Doxorubicin cardiomyopathy is ameliorated by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules

Abstract Doxorubicin cardiotoxicity is frequently reported in patients undergoing chemotherapy. The present study investigates whether cardiomyopathy induced by doxorubicin can be improved by the natural flavone acacetin in a mouse model and uncovers the potential molecular mechanism using cultured rat cardiomyoblasts. It was found that the cardiac dysfunction and myocardial fibrosis induced by doxorubicin were significantly improved by acacetin in mice with impaired Nrf2/HO‐1 and Sirt1/pAMPK molecules, which is reversed by acacetin treatment. Doxorubicin decreased cell viability and increased ROS production in rat cardiomyoblasts; these effects are significantly countered by acacetin (0.3‐3 μM) in a concentration‐dependent manner via activating Sirt1/pAMPK signals and enhancing antioxidation (Nrf2/HO‐1 and SOD1/SOD2) and anti‐apoptosis. These protective effects were abolished in cells with silencing Sirt1. The results demonstrate for the first time that doxorubicin cardiotoxicity is antagonized by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules, indicating that acacetin may be a drug candidate used clinically for protecting against doxorubicin cardiomyopathy.

mitochondrial dysfunction, calcium overload, apoptosis and disturbance of energy metabolism in cardiomyocytes. 9 Reactive oxygen species (ROS) are considered to be the main upstream factor in generating myocardial damage by doxorubicin, which generally decreases endogenous antioxidative function and induces cell apoptosis. 10 In our previous studies, the natural flavone acacetin from the traditional Chinese medicinal herb snow lotus is found to be effective in treating atrial fibrillation. 11,12 Our recent studies have demonstrated that acacetin confers myocardial protection against ischaemia/reperfusion or hypoxia/reoxygenation injury by inhibiting ROS production, decreasing inflammation and apoptosis via activating antioxidative signalling AMPK/Nrf2 pathway. 13,14 The present study determined whether acacetin could protect against doxorubicin cardiotoxicity in in vivo moue model and cellular mode with multiple experimental approaches. The results demonstrated that acacetin effectively improved doxorubicin cardiomyopathy by reducing ROS production and apoptosis via Sirt1mediated activation of AMPK/Nrf2 signal molecules.

| Cardiotoxicity induced by doxorubicin
Male C57BL/6 mice (6-8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co. (Beijing, China). The animal experiment protocol was approved by the Animal Care and Ethics Committee of Xiamen University. The animals were cared following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Mice were randomly assigned to control, doxorubicin and doxorubicin with acacetin treatment groups.
Doxorubicin dissolved in saline was administered intraperitoneally at a cumulative dose of 15 mg/kg over a period of 12 days (2.5 mg/ kg/day, every other day) followed by 16 days of observation. For the acacetin treatment group, animals were subcutaneously injected with the acacetin prodrug (15 mg/kg, b.i.d.) for 3 days before receiving doxorubicin treatment and then throughout the experimental period in this group of animals. As acacetin prodrug is converted in vivo into acacetin which exerts effective pharmacological actions, 13,15 the term prodrug is omitted in the following experiments. The animals served for control received equivalent volume of saline. After determining echocardiography, the animals were killed by cervical dislocation at the end of experiment; their hearts were then excised and processed for further histology and biochemical analysis.

| Transthoracic echocardiography
In vivo heart function was assessed by transthoracic echocardiography in sedated C57BL/6 mice fixed on a heated platform using a Vevo 2100 high-resolution imaging system (VisualSonics, Toronto, ON, Canada, 40 MHz transducer) at the end of the experiment.
Series of M-mode images at the level of papillary muscles were obtained; left ventricular internal dimensions, left ventricular ejection fraction and left ventricular fraction shortening were measured using at least three consecutive cardiac cycles.

| Histopathology
The ventricular tissue was fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned into 5-μm slices. Sections were stained with Masson's trichrome staining kit following manufacturer's instruction. Images were obtained by a microscope equipped with a camera.

| TUNEL staining
Myocardial sections were incubated with TUNEL reaction mixture (Cat. No. 11684795910, In Situ Cell Death Detection Kit, Fluorescein, Roche, Mannheim, Germany) for 1 hour at 37°C following the manufacturer's instructions. Subsequently, the sections were washed with PBS and stained with DAPI. Images were obtained by using a confocal microscope (TCS SP5, Leica, Wetzlar, Germany), and the numbers of TUNEL-positive cells were counted in 5 random fields for each sample.

| Measurement of cardiac ROS
ROS level of ventricular tissue was determined in myocardial sections incubated with 10 μM dihydroethidium (DHE) at 37°C for 20 minutes in the dark then washed three times with PBS using a confocal microscope (TCS SP5, Leica) to obtain the fluorescence images which were then quantified by Image J as described previously. 14
For viability assays, cells were seeded in 96-well plates and viability was determined with MTT assay as described previously. 14 Briefly, the cells were treated with or without doxorubicin in the absence or presence of acacetin, then incubated with 0.5 mg/mL MTT for 4 hours and resuspended in 150 μL of DMSO. Absorbance was measured at 575 nm using an Infinite M200 Pro Nanoquant (TECAN, Switzerland).

| Flow cytometry analysis
Cell viability, apoptosis and intracellular ROS level were determined with flow cytometry analysis (Beckman Coulter, USA) as described previously. 16 Rat cardiomyoblasts were seeded into 6-well plates and cultured with doxorubicin in the absence or presence of acacetin for cell viability and apoptosis assay. The detached cells were resuspended in binding buffer containing Annexin V and SYTOX, incubated for 15 minutes at room temperature in the dark and then analysed by flow cytometry within 1 hour. ROS level was determined in rat cardiomyoblasts with or without acacetin and subjected to doxorubicin treatment. The cells were then incubated with DCFH-DA (10 μM) at 37°C for 30 minutes and fluorescence was determined by Flow Cytometer (Beckman Coulter) to obtain ROS levels. Unstable flow cytometry data due to cell population and detachment procedure were discarded from data analysis.

| Western blot analysis
The related proteins, that is Nrf2, HO-1, SOD1, SOD2, Bcl2, Bax, caspase-3, AMPK, pAMPK, Sirt1, β-actin or GAPDH, were determined in rat cardiomyoblasts or mouse ventricular tissues by Western blot analysis as described previously. 14 The samples were lysed by RIPA buffer with protease inhibitors, and the protein concentrations were determined by BCA assay. Equal amounts of total proteins were separated by using SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA) which were blocked by 5% skim milk and incubated with primary antibodies (1:1000) at 4°C overnight. Then, membranes were washed three times with TBST and incubated with secondary antibody (1:10 000) at room temperature for 1 hour. The membranes were visualized by enhanced chemiluminescence (Advansta, Menlo Park, CA, USA) and were exposed to FluoChem E chemiluminescence detection system (ProteinSimple, San Jose, CA, USA). The relative densities of protein bands were analysed by image analysis software. The Western blots used in the results figures are included in Supplemental original data.

| RNA interference
The small interference RNAs (siRNAs) were used to silence Nrf2 and Sirt1 in rat cardiomyoblasts as described previously. 16 Briefly, when cells grew to 60%-70% confluence, the cells were transfected by siRNA molecules targeting Nrf2 or Sirt1 using Lipofectamine 2000 for 48 hours, and then, the silencing efficiency was determined with Western blot analysis.

| RNA isolation and reverse transcription PCR analysis
Total RNA was isolated from cardiomyoblasts using TRIZOL reagent

| Statistical analysis
Data analysis was performed with GraphPad Prism 5.0 (GraphPad Software, Inc, San Diego, CA, USA). Results are presented as mean ± SEM. One-way ANOVA followed by Tukey's post hoc test was used for comparisons of multiple groups. A value of P < 0.05 was considered as statistically significant.
Myocardial histological examination revealed that doxorubicin-induced increase of ventricular fibrosis was reduced in animals treated with acacetin ( Figure 1E). Western blot analysis showed that doxorubicin increased myocardial collagen I and collagen III protein levels (P < 0.01 vs control), these increases are significantly inhibited by acacetin (n = 5, P < 0.05 vs doxorubicin alone; Figure 1F,G).
Moreover, the overproduction of ROS by doxorubicin was observed in myocardium of C57BL/6 mice, which was decreased in animals treated with acacetin ( Figure 1H,I).
In addition, the myocardial antioxidation proteins (Nrf2, HO-1, SOD1, SOD2), Sirt1 (a regulator of cellular ageing, apoptosis, stress, etc) and pAMPK (a regulator of cellular energy homeostasis) were down-regulated in mouse hearts with doxorubicin cardiomyopathy ( Figure 2). Myocardial sections stained with TUNEL revealed apoptotic cardiomyocytes were significantly increased in mice with doxorubicin and partially decreased in mice treated with acacetin ( Figure 3A,B). The increase of apoptotic cardiomyocytes was associated with reduced anti-apoptotic protein Bcl-2 and also with up-regulated pro-apoptotic proteins Bax and cleaved caspase-3 in mice with doxorubicin ( Figure 3C,D). Interestingly, acacetin treatment countered the reduction of Nrf2, HO-1, SOD1, SOD2, Bcl-2, Sirt1 and pAMPK and the increase of apoptotic cardiomyocytes, Bax and cleaved caspase-3 (Figures 2 and 3). These results indicate that acacetin significantly protects against doxorubicin cardiotoxicity in mice.

| Cellular mechanisms of acacetin protection against doxorubicin cardiotoxicity
To investigate the potential molecules mediating the protection of acacetin against doxorubicin cardiotoxicity in mice, the effects of acacetin

| Molecular mechanisms of acacetin against doxorubicin cardiotoxicity
To determine whether antioxidation is involved in the protective effect of acacetin against doxorubicin cardiotoxicity, ROS production and the proteins related to antioxidation were determined in rat cardiomyoblasts treated with doxorubicin or doxorubicin plus acacetin.

| Nrf2 activation and acacetin protection against doxorubicin cardiotoxicity
It is well recognized that Nrf2 plays a central role in regulating an-

| Sirt1 mediates AMPK/Nrf2 activation induced by acacetin
In rat cardiomyoblasts acacetin increased the relative level of Sirt1 protein in a concentration-dependent manner ( Figure 8A) and  Figure 8B,C). It is generally believed that Sirt1 increases pAMPK by activating pLKB1. 17 It is also the case for acacetin-induced increase of Sirt1 and pAMPK, because acacetin also reversed doxorubicin-induced down-regulation of pLKB1 in a concentration-dependent manner ( Figure 8D). Whether Sirt1 mediates the acacetin-induced activation of pLKB1, pAMPK and Nrf2 was further determined in rat cardiomyoblasts transfected with siRNA molecules targeting Sirt1. Silencing Sirt1 did not affect total LBK1 and AMPK expression, but decreased pLBK1 and pAMPK and abolished acacetin-induced enhancement of pLBK1 ( Figure 8E) and pAMPK ( Figure 8F). Moreover, silencing Sirt1 also significantly decreased the expression of both whole-cell Nrf2 ( Figure 8G) and nuclei Nrf2 ( Figure 8H) and abolished acacetin-induced increase of both whole-cell Nrf2 ( Figure 8G) and nuclei Nrf2 ( Figure 8H). The alteration of Nrf2 was correlated to its mRNA expression ( Figure 8I).
These results demonstrate that doxorubicin cardiotoxicity is related to the overproduction of ROS and inhibition of Sirt1, thereby leading the reduction of antioxidation and increased myocardial apoptosis.

Acacetin protects against doxorubicin cardiotoxicity by increasing
Sirt1 and the Sirt1-dependent activation of pAMPK, the antioxidative regulator Nrf2 and the antioxidative proteins, thereby decreasing ROS production, and inhibiting apoptosis. All the findings support the notion that protection of acacetin against doxorubicin cardiotoxicity is mediated by Sirt1-dependent regulation of AMPK/Nrf2 signal molecules.

| D ISCUSS I ON
Doxorubicin and its derivative epirubicin are widely used anthracyclines to treat breast, endometrial and gastric cancers, childhood solid tumours, soft tissue sarcomas, and aggressive lymphoblastic or myeloblastic leukaemia. 18 However, the use of anthracyclines is F I G U R E 6 Acacetin protection against doxorubicin cardiotoxicity was abolished in cells with silenced Nrf2. A, Flow cytometry graphs showing cell viability, early apoptosis, late apoptosis and dead cells in rat cardiomyoblasts transfected with control siRNA or Nrf2 siRNA for 48 h and then subjected to 1 μM doxorubicin exposure in the absence or presence of 3 μM acacetin. B, Mean per cent values of cell viability, early apoptosis, late apoptosis and dead cells from flow cytometry graphs as shown in A. C, Flow cytometry graphs showing ROS levels in rat cardiomyoblasts transfected with control siRNA or Nrf2 siRNA and then subjected to 1 μM doxorubicin in the absence or presence of 3 μM acacetin. D, Summarized ROS levels in rat cardiomyoblasts transfected with control siRNA or Nrf2 siRNA from flow cytometry graphs as shown in C (n = 5 individual experiments, * P < 0.05, ** P < 0.01 vs control siRNA; # P < 0.05, ## P < 0.01 vs control siRNA with acacetin) associated with dose-dependent cardiotoxicity. 4 Doxorubicin cardiotoxicity is manifested as arrhythmias, ischaemia, systolic dysfunction, due to cardiac cell death and necrosis. 19 Although dexrazoxane is effective in antagonizing doxorubicin cardiomyopathy, 5 the potential of increasing secondary malignant neoplasms is reported. 6 The present study demonstrates that acacetin is very effective in protecting against doxorubicin cardiotoxicity.
Acacetin is a natural flavone compound that exists widely in plant pigments. We have previously reported that acacetin isolated from the traditional Chinese medicinal herb snow lotus possesses unique effects of preferentially inhibiting atrial potassium channels including I Kur (ultra-rapidly activating delayed rectifier potassium current), I K.ACh (acetylcholine-activated potassium current), I to (transient outward potassium current) and SK Ca current (small conductance Ca 2+ -activated potassium current), which contribute to its selective anti-atrial fibrillation properties. 11,15,20 Studies from other groups demonstrated that extensive beneficial effects of flavonoids were related to the scavenging free radicals and antioxidant effects. 21,22 However, free radical scavenging activities of flavonoids depend on benzene ring hydroxyl group and number of their chemical structure. [21][22][23] No free radical scavenging activity is observed for acacetin, 21 because of its structure lacks a hydroxyl group in its B-ring.
Other reports showed that acacetin has anti-inflammation, anti-tumour and antioxidative properties. 24,25 Our recent studies reported that acacetin and its water-soluble prodrug confer cardioprotection against hypoxia/reoxygenation insult in cardiomyocytes 14 and ischaemia/reperfusion injury in in vivo and ex vivo hearts. 13 Acacetin prodrug significantly improves myocardial function and inhibits ventricular arrhythmia induced by ischaemia/ reperfusion injury in rats; acacetin prevents ischaemia/reperfusion by increasing myocardial antioxidation and inhibiting inflammation and apoptosis. 13 Cellular experiments reveal that AMPK-mediated Nrf2/HO-1 activation is involved in myocardial protection through multiple effects including antioxidation, anti-inflammation and anti-apoptosis. 14 The present study showed that acacetin significantly improved doxorubicin-induced cardiac dysfunction and cardiotoxicity via reducing oxidative stress by Sirt1-dependent activation of AMPK/Nrf2 signals.
It is well recognized that endogenous antioxidant defensive system has evolved in aerobic organisms including human beings to counteract the harmful effects of free radicals and reactive F I G U R E 7 Effects of silencing Nrf2 on antioxidation and apoptosis-related proteins in cells with doxorubicin exposure. A, Western blots and relative levels of HO-1 in H9C2 cardiomyoblasts transfected with control siRNA or Nrf2 siRNA and subjected to doxorubicin injury in the absence (V, vehicle) or presence of 3 μM acacetin (Aca). B, Western blots and relative levels of SOD1 in H9C2 cardiomyoblasts with the same treatment as in A. C, Western blots and relative levels of SOD2 in H9C2 cardiomyoblasts with the same treatment as in A. D, Western blots and relative levels of Bcl-2 in H9C2 cardiomyoblasts with the same treatment as in A. E, Western blots and relative levels of Bax in H9C2 cardiomyoblasts with the same treatment as in A. F, Western blots and relative levels of cleaved caspase-3 in H9C2 cardiomyoblasts with the same treatment as in A (n = 5 individual experiments, * P < 0.05, ** P < 0.01 vs vehicle of control siRNA; ## P < 0.01 vs control siRNA with acacetin) oxygen species (ROS) and maintain redox homeostasis. 26  Our recent study showed that Nrf2 activation by acacetin is dependent on AMPK activation. 14 25 and inhibits the invasion and migration of DU145 cells by suppressing p38 MAPK signal pathway. 36 Acacetin was found to induce Bax activation and mitochondrial damage-mediated apoptosis in Jurkat T cells 37,38 and also show selective anti-cancer activity against chronic lymphocytic leukaemia. 39 Doxorubicin is widely used in the treatment of lung cancer, breast cancer, prostate cancer, leukaemia, etc. 1,2,9 Interestingly, a recent study showed that acacetin enhances the therapeutic efficacy of doxorubicin in non-small-cell lung carcinoma cells. 40 Therefore, in addition to the protection against doxorubicin cardiotoxicity, acacetin may have synergetic effect with doxorubicin for treating cancers.
Several natural active compounds, for example dioscin, sulforaphane, resveratrol and quercetin, have been reported also to confer protection against doxorubicin cardiotoxicity by reducing ROS production and regulating apoptosis-related proteins via activating Sirt1, 32 Nrf2, 41 Bmi-1 expression 42 or adjusting microR-NA-140-5p. 43 Although these natural compounds show promising therapeutic potential, their low solubility and low bioavailability are barriers for drug development. Acacetin is a novel Sirt1 activator that mediates activation of AMPK/Nrf2 signals. The Sirt1 activation by acacetin (unpublished observation) is similar to resveratrol (related to increasing nicotinamide adenine dinucleotide-dependent deacetylases and nicotinamide phosphoribosyltransferase). 34 Importantly, the water-soluble prodrug of acacetin y 13,15 allows it to be administered intravenously with doxorubicin to prevent cardiotoxicity in patients undergoing chemotherapy in the near future.
In the present study, myocardial fibrosis and collagen proteins were remarkably increased in mice with doxorubicin, which were significantly reversed in animals treated with acacetin. The limitation was that the potential mechanism of anti-fibrotic effect of acacetin was not explored in the present study, which remains clarified in the future study. However, this would not affect the conclusion that acacetin confers the protection against doxorubicin cardiotoxicity.
Collectively, the present study demonstrates for the first time

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