Honokiol protects against doxorubicin cardiotoxicity via improving mitochondrial function in mouse hearts

Honokiol is a key component of a medicinal herb, Magnolia bark. Honokiol possesses potential pharmacological benefits for many disease conditions, especially cancer. Recent studies demonstrate that Honokiol exerts beneficial effects on cardiac hypertrophy and doxorubicin (Dox)-cardiotoxicity via deacetylation of mitochondrial proteins. However, the effects and mechanisms of Honokiol on cardiac mitochondrial respiration remain unclear. In the present study, we investigate the effect of Honokiol on cardiac mitochondrial respiration in mice subjected to Dox treatment. Oxygen consumption in freshly isolated mitochondria from mice treated with Honokiol showed enhanced mitochondrial respiration. The Dox-induced impairment of mitochondrial respiration was less pronounced in honokiol-treated than control mice. Furthermore, Luciferase reporter assay reveals that Honokiol modestly increased PPARγ transcriptional activities in cultured embryonic rat cardiomyocytes (H9c2). Honokiol upregulated the expression of PPARγ in the mouse heart. Honokiol repressed cardiac inflammatory responses and oxidative stress in mice subjected to Dox treatment. As a result, Honokiol alleviated Dox-cardiotoxicity with improved cardiac function and reduced cardiomyocyte apoptosis. We conclude that Honokiol protects the heart from Dox-cardiotoxicity via improving mitochondrial function by not only repressing mitochondrial protein acetylation but also enhancing PPARγ activity in the heart. This study further supports Honokiol as a promising therapy for cancer patients receiving Dox treatment.

mitochondrial function in the hearts with in vivo treatment. The most recent finding that Honokiol protects the heart from Dox-cardiotoxicity 13 emphasizes the importance of further defining the biological action of Honokiol in the heart to exploit its potential clinical applications.
In the present study, we focus on investigating how Honokiol treatment protects the mouse heart from Dox-induced mitochondrial dysfunction, oxidative stress, and inflammation via activating PPARγ.

Results
Honokiol protects mitochondrial respiration capacities in mice suffering Dox-induced cardiotoxicity. To investigate the in vivo effects of Honokiol treatment on mitochondrial respiration, we freshly isolated mitochondria from mice of the four experimental groups as indicated (Fig. 1) and measured real-time oxygen consumption on these mitochondria in response to specific substrates and inhibitors using an Oroboro Oxygraph system ( Fig. 2A). Routine mitochondrial respiration was established by the concomitant addition of malate (5 mM) and pyruvate (5 mM), followed by ADP (1 mM) and glutamate (5 mM), to measure the oxidative phosphorylation capacity of complex I (OXPHOS CI), driven by the NADH-related substrates 17 . The cardiac mitochondria of Dox showed no difference compared to control in Routine mitochondrial respiration (Fig. 2B). However, Honokiol-treatment markedly upregulated oxygen consumption compared to control and Dox-treated mice under the Routine condition (Fig. 2B). We then measured Maximal coupling respiration by adding a saturating concentration of ADP to assess maximal oxidative phosphorylation (OXPHOS CI + CII). The Maximal coupling respiration in cardiac mitochondria isolated from the Dox + Honokiol group was upregulated compared with vehicle control, and Honokiol treatment prevented the Dox-induced downregulation (Fig. 2C). The maximal uncoupled respiration of cardiac mitochondria was evaluated by adding FCCP (ETS CI + CII). Dox + Honokiol group showed similar upregulation of oxygen consumption in controlled mitochondria, and Honokiol treatment reduced the downregulation induced by Dox in cardiac mitochondria (Fig. 2D). LEAK CI + CII respiration measured by adding oligomycin was significantly increased in cardiac mitochondria from both groups of mice treated with Honokiol (Fig. 2E). We further analyzed the respiratory control ratios (RCR) to evaluate the structural integrity of the inner mitochondrial membrane (IMM) and OXPHOS efficiency. Consistently, Honokiol raised basal RCR, and attenuated Dox-induced RCR downregulation (Fig. 2F). These results demonstrate for the first time that Honokiol promotes cardiac mitochondrial respiration and improves impaired cardiac mitochondrial respiration by Dox in mice.
Honokiol activates PPARγ signaling in cardiomyocytes. Honokiol has been reported as a natural PPARγ activator, a potential mechanism underlying the effect of Honokiol on cardiac mitochondria. To determine if Honokiol could activate PPARγ in cardiomyocytes, we first analyzed the effects of Honokiol on promoter activity via the PPAR response element (PPRE). In cultured embryonic rat cardiomyocytes (H9c2), luciferase reporter assay revealed that Honokiol increased the PPRE luciferase promoter activities at a dose of 2.5 μM (Fig. 3A). Furthermore, Honokiol treatment at both doses of 2.5 and 5 μM in the cultured H9c2 cells modestly enhanced the promoter activity of PPARγ (Fig. 3B). Moreover, in vivo treatment of Honokiol enhanced the transcript expression of PPAR γ in the heart (Fig. 3C). In mice with chronic treatment of Dox, cardiac PPARγ transcript was reduced by about 30% in Dox-treated hearts (Fig. 3C), which was rescued by Honokiol treatment (Fig. 3C). The expression of PPARγ protein in the heart showed the same pattern (Fig. 3D,E). We further examined the cardiac expression of PPARγ target genes, such as manganese super-oxide dismutase (SOD2) and Fatty acid translocase (CD36) [18][19][20] . Both SOD2 and CD36 were upregulated in the heart of Honokiol treated mice and Honokiol rescued the impaired SOD2 and CD36 expression in Dox-treated hearts (Fig. 3F,G). Supporting a recent report 12 , while Honokiol treatment had no effect on based protein acetylation, it did repress Dox-induced protein acetylation (Fig. 3H,I). These results indicate that Honokiol activates PPARγ pathway in the heart in addition to repressing stress-induced protein acetylation. Honokiol reduces myocardial reactive oxygen species levels in mice suffering chronic Dox-induced cardiotoxicity. We next investigated the potential anti-oxidant effects of Honokiol as a PPARγ activator.
Quantification of dihydroethidium (DHE) staining on heart sections showed that the intensity of DHE staining on heart sections of Dox-treated mice was markedly increased and the Dox-induced increase of DHE intensity was substantially repressed in heart sections from mice pretreated with Honokiol (Fig. 4A,B). Consistent with the DHE staining result, the Dox-induced decrease of GSH/GSSG rate, a result of rising oxidative stress 21 was ameliorated by Honokiol pretreatment in mouse hearts (Fig. 4C). Therefore, these findings support that Honokiol exerts anti-oxidant effects to reduce Dox-mediated cardiotoxicity.
Honokiol reduces chronic Dox-induced cardiac inflammation. We then investigated the effect of Honokiol, as a PPARγ activator and anti-oxidant, in alleviating Dox-induced cardiac inflammatory responses. Immunohistological staining of an inflammatory marker, CD68, on mouse heart sections revealed that Honokiol pretreatment largely abolished Dox-induced increase of CD68 positive stains on heart sections from Dox-treated mice (Fig. 5A,B). These results support that Honokiol exerts anti-inflammatory effects on mice with Dox-cardiotoxicity.
Honokiol protects against side effects from both acute and chronic Dox treatments. We next validated the previous finding of protection by Honokiol against Dox-cardiotoxicity. In the Acute protocol study, Honokiol treatment rescued the body weight loss induced by Dox in mice (Fig. 6A). Acute Dox-induced cardiac atrophy was also rescued, evident by the restoration of heart weight to body weight ratio and heart weight to tibial length ratio (Fig. 6B,C). Echocardiography assessments confirmed that Honokiol treatment rescue the depression of cardiac contraction indicated by EF% and FS% (Fig. 6E,F). Furthermore, Honokiol treatment reduced the   (Table 1).
To gain clinically relevant insights, we focused on assessing mice with chronic Dox treatment. All mice from the chronic Dox treatment survived but with reduced body weight (Fig. 7A). The heart-to-body weight ratio (HW/BW) were similar among all the experimental mice (Fig. 7B). However, when comparing the heart weight to tibial length (HW/TL) ratio, mice with Honokiol treatment mitigated the Dox-induced HW/TL ratio decline (Fig. 7C). Histological and echocardiographic results support that Honokiol treatment reduced Dox-induced cardiac atrophy (Fig. 7D,E). Echocardiography showed that the Dox-induced decrease of ejection fraction (%EF) and fractional shortening (%FS) were significantly ameliorated in the Honokiol + Dox group (Fig. 7F,G).
TUNEL assays on heart sections revealed that honokiol significantly reduced Dox-induced cardiomyocyte apoptosis (Fig. 8A,B). Furthermore, Western blot analysis revealed that cleaved Caspase 3 in heart samples was increased in Dox-treated mice but was not as pronounced in mice with Honokiol treatment (Fig. 8C to E). Honokiol treatment prevented the Dox-induced reduction of left ventricular posterior wall thickness in diastole (LVPWd) and systole (LVPWs) ( Table 2). Therefore, our results support that Honokiol protects the heart against Dox-induced cardiac dysfunction and pathological development.

Discussion
The present study investigates the mechanisms of the cardio-protective effect of Honokiol against Dox-induced cardiotoxicity in mice. We provide evidence that Honokiol facilitates cardiac PPARγ expression and its activity, contributing at least partly to Honokiol's role in improving mitochondrial respiration and reducing oxidative stress, inflammation and apoptosis in mouse hearts with Dox-cardiotoxicity.  Honokiol is a key component of a medicinal herb, Magnolia bark, which has been extensively used for thousands of years in traditional Chinese medicine. Previous research has shown that Honokiol is responsible for many pharmacological activities that may be beneficial for disease conditions such as cancer and cardiovascular disease. A recent study reported that Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial SIRT3, subsequently increasing mitochondrial protein deacetylation 12,13 . Mitochondrial respiration in cultured cardiac fibroblasts was measured in the previous study 12 , but the cellular energetics in those fibroblasts did not optimally respond to oligomycin and FCCP. Moreover, majority of mitochondria in the heart are in cardiomyocytes. Therefore, our finding provide evidence supporting the in vivo role of Honokiol on cardiac mitochondrial respiration.
Dox is known to induce cardiac mitochondrial damage followed by oxidative damages. Lo et. al reported that Honokiol attenuated mitochondrial lipid peroxidation and reduced free radical scavenging activities 16 . On the other hand, Honokiol has been shown to induce mitochondrial dysfunction and swelling in isolated mitochondria 22 . However, this study was conducted by treating directly the isolated mitochondria extracted from rat liver with Honokiol, and the doses of Honokiol were relatively high. Recognizing the still obscured effect of Honokiol on mitochondrial function in the heart, we conducted a comprehensive analysis of the effects of Honokiol on cardiac mitochondrial energetics in mice with or without Dox treatment using the real time oxygraphy assessment. Another novel finding here is that Honokiol protects mitochondrial respiration capacities in mice suffering Dox-induced cardiotoxicity. By titration of various substrates, we stimulated the tricarboxylic acid (TCA) cycle and the different complexes of the electron transport chain. Routine and ADP-stimulated oxygen consumption rates, as well as maximal uncoupled oxidative capacity induced by FCCP (making oxygen consumption independent of ATP production), were ameliorated in cardiac mitochondria from Honokiol treated mice. These findings support that Honokiol enhances mitochondrial function in the in vivo animal, which in turn protects against Dox toxicity to mitochondria.
While Honokiol may protect mitochondria in Dox-treated hearts via de-acetylating mitochondrial proteins, other mechanisms may also be involved. A previous study showed that Honokiol binds to the PPARγ ligand-binding domain (LBD) and acts as a partial agonist in a PPARγ-mediated luciferase reporter assay 23 . The study further showed that Honokiol might work as a modest PPARγ activator without inducing adipogenesis 23 .
A most recent report also shows that Honokiol attenuates diet-induced nonalcoholic steatohepatitis by regulating macrophage polarization through activating PPARγ 24 . Our study on cultured rat embryonic cardiomyocytes (H9c2 cell) confirms a mild, but significant, effect of Honokiol in activating the transcriptional activity of PPARγ. Recognizing the limitations of interpreting results from cultured H9c2 cells, which are not fully differentiated cardiomyocytes and with low PPARγ expression 25-28 , we further assessed the effect of Honokiol on cardiac expression of PPARγ with the treatment of Honokiol in mice. Consistently, both transcript and protein expression of cardiac PPARγ were upregulated in mice with Honokiol treatment. The upregulation of cardiac PPARγ by Honokiol is not attenuated by Dox treatment. The modest effects of Honokiol on the PPRE reporter assay in cultured H9C2 cell may be due to the relative low expression of PPARγ in H9C2. However, it appears that Honokiol treatment in vivo is sufficient to induce substantial upregulation of cardiac PPARγ transcript and protein. Although other signaling pathways, such as epigenetic modifications of Sirt3, may be involved, the PPARγ upregulation and activation effects of Honokiol appear to be the key factor in enhancing the cardiac PPARγ expression and activity. It is well established that PPARγ activation could increase gene expression of mitochondrial metabolic genes, thus facilitating mitochondrial respiration. Therefore, PPARγ activation may contribute to the mitochondrial effect of Honokiol treatment.
Given that mitochondria are the major organelles that produce ROS 29 , Dox-induced mitochondrial dysfunction causes the generation of excessive ROS in the cardiac tissue 30 . Yu et al. reported that Honokiol protects against renal ischemia/reperfusion injury by suppressing oxidative stress, iNOS, inflammation, and STAT3 in rats 31 . In the present study, Honokiol treatment depressed total ROS levels, which illustrated by the less pronounced decreased ratio of GSH/GSSG in mice suffering from Dox-induced cardiotoxicity. Furthermore, the anti-oxidative effects of Honokiol appear to attribute to its mitochondrial respiration enhancing and uncoupling capabilities 29 . As a result, cardiac inflammation-induced by Dox was attenuated by Honokiol pretreatment. The anti-oxidative and anti-inflammation capacities of Honokiol apparently contribute to its cardiac protective effect against Dox-cardiotoxicity. Moreover, the anti-oxidative stress effects of Honokiol may also derive from its transcriptional regulation of endogenous anti-oxidants as a PPARγ ligand in the heart as we reported previously 18 .  On the other hand, it has been well documented that PPARγ-specific ligands exert anti-inflammatory effects in the cardiovascular system 32 . Activating PPARγ should contribute to Honokiol anti-inflammatory effects in mice treated with Dox. In addition to the cardiac protective effects, the potential beneficial effects of Honokiol on multiple tissues in the body are possible, which are especially obvious in those mice subjected to the acute Dox treatment. On the other hand, previous studies suggest Honokiol may be a promising therapeutic anti-cancer agent. Lu et al. reported that Honokiol induces cell cycle arrest and apoptosis in vitro and in vivo in human thyroid cancer cells 33 . Hua et al. reported that Honokiol augments the anti-cancer effects of oxaliplatin in colon cancer cells 34 . Therefore, the potential dual effects of Honokiol on anti-cancer and cardiac protection indicate the promising clinical benefits of Honokiol treatment among cancer patients. Further preclinical and clinical studies are warranted.
In conclusion, our study demonstrates for the first time that Honokiol treatment protects the heart from Dox-cardiotoxicity via facilitating mitochondrial respiration and exerting anti-oxidant and anti-inflammation   No: 35354-74-6, MedChem Expression, USA) treatment (0.2 mg/kg/day for 35 days, i.p.) started one week before the start of Dox treatment. The detailed protocol is indicated (Fig. 1). Dox was dissolved in 0.9% normal saline, and Honokiol was dissolved in corn oil. In control mice, vehicle (corn oil) was used. After echocardiographic assessment, all animals were sacrificed, and organs were collected and snap frozen in liquid nitrogen followed by storage at −80 °C. Hearts were also perfused with cardioplegia solution (25 mM KCl and 5% glucose) and fixed with formalin for histological analysis as described previously 35-37 . Echocardiographic assessment. Echocardiography was performed in mice anesthetized with 1.5% isoflurane as previously described 37 using a Vevo 1100 Imaging System (Visual Sonics, Toronto, Canada) equipped with a 30 MHz linear-array transducer. The following parameters were obtained: LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD), the percentage of fractional shortening (FS, %), ejection fraction (EF, %), and other parameters were measured from the M-mode images and two-dimensional obtained in the longand short-axis views by the corresponding matching software. All measurements were performed from leading edge according to the American Society of Echocardiography guidelines.
Mitochondrial isolation. Cardiac mitochondria were isolated from male C57BL/6 J mice. Briefly, blood and main vasculature were dissected, and heart tissue was minced on ice, then suspended in buffer A (250 mM sucrose, 10 mM Tris/Cl, 0.5 mM EDTA) and homogenized using a 2 ml Potter-Elvehjem Teflon-glass homogenizer. The resulting samples were centrifuged at 1000 g for 10 minutes, and the supernatant with mitochondria was poured into another ice-cold tube, followed by centrifugation at 8000 g for 5 minutes. The mitochondrial enriched sediments were resuspended in buffer A. Mitochondrial protein content was determined by the Lowry method.
Assessment of mitochondrial respiration. Mitochondrial respiration was measured as previously described 17 . Briefly, 200 μg of freshly isolated mitochondria were measured in 2 ml of MirO5 mitochondrial respiration medium (3 mM MgCl 2 , 60 mM Lactobionic acid, 20 mM Taurine, 10 mM KH 2 PO 4 , 20 mM HEPES,110 mM D-sucrose, 1 g/L BSA and 0.5 mM EGTA) using an Oroboros 2k-Oxygraph (Oroboros Instruments, Innsbruck, Austria). Mitochondrial respiration was stimulated by basal substrates (5 mM pyruvate, 5 mM malate, 10 mM glutamate and 1 mM ADP) for complex I activity. Next 10 mM succinate was added to measure combined respiration rates of complex I and complex II. After that, 2 μg/ml oligomycin was added for the estimate of the overall mitochondrial related respiration. Further, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added to determine the maximal coupling respiration. Finally, the addition of antimycin A allowed for the measurement of non-mitochondrial oxygen consumption.
LDH activity. Serum was separated from the blood for the measurement of tissue injury marker lactate dehydrogenase (LDH) 38 using an LDH assay kit (Applygen Technologies Inc, Beijing, China). The assay is based on LDH-dependent and NADH-catalyzed reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diph enyltetrazolium bromide to a reduced form. Absorbance was measured at 440 nm using a BioTek plate reader (BioTek), and the values were directly proportional to the enzyme activity.
Detection of ROS production. Dihydroethidium (DHE, Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) was applied to frozen section samples (7 μm-thick sections). The heart sections were stained with 5 μM DHE and incubated in a light-protected humidified chamber at 37 °C for 30 min. Fluorescence intensity was examined by fluorescence microscopy (Nikon DXM1200 fluorescence microscope) and images were analyzed with the ImageJ software.
GSH/GSSG ratio assay. The ratios of glutathione and oxidized glutathione (GSSG) were measured using a GSH and GSSG assay kit (S0053, Beyotime Institute of Biotechnology), according to the manufacturer's protocol.
Cell culture. H9c2  Kit (TRANSGEN, Beijing, China) according to the manufacturer's protocol. PCR products were separated on 2% agarose gels and documented with BioRad Gel Doc. Results from each gene/primer pair were normalized to β-actin and compared across conditions. The sequences of the primers are listed as follows: P PA R γ f o r w a r d p r i m e r 5 ′ -A A AG AC C C AG C T C TAC A AC A-3 ′ a n d r e v e r s e p r i m e r 5′-TCGTAGATGACAAATGGTGA-3′, SOD2 forward primer 5′-GCCTCCCAGACCTGCCTTAC-3′ and reverse primer 5′-TCGGTGGCGTTGAGATTGT-3′, CD36 forward primer 5′-AGATGACGTGGCAAAGAACAG-3′ and reverse primer 5′ -CCT TGGCTAGATAACGAACTCTG-3′ β -actin for ward primer 5′ -CTGTCCCTGTATGCCTCTG-3′ and reverse primer 5′-ATGTCACGCACGATTTCC-3′. At least three independent experiments were conducted to ensure the reproducibility of the data.
Western blotting. The frozen cardiac tissues were lysed in a RIPA buffer (Applygen Technologies Inc, Beijing, China). The BCA Protein Assay Kit (Boster Biological Engineering Co., Ltd, Wuhan, China) was used to measure protein concentrations. Thirty microgram protein samples were separated by 10% sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). Furthermore, 5% non-fat dried milk was used to block the membrane for 2 h at room temperature and then incubated with primary antibodies (PPARγ 1:1,000, Santa Cruz, USA; Acetylated-Lysine 1: 1,000, Cell Signaling) at 4 °C overnight. After being washed with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody anti-mouse IgG (1: 2,500, Boster Biological Engineering Co., Wuhan, China) and peroxidase-conjugated secondary antibody anti-rabbit IgG (1:10,000, Santa Cruz, USA) for 1 h at room temperature. Enhanced chemiluminescence reagents were used to exposed the bands. Equivalent protein loads were verified and normalized using GAPDH (1:1,000, Santa Cruz, USA) or actin (1:1,000, Sigma-Aldrich, USA) blots. Finally, bands were then quantified by densitometry using ImageJ software.
Histological analysis. Heart tissue samples were fixed in 4% formalin for immunohistochemistry. Briefly, samples were embedded in paraffin, cut into 4 μm-thick sections, and stained with rabbit anti-CD68 (1:100, Boster, Wuhan, China). Staining was visualized with biotin labeled Goat anti-rabbit Ig-G secondary antibody. Staining with the secondary antibody alone was performed as a negative control. The number of CD68-positive cells was similarly counted in 5 randomly selected fields at 400x magnification and used to calculate the mean number of positively stained cells per microscopic field. Images were taken with a light microscope.

Statistical Analyses. Data for 2-group comparisons were analyzed with the nonparametric Student t-test;
otherwise, data were analyzed by one-factor or mixed, 2-factor ANOVA and multiple comparisons test using the GraphPad Prism 6 software (GraphPad Software Inc.). Values of Quantitative results were expressed as mean ± SEM. Differences between groups and treatments were regarded as significant at p < 0.05. Data availability. All data generated or analysed during the current study are available from the corresponding author on reasonable request.