hearts were embedded with paraffin, followed by transverse slicing into 4μm sections. The slices were stained with haematoxylin and eosin, and Masson's trichrome following standard methods. 23,24 Ventricular sections were stained with wheat germ agglutinin (WGA) labelled with fluorescein isothiocyanate (FITC) to examine the cross-sectional area of cardiomyocytes. The cell area and myocardial fibrosis were measured using Image J software (NIH).

| Neonatal rat cardiomyocyte culture and treatment
Primary Neonatal rat cardiomyocyte (NRCMs) were isolated from neonatal (1-3 day) Sprague Dawley rats using a Neonatal Heart Dissociation Kit (Miltenyi Biotechnology) according to the manufacturer's instructions. Subsequently, the cardiomyocytes were kept in DMEM culture media containing 10% fetal bovine serum for 24 h under normal conditions (at 37°C with 5% CO 2 ). This was followed by a change to serum-free DMEM for 18 h. The cells were then preincubated with the AMPK inhibitor CpC (5 µM) for 1 h and simultaneously treated with phenylephrine (PE, 50 µM) and TQ (5 µM) for another 24 h before they were harvested.

| RNA isolation and quantitative real-time PCR
Trizol reagent (Takara) was used to extract total RNA from ventricular tissues and NRCMs. HiScript II Q RT SuperMix (Vazyme) was used to perform reverse transcription. To quantify mRNA levels of genes, such as ANP, BNP and collagen I, qRT-PCR was conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The PCR primer sequences of the target gene are reported in Table S1.
GAPDH was used as an internal control. The comparative Ct(2 −ΔΔC T ) method was employed for subsequent analysis.

| Western blot analysis
Total protein was isolated from ventricular tissues and NRCMs in lysis buffer (Beyotime Biotechnology). Equal amounts of protein (20-30 µg) were separated on 10% SDS/PAGE by electrophoresis. The proteins were subsequently transferred to polyvinylidene difluoride membranes (Millipore) and incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. All bands on blots were detected by chemiluminescence and analysed with ImageJ software.

| Determination of oxidative stress
Dihydroethidium (DHE) staining was used to detect ROS in cardiac tissues as previously described. 25 In brief, the ventricular tissues were frozen in liquid nitrogen immediately after isolation, and sliced into 5μm-thick frozen sections. The slices were then stained with DHE and placed in a humidified room for 15 min at 37°C in the dark. A DCFH-DA fluorescent probe (Beyotime Biotechnology) was used to measure ROS generation in NRCMs. The probe was diluted to 5 μM in serum-free medium before use. After treatment, the cardiomyocytes were incubated with the probe for 20 min at 37°C in a CO 2 incubator. Images of DHE staining and intracellular DCF fluorescence were captured by fluorescence microscopy (Olympus IX83). ImageJ software was employed to quantify the fluorescence intensity.

| Measurement of cell surface area
NRCMs were stained with FITC-Phalloidin (Yeasen Biotechnology) for myocyte size detection according to the manufacturer's protocol. To identify nuclei, fixed cells were counterstained with DAPI (Dawen Biotechnology). Immunofluorescent images were captured by an LSM 710 confocal microscope (Zeiss). Cell area was measured using ImageJ software.

| Statistical analysis
Statistical analyses were conducted using the GraphPad Prism program (GraphPad). When data were normally distributed, one-way ANOVA followed by Bonferroni post hoc test was performed to compare data from more than two groups. All data are presented as the mean ±SEM, and p < 0.05 was considered statistically significant.

| TQ attenuated pressure overload-induced cardiac failure and hypertrophy
TAC surgery was performed to establish a mouse model of cardiac dysfunction and hypertrophy. Based on a previous study, 26

| TQ ameliorated cardiac fibrosis following TAC surgery
It is well-documented that cardiac fibrosis typically occurs together with pathological hypertrophy and acts as an essential factor driving the progression of cardiac dysfunction. 27 Here, myocardial interstitial and perivascular fibrosis in the ventricular tissues was evaluated in Masson's trichrome-stained sections 6 weeks after the operation.
TAC operation, as expected, promoted the progression of cardiac fibrosis, while treatment with TQ counteracted this alteration significantly (Figure 2A-B). In line with our previous results, the mRNA expression of collagen Ⅰ, collagen Ⅲ and CTGF were significantly increased in mice of the TAC group compared with their matched controls, but remarkably decreased in TQ-treated mice in the TAC group ( Figure 2C).

| TQ inhibited TAC surgery-induced oxidative stress.
Cardiac hypertrophy leads to increased intracellular ROS levels, which in turn exacerbate hypertrophy and fibrosis. 28  and CTGF (n = 6). *p < 0.05; One-way ANOVA followed by Bonferroni post hoc tests DHE-stained samples, we found that pressure overload for 6 weeks resulted in a substantial increase in ROS levels in ventricular tissues, while TQ treatment reduced levels close to those of the shamoperated group (Figure 3A-B). In addition, the qRT-PCR analysis revealed that TQ restored the mRNA expression of oxidant stressrelated genes (NOX4, SOD1 and SOD2) in mice of the TAC group ( Figure 3C). Accumulating evidence has suggested that AMPK is an inhibitor of oxidative stress in cardiac hypertrophy. 9,10,29 To confirm AMPK involvement in the mechanism of TQ, we next examined the phosphorylation level of AMPK using immunoblotting analysis. Our results showed that while TAC surgery remarkably inhibited AMPK phosphorylation, TQ administration restored AMPK phosphorylation to some extent in this pathological condition ( Figure 3D-E).

| TQ administration mitigated PE-induced hypertrophic response in NRCMs
We have demonstrated the efficacy of TQ in vivo, and next performed in vitro experiments using cardiomyocytes isolated from neonatal rats. First, a cell viability assay was performed to assess the effect of different concentrations of TQ on NRCMs ( Figure S2).
A concentration of 5 µM was chosen for subsequent in vitro experiments. The cells were simultaneously exposed to PE (50 µM) and TQ (5 µM) for 24 h. Total protein and RNA were isolated, and phalloidin staining as well as DCFH-DA incubation (for ROS detection) were performed. We observed a protective effect of TQ on PE-induced increased cell surface area ( Figure 4A-B), and on expression of ANP and BNP ( Figure 4C-D) and excessive ROS production ( Figure 4E-F).
Similar to what was observed in vivo, TQ treatment resulted in restoration of PE-mediated decrease of p-AMPK ( Figure 4G-H).

| TQ lost its inhibitory effects on cardiac hypertrophy in the presence of CpC in vivo and in vitro
Given our observation that TQ enhanced the phosphorylation of AMPK under pathological conditions, we next used CpC, a specific AMPK inhibitor, 30 to test the role of AMPK in mediating the beneficial effects of TQ on cardiac hypertrophy.
As shown in Figure 5A Table S3. In addition, the beneficial effects of TQ on cardiac fibrosis and oxidative stress were also partially abolished in mice treated with CpC ( Figure 5K-N, Figure S3). Altogether, these results support the hypothesis that TQ exerted its cardioprotective effect in an AMPK-dependent manner.

| TQ inhibited the MAPK signalling pathway in TAC-and PE-induced cardiac hypertrophy, but CpC abolished this effect
To further elucidate the underlying molecular mechanisms of TQ, we evaluated signalling pathways playing a critical role in the hypertrophic process.
First, we examined the phosphorylation level of ACC in mice hearts. ACC is one of the direct targets of AMPK and mirrors the activity of AMPK. 31 Consistent with what was observed for p-AMPK, phosphorylation of ACC was reduced in TAC-injured hearts, and significantly restored in the TAC + TQ group, which further supported that TQ activated AMPK in the pressure-overload model.
In addition, we investigated whether TQ affects the mammalian target of rapamycin (mTOR) and its effector p70S6K, members of a key downstream signalling pathway of AMPK in the progression of cardiac hypertrophy. 32,33 Here, we observed a marked increase in phosphorylation levels of mTOR and p70S6K in TAC-injured heart and PE-treated NRCMs. However, phosphorylation of these molecules was not significantly altered after TQ treatment ( Figure S4).
In summary, these results suggested that AMPK/MAPK signalling plays a major role in the cardioprotective benefits of TQ treatment.

| DISCUSS ION
The present study demonstrates that TQ protects against cardiac remodelling and dysfunction in mice subjected to TAC. These ob  However, whether TQ prevents pathological cardiac hypertrophy remains elusive. In the present study, TAC operation was employed to mimic the disease process of cardiac hypertrophy. In this model, sustained excessive pressure load results in hypertrophy, cardiomyocyte apoptosis, oxidative stress and fibrosis. 10,22,37 Here, we observed that the fetal gene expression program was re-activated, cross-sectional cell areas in ventricular tissues  20 It was also found that supplementation of NS (800 mg/kg) to exercise training promoted global cardiac hypertrophy. 21 In the present study, emphasis was placed on pathological hypertrophy. Our findings regarding enhanced cardiac function are consistent with the findings of the previous study, but the conclusions concerning myocardial hypertrophy differ from those of previous authors. We attributed this discrepancy to the different mechanisms of the two types of myocardial hypertrophy 1 and because the two substances (NS and TQ) are not exactly the same compound.
Cardiac fibrosis is a hallmark of cardiac remodelling induced by sustained pressure overload. It is a late-stage, irreversible process that increases heart wall stiffness, deteriorates contractile function and leads to chronic heart failure. 38,39 The anticardiac fibrotic effect of TQ has been suggested in previous studies in which animals were exposed to lipopolysaccharides (LPS) or lead. 40,41 Similarly, our observations demonstrated that TQ treatment slowed the progression of interstitial and perivascular fibrosis in mice hearts following TAC surgery.
Excessive ROS accumulation has been associated with a progression to cardiac fibrosis and end-stage heart failure. 12,13,42,43 Targeting oxidative stress has been the subject of investigation in the preclinical setting and demonstrated highly promising results. [44][45][46] As a bioactive natural compound, TQ is one of the agents that has been considered as potent antioxidants and has been extensively studied in the past decade. The current study demonstrated the protective effect of TQ on oxidative stress in vivo and in vitro, as shown by the detection of ROS-related gene expression in addition to DHE/DCFH-DA fluorescent microscopy.
The free radical scavenger property of TQ could be ascribed to its quinone moiety and its capacity to pass cell membranes and enter subcellular organelles. 14 The rebalance of the oxidative status following TQ treatment is presumed to translate into the recovery from pathological cardiac hypertrophy.
AMPK is a stress-activated kinase that regulates cardiac metabolism, protein synthesis and the ROS/redox balance under physiological and pathological conditions. 8,47 There is ample evidence suggesting that AMPK is an inhibitor of pathological cardiac hypertrophy. [48][49][50] On the other hand, previous studies have reported that AMPK activation can be regulated by TQ. 51,52 Therefore, we tested the hypothesis that AMPK plays a crucial role in mediating the cardioprotective benefits of TQ. Our results showed that TQ restored the phosphorylation of AMPK in our experimental models of cardiac hypertrophy, both in vivo and in vitro. However, when CpC (a selective  3-4). *p < 0.05; ns, no significance; One-way ANOVA followed by Bonferroni post hoc tests AMPK inhibitor) was administered, TQ no longer protects the heart from hypertrophy, fibrosis and oxidative stress.
The MAPK family has been recognized as one of the downstream signal pathways of AMPK. 53,54 The family includes ERK1/2, p38 and JNK1/2, which all play a critical role in the progression of pathological cardiac hypertrophy. 55 Earlier reports have shown that TQ inhibited MAPK signalling in models of cardiac damage and acute liver injury. 56,57 In line with these findings, we observed activation of ERK1/2, p38 and JNK1/2 MAPK in the post-TAC cardiac tissues and PE-treated NRCMs. TQ administration markedly inhibited these effects. However, TQ lost its inhibitory effects on MAPK signalling when AMPK was inhibited in vivo and in vitro, illustrating that TQ exerts its protective effect in an AMPK-dependent manner. Unexpectedly, CpC did not enhance the phosphorylation level of JNK1/2 in vitro. We

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the supplementary material of this article.