Yellow Wine Polyphenolic Compounds prevents Doxorubicin‐induced cardiotoxicity through activation of the Nrf2 signalling pathway

Abstract Doxorubicin (DOX) is considered as the major culprit in chemotherapy‐induced cardiotoxicity. Yellow wine polyphenolic compounds (YWPC), which are full of polyphenols, have beneficial effects on cardiovascular disease. However, their role in DOX‐induced cardiotoxicity is poorly understood. Due to their antioxidant property, we have been suggested that YWPC could prevent DOX‐induced cardiotoxicity. In this study, we found that YWPC treatment (30 mg/kg/day) significantly improved DOX‐induced cardiac hypertrophy and cardiac dysfunction. YWPC alleviated DOX‐induced increase in oxidative stress levels, reduction in endogenous antioxidant enzyme activities and inflammatory response. Besides, administration of YWPC could prevent DOX‐induced mitochondria‐mediated cardiac apoptosis. Mechanistically, we found that YWPC attenuated DOX‐induced reactive oxygen species (ROS) and down‐regulation of transforming growth factor beta 1 (TGF‐β1)/smad3 pathway by promoting nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) nucleus translocation in cultured H9C2 cardiomyocytes. Additionally, YWPC against DOX‐induced TGF‐β1 up‐regulation were abolished by Nrf2 knockdown. Further studies revealed that YWPC could inhibit DOX‐induced cardiac fibrosis through inhibiting TGF‐β/smad3‐mediated ECM synthesis. Collectively, our results revealed that YWPC might be effective in mitigating DOX‐induced cardiotoxicity by Nrf2‐dependent down‐regulation of the TGF‐β/smad3 pathway.


| INTRODUC TI ON
Doxorubicin (DOX), an effective anthracycline, is a chemotherapeutic drug commonly used to treat a wide range of cancers. However, its use is limited due to the potential risk of cardiotoxicity. 1 Increased mortality associated with the total accumulated dose of DOX has been shown in chemotherapy cycles. 2 Under this circumstance, researchers have considered designing DOX analogues with reduced acute toxicity; however, this has been confounded by a paralleled loss of anti-cancer efficacy. 3 On the contrary, cardioprotective adjuvants, such as dexrazoxane 4 and vitamin C, 5 were co-administered to boost chemotherapeutic efficacy with the purpose of suppressing cardiotoxicity. 6 Nevertheless, limited evidence supports the cardioprotective effects of these natural compounds and synthetic drugs against DOX-induced cardiotoxicity. 7,8 There has been growing interest in the possible benefits of polyphenols due to their antioxidant as well as anti-apoptotic properties. 9 Emerging evidence has demonstrated that polyphenols can prevent DOX-induced cardiotoxicity by exerting their anti-cancer and cardioprotective effects. 10 We previously investigated the beneficial effects of yellow wine, which is full of polyphenols, and found that yellow wine polyphenolic compounds (YWPC) could delay the development of atherosclerosis. 11,12 However, whether YWPC inhibit DOX-induced cardiotoxicity remains unclear.
To date, multiple molecular mechanisms have been indicated in DOX-induced cardiotoxicity, and oxidative stress is believed to play a central role, 1,13 as witnessed by reactive oxygen species (ROS)induced injury, including lipid peroxidation and reduced levels of endogenous antioxidant defences. 14 Oxidative stress also induced activation of several pro-fibrogenic factors to the subsequent extracellular matrix (ECM) accumulation, leading to the development of cardiac fibrosis. 15 Therefore, understanding how the oxidant-antioxidant balance is related to the control of oxidative stress may provide insights into effective therapies for DOX-induced cytotoxicity. To control oxidative homoeostasis, cells are equipped with an efficient defence system against oxidants. A plenty of studies have identified that nuclear factor-E2-related factor-2 (Nrf2) is implicated as a master regulator in the cellular response to oxidative stress. 16 Moreover, emerging evidence suggests the importance of inflammation in the pathogenesis of DOX-induced cardiotoxicity 17,18 and Nrf2 also has an anti-inflammatory effect. 19 Although several studies have revealed that the deactivation of Nrf2 signalling was involved in DOX-induced cardiotoxicity, 17,20 whether Nrf2 activation could help to alleviate DOX-induced cardiac fibrosis are rarely studied. 21 In this study, we hypothesized that YWPC could ameliorate DOX-induced cardiotoxicity by inhibiting oxidative stress-induced cell apoptosis and oxidative stress-mediated ECM accumulation.
To reveal the mechanisms underlying the action of YWPC, we also examined whether YWPC could suppress DOX-induced oxidative stress through the regulation of Nrf2 signalling pathway.

| Histological assessment of myocardial injury and collagen content
After treatment for 4 weeks, all rats were anaesthetized to remove the heart. The heart was quickly photographed and followed by dissecting into small portions and immediately snap-frozen in liquid nitrogen until analysis. Part of the heart was fixed in 10% formalin, embedded in paraffin, and sectioned at 5-μm thickness.
Haematoxylin and eosin (H&E) staining was used to evaluate the general morphology of the myocardium. The sections were imaged using the Leica DM3000 biological microscope (Leica, Wetzlar, Germany) at 200× magnification. For collagen analysis, the heart sections underwent Masson's trichrome staining. Collagen deposition was quantified using Image-ProPlus6 (Media Cybernetics, Rockville) as previously described. 27

| Immunohistochemistry
Sections were stained with primary antibodies against TGF-β1 at room temperature. The detailed procedure has been previously described. 28

| Wheat germ agglutinin (WGA) staining
Part of each heart form rats was quickly stored at −80°C. The 5-μm thick frozen heart sections were fixed with 4% PFA for 15 minutes and washed with PBS for three times. Subsequently, the sections were incubated with 5 μg/mL WGA (L4895; Sigma-Aldrich) solubilized in PBS for 20 minutes at room temperature in darkness. After washeing three times with PBS, the sections were stained with DAPI (P36941; Invitrogen) for 5 minutes at room temperature in dark. A Nikon Eclipse Ti-U fluorescence microscope (Minato-ku, Tokyo, Japan) was used to acquire images and a total of 30 fields per section were observed at 400× magnification and analysed with ImageJ software (National Institutes of Health). The cardiac myocyte size was determined by the measurement of total area divided by the number of cardiac myocytes.

| Measurement of TNF-β1, IL-1β and IL-6
Heart tissues were lysed with protein lysis buffer, and the lysates were subject to determine the levels of pro-inflammatory cytokines (TNF-β1, IL-1β and IL-6) using ELISA Kits (Qiagen, Inc, Hilden, Germany), according to the manufacturer's instructions. Optical densities were measured using a SpectraMax Plus Absorbance microplate reader (Molecular devices, CA) at 450 nm.

| Transmission electron microscopy
A portion of the left ventricle was from each rat cut into 1 mm fragments and was fixed in 2.5% glutaraldehyde overnight for electron microscopic examination. The tissues were fixed in 1% osmic acid followed by dehydrated in a series of acetone washes. The tissues were then embedded in Araldite for coronal sections and sectioned at 1 μm. The slides were stained with toluidine blue and ultra-thin sections were cut in from this block and were observed under a Cs-corrected TEM with Monochromator (Titan G2 60-300, FEI, Hillsboro).

| Apoptosis assay
An in situ cell death detection kit, POD (Roche Co. Ltd., Basel, Switzerland), was used to detect DNA fragmentation of individual cells. Paraffin-embedded sections of the heart tissues were deparaffinized with xylene and rehydrated in a graded ethanol series. The sections were incubated with proteinase K at 37°C for 20 minutes.
Tissues then were incubated with the TdT-mediated dUTP nick end labeling (TUNEL) reaction mixture at 37°C for 1 hour in the dark.
Subsequently, the sections were covered with converter-POD at 37°C for 30 minutes, followed by incubation with DAB for 3 minutes. After counterstaining with the nuclear counterstain hematoxylin for 30 seconds, the number of TUNEL-positive cells was counted with a Leica microscope at 100× magnification.

| Immunofluorescence
Frozen tissue sections were used for immunofluorescence assay to visualize the expression of caspase 3. Briefly, the cryostat sections were thawed at room temperature for 20 minutes and followed by incubation in blocking buffer (4% BSA) for 30 minutes. Subsequently, immunostaining was performed with an anti-caspase 3 antibody (ab32351, Abcam) and an Alexa Fluor 594 WGA (Life Technologies).

| Western blot analysis
Total protein was extracted from stored frozen tissues using RIPA

| Real-time reverse-transcription PCR
Total RNA was isolated from hearts tissues using TRIzol reagent

| Assessment of mitochondrial membrane potential
JC-1 method was used to measure mitochondrial membrane potential as described previously. 32 In brief, following treatment, H9C2 cells were incubated with 2.5 mmol/L JC-1 dye (Solarbio, Beijing, China) for 30 min in the dark at 37°C. Red JC-1 aggregates represented a normal hyperpolarized membrane potential, while Green JC-1 monomers indicated a loss of mitochondrial membrane potential. Images were obtained by the fluorescence microscope at 400× amplification.

| Statistical analysis
Results are shown as mean ± standard deviation (SD). Differences among groups were analysed using one-way ANOVA, followed by Tukey's post hoc analysis. Data were analysed with the SPSS version 20.0 software (SPSS Inc, Chicago, IL). P < 0.05 was considered statistically significant.

| YWPC improve DOX-induced cardiac hypertrophy
As shown in Figure 1B, body weight gain was significantly decreased in the DOX group as compared to the control group.
However, co-administration of DOX and YWPC could reverse this trend. At the fourth week, the heart weight/body weight was higher in the DOX group than in the DOX + YWPC and control groups ( Figure 1C). The global appearance of the heart in the indicated four groups has been shown in Figure 1D. The rats in the DOX group had an enlarged heart, whereas the remaining rats in the other three groups had similar normal heart. H&E staining Interestingly, in rats that were co-treated with YWPC, the normal myocardial architecture was preserved and cell injury significantly reduced ( Figure 1E). Further, we found that DOX-associated enlargement in cardiomyocyte size was attenuated in YWPC-treated rats as assessed by WGA staining ( Figure 1F). Collectively, these

| YWPC improve DOX-induced cardiac dysfunction
Echocardiography was then used to monitor the improvement in cardiac function (Figure 2A). Results suggested that LVEF ( Figure 2B) and LVFS ( Figure 2C) were similar in the YWPC and control groups, but significantly reduced in the DOX group, which was then enhanced by YWPC treatment. Compared to control group, DOX treatment markedly increased LVIDs ( Figure 2D) and LVIDd ( Figure 2E), which were ameliorated by YWPC treatment. However, the results of electrocardiography showed no significant difference in heart rate between the groups ( Figure 2F).

| YWPC prevent DOX-induced oxidative stress, in vivo
To further understand the molecular mechanisms associated with the beneficial effects of YWPC, the levels of oxidative stress and endogenous antioxidant enzyme activities in the cardiac tissue were measured. As shown in Figure 3A, DHE staining showed increased number of DHE-positive cells in the DOX group, indicating the generation of ROS. YWPC effectively reduced the DOX-induced generation of ROS. In addition, high levels of lipid peroxidation production MDA were observed in DOX-stimulated rats, which were significantly decreased with the treatment of YWPC ( Figure 3B).
Subsequently, a significant decrease in the activity of antioxidant enzymes, including SOD and catalase was observed in DOX-treated rats as compared to control and YWPC-treated rats ( Figure 3C and   3D). YWPC intake dramatically alleviated the reduction in antioxidant enzyme activity in the DOX + YWPC group. Furthermore, the levels of pro-inflammatory cytokines, TNF-β1, IL-1β, and IL-6 were significantly increased in the DOX group compared with the control and YWPC groups; however, their levels were reduced in the DOX + YWPC group compared to the DOX group ( Figure 3E-G).
These results indicated that YWPC significantly alleviated DOX-induced oxidative damage through modulating the balance of oxidation and antioxidation.

| YWPC attenuate DOX-induced myocardial apoptosis
Because apoptosis plays a key role in DOX-associated cardiotoxic-  Figure 5A). In H9C2 cells incubated with DOX for 24 hours, a remarkable increase in cell apoptosis was observed.

| YWPC attenuate DOX-induced reactive oxygen species (ROS) and down-regulation of transforming growth factor beta (TGF-β)/smad3 pathway dependent on Nrf2 status
ROS scavenging is a powerful way to protect against DOX-induced cardiac damage. 33 To explore the molecular mechanisms underlying the antioxidant effects of YWPC, we examined the effect of YWPC on the intracellular ROS level of (5 μmol/L for 24 hours) DOX-stimulated H9C2 cells using DCFDA. As illustrated in Figure 6A, treatment with 50 mg/L YWPC suppressed the intracellular ROS production of H9C2 cells, which triggered by DOX. A large number of studies have manifested the critical role of TGF-β1/smad3 signalling pathway in cardiac fibrosis. Using antibodies that recognize the unphosphorylated and phosphorylated form of smad3, we found that phosphorylation of smad3 induced by DOX was significantly attenuated in the presence of YWPC ( Figure 6B). In addition, due to the critical role of Nrf2 in regulation of oxidative stress, we subsequently explored whether YWPC were involved in the regulation of Nrf2 expression.
Intriguing, when compared to the control group, 6 hours of DOX treatment notably up-regulated the cytosol expression levels of Nrf2, while down-regulated its nucleus levels. As expected, YWPC treatment obviously promoted Nrf2 translocation from cytoplasm to  Figure 6C). To confirm the role of ROS and Nrf2 in YWPCinduced suppression of TGF-β/smad3 pathway, H9C2 cells were treated with transfected with Nrf2 siRNAs ( Figure 6D). Using RT-qPCR, we identified that treatment of H9C2 cells with DOX resulted in a significant decrease in levels of downstream genes Nrf2, including HO-1, NQO-1 and GCLM. However, this effect could be reversed by YWPC treatment. We subsequently found that CDDO-EA (a Nrf2 activator) could further up-regulate YWPC-enhanced antioxidant genes, while knockdown of Nrf2 could abolish YWPC-enhanced antioxidant genes ( Figure 6E-G). Importantly, we found that YWPC reduced the DOX-induced TGF-β1 levels in the culture supernatants of H9C2 cells. CDDO-EA treatment further decreased TGF-β1 levels while Nrf2 silencing increased TGF-β1 levels ( Figure 6H). In summary, these data indicated that YWPC attenuate DOX-induced ROS via TGF-β1/smad3 pathway in a Nrf2 dependent manner.

| YWPC alleviate DOX-induced cardiac fibrosis, in vivo
To validate the anti-fibrosis effects of YWPC, we then measured the changes of fibrotic proteins in DOX-injected rats with or without YWPC or CDDO-EA treatment. As shown in Figure 7A, C, the results of Masson's trichrome staining showed that compared with control group, the collagen content in the heart tissues of DOXtreated rats was increased, which was then significantly reduced by YWPC or CDDO-EA treatment. In addition, the pro-fibrotic factor TGF-β1, the ECM content collagen I and the cardiac fibrosis-associated protein α-SMA were remarkably increased in DOX-treated rats compared to those in control group, as detected by IHC. However, the levels of TGF-β1, Collagen I and α-SMA were reduced by the addition of YWPC or CDDO-EA ( Figure 7B, D). Western blot analysis then showed that DOX significantly increased p-smad3 expression, which was reduced by YWPC or CDDO-EA treatment ( Figure 7E).
Above Generally, Figure 8 showed the protective effects and potential mechanisms of YWPC on DOX-induced cardiotoxicity.

| D ISCUSS I ON
In this study, we demonstrated that YWPC effectively alleviated YWPC used in our study were derived from Shaoxing rice wine and have been identified owning the cardiovascular protective effects through inhibiting matrix metalloproteinase-2, -9 expression during atherogenesis. 11,12,34 The main mechanisms by which polyphenols are cardioprotective are related to the improvement of vascular function and their anti-atherogenic effect. 35 Of note, the use of polyphenols is currently being investigated as a possible adjunctive therapy in combination with DOX to protect the myocardium from DOX-induced oxidative stress. 36 DOX accumulates in the mitochondria and disrupts the mitochondria electron chain resulting in an enhanced production of ROS. 37 This enhanced production of reactive radicals can react with proteins, lipids and DNA molecules, causing DNA damage and lipid peroxidation. 38,39 Oxidative stress through increased generation of ROS and compromised antioxidant defence are well documented in DOX-related myocardial apoptosis. 5,40 Consistent with previous studies, 41 we found DOX treatment markedly induced ROS generation and reduced the activity of endogenous antioxidant enzymes, such as SOD and catalase in the heart tissues. This DOX-induced oxidative stress in the heart could be attenuated by 30 mg/kg YWPC treatment, which may be largely due to the antioxidant properties of polyphenols. 7 Currently, there are plenty of polyphenol monomers identified have the cardioprotective effects of DOX-induced cardiotoxicity because of their antioxidative effects. For instance, resveratrol has the ability to ameliorate DOXinduced myocardial structure damage and cardiac dysfunction in rats through inhibiting free radical generation, improving mitochondrial dysfunction and suppressing apoptosis. 42 Epigallocatechin-3-gallate possesses cardioprotective action against DOX by suppressing oxidative stress and inflammation, as well as activation of pro-survival pathway. 43 Consistently, we identified that YWPC mitigated cellular However, the potential mechanisms underlying the action of YWPC remain to be elucidated.
Excessive ROS generation has been identified as an important pathogenic factor of DOX-induced cardiotoxicity. DOX-derived ROS causes an imbalance between pro-and anti-apoptotic proteins (such as Bcl-2 family 44 ), disrupting mitochondrial membrane potential, causing cytochrome c release and subsequently causing cell apoptosis. 45 The heart has high energy requirement and a high mitochondrial density, which is more susceptible to DOX-induced toxicity. 46 With the treatment of YWPC, we found great improvement in the disordered and swelled mitochondria induced by DOX. Researchers have confirmed that DOX treatment affects mitochondrial gene expression and suppresses cardiac mitochondrial metabolism and biogenesis, resulting in mitochondrial dependent apoptosis. 47 Here, we found that YWPC reduced the level of DOX-induced mitochondria-mediated apoptosis of cardiac myocytes, with the up-regulation of Bcl-2 and down-regulation of Bax, and release of cytochrome C and cleaved caspase-3.
Cardiac fibrosis is the common feature of many cardiac pathophysiological conditions, and has been considered to be involved in cardiac stiffness and dysfunction in DOX-induced cardiotoxicity. 21 In the present study, we found that co-administration with YWPC caused a remarkable inhibition of TGF-β1/smad3 pathway, reduction of ECM and mitigatory fibrosis in DOX-treated rats. TGF-β1/ smad3 pathway is considered to be the most potent and ubiquitous pro-fibrogenic cytokines that involved in the development of fibrosis. 15,48 It also reported that DOX triggered fibrosis through α-SMA, Collagen I and III regulation. Consistently, we found the protein levels of Collagen I and α-SMA were dramatically increased in DOX-treated rats, indicating cardiac fibroblasts activation and excessive ECM deposition. After YWPC treatment, ECM proteins were reduced. A rapidly expanding body of evidence supports that Nrf2 overexpression alleviates ROS accumulation and oxidative damage by mediating TGF-β1/Smad3 signalling inhibition. [49][50][51] Once exposure to ROS, Nrf2 dissociates from Keap1 and translocates into the nucleus to activate the transcription of various cytoprotective genes including HO-1, NQO-1 and GCLM. 52 Because Nrf2 exerts its cytoprotective effects against oxidative stress after translocation into the nucleus from the cytoplasm, 53 we examined the effects of YWPC on the expression of Nrf2 in H9C2 cells exposed to DOX.
Suppression of Nrf2 signalling pathway was observed after 24 hours' DOX stimulation, indicating DOX induced more reactive oxidants production than antioxidants, causing net oxidative stress in DOXtreated cardiac myocytes. Our results demonstrated that treatment with YWPC could activate the Nrf2 pathway to prevent oxidative injury in DOX-treated H9C2 cells. Our results further showed that knockdown of Nrf2 could reverse the inhibitory effects of YWPC on DOX-induced TGF-β1 levels. A recent study revealed that siRNAmediated knockdown of Nrf2 could down-regulate Tanshinone IIAinduced Nrf2 activation and reverse the effect of Tanshinone IIA on the DOX-induced suppression of cell viability. 53 Furthermore, Li et al identified that knockout of Nrf2 could exaggerate DOX-induced cardiotoxicity and cardiac dysfunction by controlling oxidative stress and autophagy. 54 On the contrary, using COOD-EA (a Nrf2 inducer) as a positive control, our study confirmed that YWPC could active Nrf2 pathway in DOX-induced rats to inhibit the progress of cardiac fibrosis, in vivo. Taken together, our data supported the idea that YWPC could activate Nrf2 signalling pathway and attenuate the DOX-induced toxicity.
Although it is still unsure whether YWPC have the ability to reinforce the chemotherapeutic efficacy of DOX or have anti-cancer effects, the current study found that YWPC supplementation alleviated DOX-induced cardiac dysfunction, as well as migrated cardiac fibrosis, and discussed its potential mechanisms. However, our study had certain limitations. Firstly, YWPC used in this experiment are a mixture of different monomer components, and which one of them plays a protective role is still unclear. Besides, findings based on DOX-induced cardiotoxicity in SD rats could not fully represent the situations and pathology in human; thus, further validations in clinical study are needed in future study.
Collectively, our data showed that YWPC supplementation protects against oxidative stress, mitochondrial damage, fibrosis, hypertrophy and apoptosis, eventually improving cardiac function in DOX-treated rats. The cardioprotective role of YWPC is contributing from its ability to inhibit DOX-stimulated oxidative stress by augmenting the Nrf2 pathway. The results suggested that YWPC might be a promising novel therapeutic strategy to reduce DOX-induced cardiotoxicity.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.