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Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis

Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative... marine drugs Article Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis 1 , 2 , † 1 , † 1 , † 1 , † 1 1 Jun-Jie Guo , Lei-Lei Ma , Hong-Tao Shi , Jian-Bing Zhu , Jian Wu , Zhi-Wen Ding , 2 1 , 3 , 1 , 3 , Yi An , Yun-Zeng Zou * and Jun-Bo Ge * Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 180 Feng Lin Road, Shanghai 200032, China; qy_junjie@163.com (J.-J.G.); mllsdjn@126.com (L.-L.M.); hongtaoshi@126.com (H.-T.S.); zhujianbing543@163.com (J.-B.Z.); everwoo@sina.com (J.W.); zhiwen.d@gmail.com (Z.-W.D.) Department of Cardiology, The Affiliated Hospital of Qingdao University, 16 Jiang Su Road, Qingdao 266003, China; any@medmail.com.cn Institute of Biomedical Science, Fudan University, Shanghai 200032, China * Correspondence: zou.yunzeng@zs-hospital.sh.cn (Y.-Z.Z.); jbge@zs-hospital.sh.cn (J.-B.G.); Tel.: +86-21-54237969 (Y.-Z.Z.); +86-21-64041990 (ext. 2745) (J.-B.G.) † The first four authors contributed equally to this work. Academic Editor: Peer B. Jacobson Received: 11 September 2016; Accepted: 9 December 2016; Published: 20 December 2016 Abstract: Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. Keywords: alginate oligosaccharide; doxorubicin cardiotoxicity; oxidative stress; endoplasmic reticulum; apoptosis 1. Introduction Doxorubicin (DOX), an anthracycline antibiotic, is a widely used and highly potent chemotherapeutic agent to treat a broad range of cancers. However, the clinical usage of DOX is greatly limited by its dose-dependent and time-dependent cardiotoxicity [1]. The mechanisms of DOX-induced cardiotoxicity have not been fully elucidated. A plethora of deleterious effects such as excessive reactive oxygen species (ROS) generation, lipid peroxidation, DNA/RNA damage, calcium mishandling, inhibition of autophagic flux and endoplasmic reticulum-mediated apoptosis have all Mar. Drugs 2016, 14, 231; doi:10.3390/md14120231 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016, 14, 231 2 of 13 been linked to DOX-induced cardiotoxicity [2–4]. Anti-oxidative therapies including using antioxidants and transgenic mice overexpressing anti-oxidative enzymes have been verified to be effective in protecting against DOX-induced cardiotoxicity [5–8]. A recent study has demonstrated that a chemical endoplasmic reticulum chaperone could obliterate DOX-induced cardiac dysfunction [9]. These observations hint that some agents may alleviate DOX-induced cardiotoxicity by inhibiting oxidative stress or endoplasmic reticulum-mediated apoptosis. Alginate, an acidic polysaccharide consisting of alternative blocks of -D-mannuronic acid (M) and -L-guluronic acid (G), is extracted from various marine brown algae and has been widely used in various fields, such as food, tissue engineering, and drug delivery, because of its non-toxic, non-immunogenic characteristics [10]. Alginate oligosaccharide (AOS), produced by depolymerizing alginate using different degradation methods including enzymatic degradation, acid hydrolysis and oxidative degradation, has been proven to exert several pharmacological activities, including anti-oxidative [11], anti-apoptotic [12], anti-inflammatory [13] and anti-proliferative effects [14]. Nevertheless, whether AOS is protective against acute DOX cardiotoxicity is not yet clear, and the underlying mechanisms need to be elucidated. For this purpose, the present study aimed to evaluate the protective effect of AOS acquired by an enzymatic degradation method on acute DOX cardiotoxicity. To explore the potential mechanisms of this protection, we examined the influences of AOS on the oxidative stress and endoplasmic reticulum-mediated apoptosis. 2. Results 2.1. AOS Pretreatment Preserves DOX-Induced Cardiac Dysfunction in Mice To evaluate the cardiac function, we used a dose of 15 mg/kg DOX because of the high morbidity induced by the dose of 20 mg/kg DOX. First, we observed whether AOS treatment (200 mg/kg/day, five days) after DOX injection could exert a protective effect on cardiac dysfunction induced by DOX insult. Unfortunately, no significant improvement was observed (Figure S5), and further research was not pursued. Then, the cardioprotection of AOS pretreatment (200 mg/kg/day, seven days) was observed. All mice remained alive on the fifth day after DOX administration, and representative echocardiograms are shown in Figure 1A. Compared with the control (CON) group, mice pre-treated with AOS alone did not exhibit abnormal cardiac function, and there was no difference in left ventricular end-diastolic dimensions (LVEDD) among the four groups (Figure 1B). Surprisingly, mice receiving a DOX injection exhibited a significant increase of 52% for left ventricular end systolic dimensions (LVESD) compared to the CON group, whereas this alteration was markedly restored in the AOS + DOX group (Figure 1C). Moreover, DOX insult induced a marked decrease of 37% and 30%, respectively, in cardiac contractility characterized by the left ventricular ejection fraction (EF) and fractional shortening (FS), and these effects were significantly attenuated by AOS pretreatment (Figure 1D,E). Mar. Drugs 2016, 14, 231 3 of 13 Mar. Drugs 2016, 14, 231  3 of 13  Figure 1. AOS pretreatment attenuates doxorubicin (DOX)‐induced cardiac contractile dysfunction.  Figure 1. AOS pretreatment attenuates doxorubicin (DOX)-induced cardiac contractile dysfunction. (A)  Cardiac  function  of  mice  receiving  DOX  injection  with  or  without  AOS  pretreatment  (200  (A) Cardiac function of mice receiving DOX injection with or without AOS pretreatment mg/kg/day, 7 days) was measured after five days, and representative echocardiographic images were  (200 mg/kg/day, 7 days) was measured after five days, and representative echocardiographic acquired; (B–E) Cardiac function was measured after five days. LVEDD: left ventricular end‐diastolic  imagesdimension; were acquir  LVESD: ed; ( Bleft –E ventr ) Caridiac cular function end‐systolic was dimension; measured EFafter : left ventri five days. cular eject LVEDD: ion fraction; left ventricular  FS:   # end-diastolic left ventri dimension; cular fractional LVESD:  shortening. left ventricular  * p < 0.05 vs end-systolic . the control (CON) dimension;  group;EF:  p <left  0.05ventricular  vs. the CONejection  +  DOX group, n = 6 in each group.  fraction; FS: left ventricular fractional shortening. * p < 0.05 vs. the control (CON) group; p < 0.05 vs. the CON + DOX group, n = 6 in each group. 2.2. AOS Pretreatment Attenuates Acute DOX‐Induced Cardiotoxicity in Mice  The general status of mice in the four groups was observed daily after the DOX insult. Mice in  2.2. AOS Pretreatment Attenuates Acute DOX-Induced Cardiotoxicity in Mice CON + DOX group appeared lethargic and weak with weight loss, and 90% of them had died by  The general status of mice in the four groups was observed daily after the DOX insult. Mice in eight days after DOX (20 mg/kg) treatment (Figure 2A). However, these signs were attenuated in the  CON + mice DOX  pre‐ gr treated oup  appear with AO ed S (2 lethar 00 mg/kg/da gic andy, weak seven with days) weight and mort loss, ality wa and s dec 90% reased of them  to 40% had  by 15 died   by days. The mice in both the CON and AOS groups were all alive over the entire observation period  eight days after DOX (20 mg/kg) treatment (Figure 2A). However, these signs were attenuated in (15 days). The heart‐to‐body weight ratio (Figure 2B) on the fifth day after DOX injection (15 mg/kg)  the mice pre-treated with AOS (200 mg/kg/day, seven days) and mortality was decreased to 40% was obviously decreased compared with the CON group, but the AOS pretreatment (200 mg/kg/day,  by 15 days. The mice in both the CON and AOS groups were all alive over the entire observation seven days) significantly improved the loss of the heart‐to‐body weight ratio compared with the CON  period (15 days). The heart-to-body weight ratio (Figure 2B) on the fifth day after DOX injection + DOX group. Similarly, cTnI—a specific biomarker of cardiac injury—was significantly increased in  (15 mg/kg) was obviously decreased compared with the CON group, but the AOS pretreatment the CON + DOX group compared with the CON and AOS groups. However, the cTnI level was  (200 mg/kg/day, seven days) significantly improved the loss of the heart-to-body weight ratio significantly decreased in the AOS + DOX group compared with the CON + DOX group (Figure 2C).  compared with the CON + DOX group. Similarly, cTnI—a specific biomarker of cardiac injury—was significantly increased in the CON + DOX group compared with the CON and AOS groups. However, the cTnI level was significantly decreased in the AOS + DOX group compared with the CON + DOX group (Figure 2C). Mar. Drugs 2016, 14, 231 4 of 13 Mar. Drugs 2016, 14, 231  4 of 13  Mar. Drugs 2016, 14, 231  4 of 13  Figure 2. AOS pretreatment attenuates acute DOX cardiotoxicity in mice. (A) Kaplan‐Meier survival  Figure 2. AOS pretreatment attenuates acute DOX cardiotoxicity in mice. (A) Kaplan-Meier survival curve analysis of mice after DOX injection with or without AOS pretreatment (200 mg/kg/day, 7 days).  curve analysis of mice after DOX injection with or without AOS pretreatment (200 mg/kg/day, 7 days). n = 10 in each group; (B) Graphs showing the heart weight to body weight ratio. HW: heart weight;  n = 10Figure in each  2. AO group S pretreatment ; (B) Graphs  attenuates showing acute the DOX heart cardiot weight oxito city body  in miweight ce. (A) Kaplan ratio.‐HW Meie:r heart surviva weight; l  BW: body weight. n = 6 in each group; (C) Evaluation of plasma cTnI levels. n = 6 in each group. * p <  curve analysis of mice after DOX injection with or without AOS pretreatment (200 mg/kg/day, 7 days).  BW: body 0.05 vs weight. . the CONn group; = 6 in   each p < 0.05 gr vs oup; . the( C CON ) Evaluation  + DOX group. of plasma   cTnI levels. n = 6 in each group. n = 10 in each group; (B) Graphs showing the heart weight to body weight ratio. HW: heart weight;  * p < 0.05 vs. the CON group; p < 0.05 vs. the CON + DOX group. BW: body weight. n = 6 in each group; (C) Evaluation of plasma cTnI levels. n = 6 in each group. * p <  2.3. AOS Pretreatment Protects DOX‐Induced Myocardial Histological Alterations and Apoptosis in Mice  0.05 vs. the CON group;   p < 0.05 vs. the CON + DOX group.  2.3. AOS Pretreatment Protects DOX-Induced Myocardial Histological Alterations and Apoptosis in Mice To examine whether AOS pretreatment (200 mg/kg/day, seven days) attenuates DOX‐induced  cardiac injury, we analyzed the heart sections with hematoxylin and eosin (HE) staining through  2.3. AOS Pretreatment Protects DOX‐Induced Myocardial Histological Alterations and Apoptosis in Mice  To examine whether AOS pretreatment (200 mg/kg/day, seven days) attenuates DOX-induced electron  microscopy.  As  shown  in  Figure  3,  AOS  by  itself  had  no  effect  on  cardiac  morphology.  cardiac injury, we analyzed the heart sections with hematoxylin and eosin (HE) staining through To examine whether AOS pretreatment (200 mg/kg/day, seven days) attenuates DOX‐induced  Consistent  with  a  previous  study  [15],  DOX‐treated  mice  exhibited  extensive  focal  cytoplasmic  electron card micr iac injur oscopy y, we . As  anashown lyzed the in heart Figur sections e 3, AOS  withby  hematoxylin itself had and no eosin effect (HE) on car stain diac ing through morphology   . vacuolization, a specific change of DOX‐induced cardiac injury, whereas this effect was significantly  electron  microscopy.  As  shown  in  Figure  3,  AOS  by  itself  had  no  effect  on  cardiac  morphology.  Consistent with a previous study [15], DOX-treated mice exhibited extensive focal cytoplasmic reduced  by  AOS  pretreatment.  Terminal  deoxynucleotidyl  transferase‐mediated  dUTP  nick  end  Consistent  with  a  previous  study  [15],  DOX‐treated  mice  exhibited  extensive  focal  cytoplasmic  vacuolization, labeling (TU a specific NEL) assa change ys wereof perform DOX-induced ed to determine cardiac  the injury effect of , wher  AOS eas on ap this opto efsis fect  in DOX was‐significantly treated  vacuolization, a specific change of DOX‐induced cardiac injury, whereas this effect was significantly  hearts. As shown in Figure 4, TUNEL‐positive cardiomyocytes were rarely observed in the heart  reduced by AOS pretreatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick end reduced  by  AOS  pretreatment.  Terminal  deoxynucleotidyl  transferase‐mediated  dUTP  nick  end  sections of mice in the CON and AOS groups. Conversely, a significantly larger number of TUNEL‐ labeling (TUNEL) assays were performed to determine the effect of AOS on apoptosis in DOX-treated labeling (TUNEL) assays were performed to determine the effect of AOS on apoptosis in DOX‐treated  positive cardiomyocytes (16.6%) were detected in the CON + DOX group, whereas this change was  hearts. As shown in Figure 4, TUNEL-positive cardiomyocytes were rarely observed in the heart hearts. As shown in Figure 4, TUNEL‐positive cardiomyocytes were rarely observed in the heart  significantly mitigated by AOS pretreatment (9.6%).  sections of mice in the CON and AOS groups. Conversely, a significantly larger number of sections of mice in the CON and AOS groups. Conversely, a significantly larger number of TUNEL‐ TUNEL-positive cardiomyocytes (16.6%) were detected in the CON + DOX group, whereas this positive cardiomyocytes (16.6%) were detected in the CON + DOX group, whereas this change was  change sign was ificant significantly ly mitigated mitigated  by AOS pretrea by AOS tment pr etr (9.6% eatment ).  (9.6%). Figure  3.  Effect  of  AOS  pretreatment  on  DOX‐induced  myocardial  histological  alterations.  Representative  histopathological  findings  at  400×  magnification  of  mouse  hearts  stained  with  hematoxylin and eosin (HE). Black arrows indicate extensive cytoplasmic vacuolization and nuclear  Figure  3.  Effect  of  AOS  pretreatment  on  DOX‐induced  myocardial  histological  alterations.  condensation or dissolution, n = 6 in each group.  Figure 3. Effect of AOS pretreatment on DOX-induced myocardial histological alterations. Representative  histopathological  findings  at  400×  magnification  of  mouse  hearts  stained  with  Representative histopathological findings at 400 magnification of mouse hearts stained with hematoxylin and eosin (HE). Black arrows indicate extensive cytoplasmic vacuolization and nuclear  hematoxylin and eosin (HE). Black arrows indicate extensive cytoplasmic vacuolization and nuclear condensation or dissolution, n = 6 in each group.  condensation or dissolution, n = 6 in each group. Mar. Drugs 2016, 14, 231 5 of 13 Mar. Drugs 2016, 14, 231  5 of 13  Figure  4.  AOS  pretreatment  decreases  DOX‐induced  myocardial  apoptosis.  (A)  Representative  Figure 4. AOS pretreatment decreases DOX-induced myocardial apoptosis. (A) Representative photomicrographs  at  400×  magnification  of  mouse  hearts  stained  with  TUNEL,  apoptotic  photomicrographs at 400 magnification of mouse hearts stained with TUNEL, apoptotic cardiomyocyte nuclei appear brown‐stained, whereas normal nuclei appear blue; (B) Quantitative  cardiomyocyte nuclei appear brown-stained, whereas normal nuclei appear blue; (B) Quantitative analysis of the percentage of TUNEL‐positive cells. * p < 0.05 vs. the CON group;   p < 0.05 vs. the  analysis of the percentage of TUNEL-positive cells. * p < 0.05 vs. the CON group; p < 0.05 vs. the CON + DOX group, n = 6 in each group.  CON + DOX group, n = 6 in each group. 2.4. AOS Pretreatment Reduces DOX‐Induced Oxidative Stress in Mice  2.4. AOS Pretreatment Reduces DOX-Induced Oxidative Stress in Mice Nicotinamide‐adenine  dinucleotide  phosphate  (NADPH)  oxidases  are  involved  in  DOX‐ induced  oxidative  stress,  and  DOX‐stimulated  NADPH‐dependent  superoxide  production  in  the  Nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are involved in DOX-induced heart is critically dependent on gp91 (phox) [8]; therefore, we measured the protein expression of  oxidative stress, and DOX-stimulated NADPH-dependent superoxide production in the heart is gp91(phox) in the hearts. As shown in Figure 5A, AOS alone did not have an obvious effect on the  critically dependent on gp91 (phox) [8]; therefore, we measured the protein expression of gp91(phox) expression of gp91(phox). However, DOX treatment significantly increased the expression of gp91  in the hearts. As shown in Figure 5A, AOS alone did not have an obvious effect on the expression (phox) by 3.0‐fold compared with the CON group, and this effect was decreased to 2.1‐fold by AOS  of gp91(phox). However, DOX treatment significantly increased the expression of gp91 (phox) by pretreatment (200 mg/kg/day, seven days). Similar results were found in assessing the expression of  3.0-fold compared with the CON group, and this effect was decreased to 2.1-fold by AOS pretreatment lipid peroxide 4‐HNE (Figure 5B). AOS by itself had no marked effect on lipid peroxidation compared  (200 mg/kg/day, seven days). Similar results were found in assessing the expression of lipid peroxide with the CON group but obviously reduced the accumulation of 4‐HNE induced by DOX. The levels  4-HNE of(Figur  the lipi ed 5 B). peroxide AOS by  4‐HN itself E in had  the no CO marked N + DOX effect  grou on p and lipid AOS per oxidation + DOX gro compar up were ed fo with und to the be CON   groupincre butaobviously sed  by  3.3‐ rand educed   1.7‐fol the d, accumulation respectively,  comp of 4-HNE ared  to  induced that  in  the by  CON DOX.   grou The p.  levels Representa of the tivelipid   western blot images are shown in Figure 5C.  peroxide 4-HNE in the CON + DOX group and AOS + DOX group were found to be increased by 3.3- and 1.7-fold, respectively, compared to that in the CON group. Representative western blot images are shown in Figure 5C. Mar. Drugs 2016, 14, 231 6 of 13 Mar. Drugs 2016, 14, 231  6 of 13  Mar. Drugs 2016, 14, 231  6 of 13  Figure 5. AOS pretreatment inhibits oxidative stress in the heart treated with DOX. (A,B) Quantitative  Figure 5. AOS pretreatment inhibits oxidative stress in the heart treated with DOX. (A,B) Quantitative analyses of gp91 phox and 4‐HNE protein expression; (C) Representative blots of gp91 phox, 4‐HNE  analyses of gp91 phox and 4-HNE protein expression; (C) Representative blots of gp91 phox, 4-HNE Figure 5. AOS pretreatment inhibits oxidative stress in the heart treated with DOX. (A,B) Quantitative  and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). * p < 0.05 vs. the CON group;   p < 0.05  and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * p < 0.05 vs. the CON group; p < 0.05 vs. analyses of gp91 phox and 4‐HNE protein expression; (C) Representative blots of gp91 phox, 4‐HNE  vs. the CON + DOX group, n = 6 in each group.  the CON + DOX group, n = 6 in each group. # and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). * p < 0.05 vs. the CON group;   p < 0.05  vs. the CON + DOX group, n = 6 in each group.  2.5. AOS Pretreatment Ameliorates DOX‐Induced Endoplasmic Reticulum‐Mediated Apoptosis in Mice  2.5. AOS Pretreatment Ameliorates DOX-Induced Endoplasmic Reticulum-Mediated Apoptosis in Mice CHOP and Caspase‐12 are known to be involved in endoplasmic reticulum‐mediated apoptosis  2.5. AOS Pretreatment Ameliorates DOX‐Induced Endoplasmic Reticulum‐Mediated Apoptosis in Mice  CHOP and Caspase-12 are known to be involved in endoplasmic reticulum-mediated apoptosis in in the context of DOX cardiotoxicity [16,17]. Our results demonstrated that AOS by itself had no  CHOP and Caspase‐12 are known to be involved in endoplasmic reticulum‐mediated apoptosis  the context of DOX cardiotoxicity [16,17]. Our results demonstrated that AOS by itself had no significant significant effect on the expressions of CHOP and Caspase‐12 compared to the CON group (Figure  in the context of DOX cardiotoxicity [16,17]. Our results demonstrated that AOS by itself had no  6A,B). CHOP and Caspase‐12 protein expressions in the CON + DOX group were significantly higher  effect on the expressions of CHOP and Caspase-12 compared to the CON group (Figure 6A,B). CHOP significant effect on the expressions of CHOP and Caspase‐12 compared to the CON group (Figure  than that of the CON group, and this increase was markedly abolished by AOS pretreatment (200  and Caspase-12 protein expressions in the CON + DOX group were significantly higher than that of 6A,B). CHOP and Caspase‐12 protein expressions in the CON + DOX group were significantly higher  mg/kg/day,  seven  days).  DOX  administration  significantly  inhibited  the  expression  of  Bcl‐2  the CON group, and this increase was markedly abolished by AOS pretreatment (200 mg/kg/day, than that of the CON group, and this increase was markedly abolished by AOS pretreatment (200  compared  to  the  CON  group,  whereas  AOS  pretreatment  restored  the  expression  of  Bcl‐2  to  the  seven days). DOX administration significantly inhibited the expression of Bcl-2 compared to the mg/kg/day,  seven  days).  DOX  administration  significantly  inhibited  the  expression  of  Bcl‐2  normal level (Figure 6D). Bax protein expression in the CON + DOX group was significantly higher  CON group, whereas AOS pretreatment restored the expression of Bcl-2 to the normal level (Figure 6D). compared  to  the  CON  group,  whereas  AOS  pretreatment  restored  the  expression  of  Bcl‐2  to  the  than  that  of  the  CON  group,  whereas  AOS  pretreatment  significantly  decreased  the  expression  Bax protein expression in the CON + DOX group was significantly higher than that of the CON group, normal level (Figure 6D). Bax protein expression in the CON + DOX group was significantly higher  (Figure 6E). Representative western blot images are shown in Figure 6C,F.  whertha easn AOS that  pr of etr the eatment   CON  gro significantly up,  whereadecr s  AOS eased   pretrea the expr tment ession   signif(Figur icantly e  decre 6E). Raepr sedesentative   the  expression western   (Figure 6E). Representative western blot images are shown in Figure 6C,F.  blot images are shown in Figure 6C,F. Figure  6.  AOS  pretreatment  inhibits  endoplasmic  reticulum‐mediated  apoptosis  in  hearts  treated  with  DOX.  (A,B)  Quantitative  analyses  of  CHOP  and  Caspase‐12  protein  expression;  (C)  Figure 6. AOS pretreatment inhibits endoplasmic reticulum-mediated apoptosis in hearts treated with Figure  6.  AOS  pretreatment  inhibits  endoplasmic  reticulum‐mediated  apoptosis  in  hearts  treated  Representative blots of CHOP, Caspase‐12 and GAPDH; (D,E) Quantitative analyses of Bcl‐2 and Bax  DOX. (A,B) Quantitative analyses of CHOP and Caspase-12 protein expression; (C) Representative blots with  DOX.  (A,B)  Quantitative  analyses  of  CHOP  and  Caspase‐12  protein  expression;  (C)  protein expression; (F) Representative blots of Bcl‐2, Bax and GAPDH. * p < 0.05 vs. the CON group;  of CHOP Representative , Caspase-12  blots and  of CHOP, GAPDH;  Caspase (D,E‐)12 Quantitative  and GAPDH;analyses  (D,E) Quantitative of Bcl-2 and  analyses Bax pr ofotein  Bcl‐2 expr and Bax ession;    p < 0.05 vs. the CON + DOX group, n = 6 in each group.  protein expression; (F) Representative blots of Bcl‐2, Bax and GAPDH. * p < 0.05 vs. the CON group;  (F) Representative blots of Bcl-2, Bax and GAPDH. * p < 0.05 vs. the CON group; p < 0.05 vs. the  p < 0.05 vs. the CON + DOX group, n = 6 in each group.  CON + DOX group, n = 6 in each group. Mar. Drugs 2016, 14, 231 7 of 13 3. Discussion Treatment progression has improved the survival rate of patients with cancers. However, the progressive, irreversible chemotherapy-induced cardiotoxicity leaves the survivors at a higher risk of death than the general population [18,19]. DOX, one of the most effective chemotherapy agents, is broadly used as one component of treatment protocols, yet its administration is greatly compromised by its cardiotoxicity. Hence, it is urgent for cardio-oncologists to identify strategies that can protect against DOX-induced cardiotoxicity without affecting its anticancer activities. The current study demonstrated that AOS was cardioprotective in the face of acute DOX cardiotoxicity. We found that inhibitions of oxidative stress and endoplasmic reticulum-mediated apoptosis were associated with the cardioprotection. This present study was the first to demonstrate that mice pre-treated with AOS were well protected against acute DOX cardiotoxicity, as evidenced by a significantly improved survival rate, heart weight to body weight ratio, cardiac contractile dysfunction, histological alterations, and decreased the myocardial apoptosis. Plasma cTnI monitoring is broadly used in clinical practice and animal experiments as a specific biomarker of heart injury [20,21]. The plasma level of cTnI was significantly elevated in the DOX-treated mice, whereas AOS pretreatment down-regulated this increase, indicating the strong cardioprotective efficacy against acute DOX cardiotoxicity. AOS has also been shown to provide protection against many diseases including Alzheimer ’s disease and asthma [22,23]. By using neuron-like PC12 cells, Tusi et al. previously showed that AOS decreased H2O2-induced oxidative stress and endoplasmic reticulum-mediated apoptosis [12]. In contrast, AOS was also found to have the capacity to trigger an oxidative burst and induce resistance against infection in a few members of brown algae [24], which reminds us that AOS maybe exhibit different bioactivity in face of different injuries. Available laboratory evidence suggests that increased oxidative stress is well known as the causative factor in acute DOX cardiotoxicity [5,6,8]. Previous studies have confirmed that DOX induces the overproduction of a cascade of reactive oxygen species (ROS) such as hydroxyl radicals, oxyanions and hydrogen peroxide, which are involved in DOX-induced cardiotoxicity [25,26]. NADPH oxidases, with the primary function of ROS generation, are activated by various stimuli that are important in cardiac remodeling and are also capable of modulating other ROS sources [27]. Using rat embryonic myocyte H9C2 cell cultures, Gilleron et al. reported that the activity of NADPH oxidases increased more than two-fold after one hour of DOX incubation [28]. As a multi-component enzyme, NADPH oxidases are the primary source of oxidative stress in acute DOX cardiotoxicity. Once stimulated, the cytosolic complex migrates to the membrane and assembles with other subunits to form an active oxidase with the ability to produce superoxide anions [29]. The previous study demonstrated that DOX-induced NADPH-dependent superoxide generation was critically dependent on NOX2 (also named gp91 (phox)). Knockout of NOX2 protected the mice against heart failure, cardiomyocyte atrophy, and apoptosis after DOX treatment [8]. Overproduction of oxidants and electrophiles during DOX treatment activates the oxidative response of membrane lipids, leading to the accumulation of 4-HNE, which has been shown to affect mitochondrial function, modify protein functions and be a specific biomarker of DOX-induced oxidative stress [30,31]. Consistent with these studies, the expression of gp91 (phox), the pivotal NADPH oxidase subunit, was up-regulated after DOX administration. Similarly, the expression of the lipid peroxide 4-HNE was also up-regulated. However, pretreatment with AOS inhibited the oxidative stress process by down-regulating the expressions of gp91 (phox) and 4-HNE in the heart. Moreover, our results showed that AOS pretreatment had no obvious effect on the oxidative stress in the heart under physiological conditions. Endoplasmic reticulum-mediated apoptotic signaling has been studied in the development of heart failure [32,33]. It is also believed that increased oxidative stress can directly induce endoplasmic reticulum stress [34,35]. Chua et al. found that DOX could activate the endoplasmic reticulum-initiated apoptotic response and further augment endoplasmic reticulum stress in mouse hearts [36]. Signal transduction pathways such as the Caspase-12 and C/EBP homologous protein (CHOP) pathways can mediate and augment endoplasmic reticulum-mediated apoptosis [37]. Mar. Drugs 2016, 14, 231 8 of 13 Mar. Drugs 2016, 14, 231  8 of 13  In the context of endoplasmic reticulum stress, Caspase-12, serving as an endoplasmic reticulum membrane-resident pro-apoptotic molecule, activates the caspase cascades which mainly induce cell resident pro‐apoptotic molecule, activates the caspase cascades which mainly induce cell death [38].  death [38]. Consistent with previous research that demonstrates that the inhibition of Caspase-12 Consistent with previous research that demonstrates that the inhibition of Caspase‐12 cleavage is  cleavage is linked with the recovery of heart function [39], our data suggested that AOS pretreatment linked with the recovery of heart function [39], our data suggested that AOS pretreatment attenuated  attenuated Caspase-12 cleavage in the face of DOX insult, leading to decreased myocardial apoptosis Caspase‐12  cleavage  in  the  face  of  DOX  insult,  leading  to  decreased  myocardial  apoptosis  and  and improved cardiac dysfunction. Many studies have demonstrated that CHOP, a marker of improved cardiac dysfunction. Many studies have demonstrated that CHOP, a marker of endoplasmic  endoplasmic reticulum-mediated apoptosis [40], can promote apoptosis by inhibiting expression reticulum‐mediated  apoptosis  [40],  can  promote  apoptosis  by  inhibiting  expression  of  the  anti‐ of the anti-apoptotic factor Bcl-2 and activating expression of the pro-apoptotic factor Bax [41]. apoptotic factor Bcl‐2 and activating expression of the pro‐apoptotic factor Bax [41]. Over‐expression of  Over-expression of CHOP decreased the expression of Bcl-2 protein and facilitated translocation CHOP decreased the expression of Bcl‐2 protein and facilitated translocation of the Bax protein from  of the Bax protein from the cytosol to the mitochondria [42]. Our data also supported this hypothesis. the cytosol to the mitochondria [42]. Our data also supported this hypothesis. Administration of DOX  Administration of DOX significantly activated the CHOP signaling pathway-mediated myocardial significantly activated the CHOP signaling pathway‐mediated myocardial apoptosis by regulating  apoptosis by regulating the expressions of Bcl-2 and Bax. However, the detrimental effect was the  expressions  of  Bcl‐2  and  Bax.  However,  the  detrimental  effect  was  ameliorated  by  AOS  ameliorated by AOS pretreatment. Importantly, our results showed that AOS pretreatment had pretreatment. Importantly, our results showed that AOS pretreatment had no marked effects on the  no marked effects on the endoplasmic reticulum mediated-apoptotic signaling pathway under endoplasmic reticulum mediated‐apoptotic signaling pathway under physiological conditions.  physiological conditions. 4. Materials and Methods  4. Materials and Methods 4.1. 4.1. Preparatio Preparationn of of AOS AOS  AOS, AOS, aa gift gift fr from om Qingdao Qingdao BZ BZ Oligo Oligo Biotech Biotech Co. Co.  Ltd Ltd (Qingdao, (Qingdao,  China), China),  was was  pr produced oduced  by by the the  method method of of enzymatic enzymatic degradation degradation as as pr previously eviously described described [4 [433] ].. Br Briefly iefly,, alginate alginate (5 (5 g) g) derived derived fr from om  Laminaria Laminaria japonica japonica with with an an M/G M/G ratio ratio of of 1.86/1 1.86/1 [4 [44 4]] wa was s d dissolved issolved in in 50 5000 mL mL of of 50 50 mmol/L mmol/L Tris Tris-HCL ‐HCL  buf buffe ferr (pH (pH 7.0), 7.0), which which was was added added to to 200 200 units units of of alginate alginate lyase lyase purified purified fr from om Pseudomonas Pseudomonas sp. sp. HZJ HZJ  216. 216. The The en enzymatic zymatic rre eaction action was was performed performed at at 30 30 °C C for for 6 6 h, h, and and th then en termi terminated nated by by boiling boiling in in water water  for for 55 min. min. The The AOS AOS was was initially initially sep separat arated ed by by adjusting adjusting the the pH pH of of the the supernatan supernatant t to to 2.85. 2.85. Next, Next, the the  methods methods of ofanion-exchange  anion‐exchange chr  chrom omatography atography and and desalting  desalting wer were e used used to further  to furt purify her purify the AOS.  the  Then, AOS.  the Then, methods   the  methods of High  of Performanc   High  Per eformance Gel Permeation   Gel  Pe Chr rmeation omatography   Chroma (HPGPC) tography (Dionex, (HPGPC Sunnyvale, )  (Dionex,  CA, Sunnyv USA) ale, and   CA, Electr   USA) ospray   and Ionization   ElectrMass ospraSpec y  Ionization troscopy (ESI-MS) Mass  Spectros (Agilentcopy Technologies,   (ESI‐MS) Santa   (Agil Clara, ent  CA, Technologies, USA) wer eSa used nta C to lar determine a, CA, USA the ) were relative  used molecular  to determine mass the and relative degree molecula of polymerization r mass and (DP) degre ofe  the of polymeriza AOS. The rtion elative  (DPmolecular ) of the AOS. weight  The of relative AOS ismolecula approximately r weight1.2  of kDa AOS(Figur  is app erS1), oxim and atelthe y 1.2 DP kD ofa  AOS (Figure mainly  S1),  ranges and the fr DP om  2ofto AOS 6 (Figur  mainly es S2 range and S3, s from Tables  2 to S1 6and  (Fig S2). ures Lastly  S2 and , the S3 1H , Tables Nuclear  S1Magnetic  and S2).  Resonance Lastly,  the (1H-NMR) 1H  Nuclear spectr   Magn oscopy etic  Resonance method was  (1 performed H‐NMR)  spectr to determine oscopy the method M/G  ratio was  of performed AOS which   to  is determine 1/2.6 (Figur  the eM/G S4). rat The io chemical of AOS wh strich uctur  is 1/ e2. of6 AOS (Figur acquir e S4).ed  The thr che ough mical enzymatic  structure degeneration  of AOS acquwas ired  shown through in en Figur zymatic e 7.  degeneration was shown in Figure 7.  Figure  7.  Schematic  representation  of  the  molecular  structure  of  alginate  oligosaccharide  (AOS)  Figure 7. Schematic representation of the molecular structure of alginate oligosaccharide (AOS) prepared by enzymatic degradation.  prepared by enzymatic degradation. 4.2. Animals and Drug Administration  4.2. Animals and Drug Administration Adult  male  C57BL/6  mice  (22–25  g;  Department  of  Laboratory  Animal  Science,  Fudan  Adult male C57BL/6 mice (22–25 g; Department of Laboratory Animal Science, Fudan University, University, (Shanghai, China) were maintained under specific pathogen‐free conditions in an animal  (Shanghai, China) were maintained under specific pathogen-free conditions in an animal room on a room  on  a  12/12  h  light/dark  cycle  with  free  access  to  water  and  food.  All  experiments  were  performed in strict accordance with the guidelines of the China Council on Animal Management. The  protocols  were  approved  by  the  Committee  on  the  Ethics  of  Animal  Experiments  of  Fudan  University.  The  model  of acute DOX  cardiotoxicity was  induced  by intraperitoneal  injection  of a  Mar. Drugs 2016, 14, 231 9 of 13 12/12 h light/dark cycle with free access to water and food. All experiments were performed in strict accordance with the guidelines of the China Council on Animal Management. The protocols were approved by the Committee on the Ethics of Animal Experiments of Fudan University. The model of acute DOX cardiotoxicity was induced by intraperitoneal injection of a single dose (15 mg/kg) of DOX (Sigma-Aldrich, St. Louis, MO, USA) [15]. The AOS was dissolved in normal saline with a concentration of 2.0% w/v. In part one, the mice were randomly divided into the following four groups according to treatment: vehicle control group (CON, n = 6), DOX group (CON + DOX, n = 6), AOS treatment group (AOS-T, n = 6) and AOS treatment + DOX group (AOS-T + DOX, n = 6). The mice were treated with AOS (200 mg/kg/day) or an equal volume of normal saline by gavage for 5 consecutive days from the day of DOX injection to the end of the study based on a previous study [21]. In part two, the mice were randomly divided into the following four groups according to treatment: vehicle control group (CON, n = 6), DOX group (CON + DOX, n = 6), AOS pretreatment group (AOS, n = 6), and AOS pretreatment + DOX group (AOS + DOX, n = 6). The mice were pretreated with AOS (200 mg/kg/day) or an equal volume of normal saline by gavage for 7 consecutive days, and on the eighth day, the mice were acutely injected intraperitoneally with DOX or an equal volume of normal saline. Next, the mice received the same daily dose of AOS (200 mg/kg/day) and normal saline by gavage for another five days. At the end of the study, cardiac function was measured. Following anesthesia, body weights were recorded, blood samples were collected, and the hearts were rapidly removed, weighed, and then snap frozen in liquid nitrogen for further examinations. For survival observation, the mice were treated with a single high dose (20 mg/kg) of DOX, and the mortality was monitored (n = 10 in each group) for 15 days after DOX injection. A total of 112 animals were used in the experiments. 4.3. Determination of Cardiac Troponin-I Level On the fifth day after DOX injection, blood samples were collected from the carotid artery with EDTA-containing syringes prior to heart harvest. The plasma was prepared by centrifugation at 4000 rpm for 30 min and 4 C immediately frozen and stored at 80 C. The plasma was thawed only once for cardiac troponin-I (cTnI) measurement. The level of plasma cTnI was quantified with a Mouse Cardiac troponin-I for plasma kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assay was implemented according to the protocol provided by the manufacturer. 4.4. Hemodynamic Measurements Transthoracic echocardiography was performed by using an animal specific instrument (Vevo707B, Visual Sonics Inc., Toronto, ON, Canada) on the fifth day after DOX and normal saline injection. The mice were anesthetized with 1%–2% isoflurane, and the heart rates were stably maintained between 450 and 500 beats per min. After the B- and M-mode images were acquired, the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured as previously described [45]. All measurements were performed by three experienced technicians who were blinded to the animal groups. 4.5. Histopathological Analysis The heart tissue was immediately placed in 10% neutral buffered formalin for 24 h. Then, the specimen was embedded in paraffin, and serial sections were cut at 4 m thickness. The sections were stained with hematoxylin and eosin (HE). Eight microscopic fields (400) from each section were captured from each slide and imaged with a color digital camera. 4.6. Detection of Myocardial Apoptosis Myocardial apoptosis was examined by using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Paraffin-embedded sections of heart tissues were stained using an In Situ Cell Apoptosis Detection Kit (POD; Roche Diagnostics Corp, Indianapolis, IN, Mar. Drugs 2016, 14, 231 10 of 13 USA) according to the manufacturer ’s protocol. Eight microscopic fields (400) from each section were analyzed by counting brown nuclei. The percentage of TUNEL-positive nuclei (brown nuclei) was calculated. 4.7. Western Blot Analyses Hearts were quickly removed and frozen in liquid nitrogen. Total protein was extracted from the homogenized heart tissues using RIPA lysis buffer (Beyotime Biotechnology, Nanjing, China) and a complete protease inhibitor cocktail. Subsequently, extracted proteins (50 g) were loaded in a 10% or 12% polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. The protein expression was detected by immunoblotting with antibodies against gp91 (phox), 4-HEN, Caspase-12 (Abcam), CHOP, Bax, and Bcl-2 (Cell Signaling Technology). After three washes, the blots were incubated with radish peroxidase-conjugated rabbit secondary antibody immunoglobulin G (Kangchen Biotechnology, Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. The protein bands were detected by chemiluminescence and quantified using a Bio-Rad equipped with image software basic Quantity One (Bio-Rad, Hercules, CA, USA). 4.8. Statistical Analysis Data were expressed as the mean  standard deviation and compared by one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test using Graph Pad Prism 6 software (San Diego, CA, SA). Survival curves were determined by the Kaplan-Meier estimator and compared by a log-rank test. A p < 0.05 was considered statistically significant. 5. Conclusions The current study is the first to demonstrate that AOS successfully prevents acute DOX cardiotoxicity in mice, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. Our data indicate that AOS may clinically serve as a novel preventive strategy against acute DOX cardiotoxicity. However, we should not ignore the question of whether AOS administration interferes with the anticancer activity of DOX, which was not illuminated in the present study. Moreover, the pharmacokinetics of AOS were also not investigated. The challenge for future studies is to focus on answering these important questions. Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/14/12/231/s1, Figure S1: The relative molecular mass of AOS analyzed by High Performance Gel Permeation Chromatography (HPGPC). The relative molecular mass of AOS is 1.2 kDa, which was determined by the different molecular weights of dextran standards from the National Institutes for Food and Drug Control, Figure S2: The degree of polymerization (DP) of AOS analyzed by Electrospray Ionization Mass Spectroscopy (ESI-MS), Figure S3: The different degree of polymerization (DP) of AOS analyzed by High Performance Gel Permeation Chromatography (HPGPC), Figure S4: The M/G ratio of AOS analyzed by 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy. The M/G ratio of AOS was approximately 1/2.6 by calculating the integral areas of the M and G residues, Figure S5: Effects of AOS administered after DOX injection on cardiac dysfunction induced by DOX insult. (A) The cardiac function of mice receiving DOX injection with or without AOS (200 mg/kg, 5 days) treatment was measured after 5 days, and representative echocardiographic images were acquired; (B–E) Data of cardiac function were collected. LVEDD: left ventricular end-diastolic dimension; LVESD: left ventricular end-systolic dimension; EF: left ventricular ejection fraction; and FS: left ventricular fractional shortening. * p < 0.05 vs. the CON group; n = 6 in each group, Table S1: Electrospray Ionization Mass Spectroscopy (ESI-MS) data analysis of AOS. The DP of AOS mainly ranged from 2 to 6, Table S2: The relative percentages of the different degrees of polymerization of AOS. The AOS mainly contains DP2~DP6; the relative contents of different DP are DP2 26.27%, DP3 32.50%, DP4 18.55%, DP5 13.98%, DP6 3.69%, and >DP7 5.01%, according to the peak area normalization method. Acknowledgments: This study was supported by the General Program (Key Program, Major Research Plan) of the National Natural Science Foundation of China (Grant No. 81230007) and the National Natural Science Funds of China (Grant No. 81470386). We thank Qingdao BZ Oligo Biotech Co. Ltd. for the preparation of AOS. Author Contributions: J.-J.G. and L.-L.M. conceived and designed the experiments and wrote the paper; H.-T.S. analyzed the data. J.-B.Z., J.W. and Z.-W.D. performed the experiments; Y.A., Y.-Z.Z. and J.-B.G. gave constructive comments for the results and discussion of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Mar. Drugs 2016, 14, 231 11 of 13 References 1. Singal, P.K.; Iliskovic, N. Doxorubicin-induced cardiomyopathy. N. Engl. J. Med. 1998, 339, 900–905. [CrossRef] [PubMed] 2. Ky, B.; Vejpongsa, P.; Yeh, E.T.; Force, T.; Moslehi, J.J. Emerging paradigms in cardiomyopathies associated with cancer therapies. Circ. 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The effects of different angiotensin II type 1 receptor blockers on the regulation of the ACE-AngII-AT1 and ACE2-Ang(1-7)-Mas axes in pressure overload-induced cardiac remodeling in male mice. J. Mol. Cell. Cardiol. 2016, 97, 180–190. [CrossRef] [PubMed] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Marine Drugs Multidisciplinary Digital Publishing Institute

Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis

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Abstract

marine drugs Article Alginate Oligosaccharide Prevents Acute Doxorubicin Cardiotoxicity by Suppressing Oxidative Stress and Endoplasmic Reticulum-Mediated Apoptosis 1 , 2 , † 1 , † 1 , † 1 , † 1 1 Jun-Jie Guo , Lei-Lei Ma , Hong-Tao Shi , Jian-Bing Zhu , Jian Wu , Zhi-Wen Ding , 2 1 , 3 , 1 , 3 , Yi An , Yun-Zeng Zou * and Jun-Bo Ge * Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 180 Feng Lin Road, Shanghai 200032, China; qy_junjie@163.com (J.-J.G.); mllsdjn@126.com (L.-L.M.); hongtaoshi@126.com (H.-T.S.); zhujianbing543@163.com (J.-B.Z.); everwoo@sina.com (J.W.); zhiwen.d@gmail.com (Z.-W.D.) Department of Cardiology, The Affiliated Hospital of Qingdao University, 16 Jiang Su Road, Qingdao 266003, China; any@medmail.com.cn Institute of Biomedical Science, Fudan University, Shanghai 200032, China * Correspondence: zou.yunzeng@zs-hospital.sh.cn (Y.-Z.Z.); jbge@zs-hospital.sh.cn (J.-B.G.); Tel.: +86-21-54237969 (Y.-Z.Z.); +86-21-64041990 (ext. 2745) (J.-B.G.) † The first four authors contributed equally to this work. Academic Editor: Peer B. Jacobson Received: 11 September 2016; Accepted: 9 December 2016; Published: 20 December 2016 Abstract: Doxorubicin (DOX) is a highly potent chemotherapeutic agent, but its usage is limited by dose-dependent cardiotoxicity. DOX-induced cardiotoxicity involves increased oxidative stress and activated endoplasmic reticulum-mediated apoptosis. Alginate oligosaccharide (AOS) is a non-immunogenic, non-toxic and biodegradable polymer, with anti-oxidative, anti-inflammatory and anti-endoplasmic reticulum stress properties. The present study examined whether AOS pretreatment could protect against acute DOX cardiotoxicity, and the underlying mechanisms focused on oxidative stress and endoplasmic reticulum-mediated apoptosis. We found that AOS pretreatment markedly increased the survival rate of mice insulted with DOX, improved DOX-induced cardiac dysfunction and attenuated DOX-induced myocardial apoptosis. AOS pretreatment mitigated DOX-induced cardiac oxidative stress, as shown by the decreased expressions of gp91 (phox) and 4-hydroxynonenal (4-HNE). Moreover, AOS pretreatment significantly decreased the expression of Caspase-12, C/EBP homologous protein (CHOP) (markers for endoplasmic reticulum-mediated apoptosis) and Bax (a downstream molecule of CHOP), while up-regulating the expression of anti-apoptotic protein Bcl-2. Taken together, these findings identify AOS as a potent compound that prevents acute DOX cardiotoxicity, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. Keywords: alginate oligosaccharide; doxorubicin cardiotoxicity; oxidative stress; endoplasmic reticulum; apoptosis 1. Introduction Doxorubicin (DOX), an anthracycline antibiotic, is a widely used and highly potent chemotherapeutic agent to treat a broad range of cancers. However, the clinical usage of DOX is greatly limited by its dose-dependent and time-dependent cardiotoxicity [1]. The mechanisms of DOX-induced cardiotoxicity have not been fully elucidated. A plethora of deleterious effects such as excessive reactive oxygen species (ROS) generation, lipid peroxidation, DNA/RNA damage, calcium mishandling, inhibition of autophagic flux and endoplasmic reticulum-mediated apoptosis have all Mar. Drugs 2016, 14, 231; doi:10.3390/md14120231 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016, 14, 231 2 of 13 been linked to DOX-induced cardiotoxicity [2–4]. Anti-oxidative therapies including using antioxidants and transgenic mice overexpressing anti-oxidative enzymes have been verified to be effective in protecting against DOX-induced cardiotoxicity [5–8]. A recent study has demonstrated that a chemical endoplasmic reticulum chaperone could obliterate DOX-induced cardiac dysfunction [9]. These observations hint that some agents may alleviate DOX-induced cardiotoxicity by inhibiting oxidative stress or endoplasmic reticulum-mediated apoptosis. Alginate, an acidic polysaccharide consisting of alternative blocks of -D-mannuronic acid (M) and -L-guluronic acid (G), is extracted from various marine brown algae and has been widely used in various fields, such as food, tissue engineering, and drug delivery, because of its non-toxic, non-immunogenic characteristics [10]. Alginate oligosaccharide (AOS), produced by depolymerizing alginate using different degradation methods including enzymatic degradation, acid hydrolysis and oxidative degradation, has been proven to exert several pharmacological activities, including anti-oxidative [11], anti-apoptotic [12], anti-inflammatory [13] and anti-proliferative effects [14]. Nevertheless, whether AOS is protective against acute DOX cardiotoxicity is not yet clear, and the underlying mechanisms need to be elucidated. For this purpose, the present study aimed to evaluate the protective effect of AOS acquired by an enzymatic degradation method on acute DOX cardiotoxicity. To explore the potential mechanisms of this protection, we examined the influences of AOS on the oxidative stress and endoplasmic reticulum-mediated apoptosis. 2. Results 2.1. AOS Pretreatment Preserves DOX-Induced Cardiac Dysfunction in Mice To evaluate the cardiac function, we used a dose of 15 mg/kg DOX because of the high morbidity induced by the dose of 20 mg/kg DOX. First, we observed whether AOS treatment (200 mg/kg/day, five days) after DOX injection could exert a protective effect on cardiac dysfunction induced by DOX insult. Unfortunately, no significant improvement was observed (Figure S5), and further research was not pursued. Then, the cardioprotection of AOS pretreatment (200 mg/kg/day, seven days) was observed. All mice remained alive on the fifth day after DOX administration, and representative echocardiograms are shown in Figure 1A. Compared with the control (CON) group, mice pre-treated with AOS alone did not exhibit abnormal cardiac function, and there was no difference in left ventricular end-diastolic dimensions (LVEDD) among the four groups (Figure 1B). Surprisingly, mice receiving a DOX injection exhibited a significant increase of 52% for left ventricular end systolic dimensions (LVESD) compared to the CON group, whereas this alteration was markedly restored in the AOS + DOX group (Figure 1C). Moreover, DOX insult induced a marked decrease of 37% and 30%, respectively, in cardiac contractility characterized by the left ventricular ejection fraction (EF) and fractional shortening (FS), and these effects were significantly attenuated by AOS pretreatment (Figure 1D,E). Mar. Drugs 2016, 14, 231 3 of 13 Mar. Drugs 2016, 14, 231  3 of 13  Figure 1. AOS pretreatment attenuates doxorubicin (DOX)‐induced cardiac contractile dysfunction.  Figure 1. AOS pretreatment attenuates doxorubicin (DOX)-induced cardiac contractile dysfunction. (A)  Cardiac  function  of  mice  receiving  DOX  injection  with  or  without  AOS  pretreatment  (200  (A) Cardiac function of mice receiving DOX injection with or without AOS pretreatment mg/kg/day, 7 days) was measured after five days, and representative echocardiographic images were  (200 mg/kg/day, 7 days) was measured after five days, and representative echocardiographic acquired; (B–E) Cardiac function was measured after five days. LVEDD: left ventricular end‐diastolic  imagesdimension; were acquir  LVESD: ed; ( Bleft –E ventr ) Caridiac cular function end‐systolic was dimension; measured EFafter : left ventri five days. cular eject LVEDD: ion fraction; left ventricular  FS:   # end-diastolic left ventri dimension; cular fractional LVESD:  shortening. left ventricular  * p < 0.05 vs end-systolic . the control (CON) dimension;  group;EF:  p <left  0.05ventricular  vs. the CONejection  +  DOX group, n = 6 in each group.  fraction; FS: left ventricular fractional shortening. * p < 0.05 vs. the control (CON) group; p < 0.05 vs. the CON + DOX group, n = 6 in each group. 2.2. AOS Pretreatment Attenuates Acute DOX‐Induced Cardiotoxicity in Mice  The general status of mice in the four groups was observed daily after the DOX insult. Mice in  2.2. AOS Pretreatment Attenuates Acute DOX-Induced Cardiotoxicity in Mice CON + DOX group appeared lethargic and weak with weight loss, and 90% of them had died by  The general status of mice in the four groups was observed daily after the DOX insult. Mice in eight days after DOX (20 mg/kg) treatment (Figure 2A). However, these signs were attenuated in the  CON + mice DOX  pre‐ gr treated oup  appear with AO ed S (2 lethar 00 mg/kg/da gic andy, weak seven with days) weight and mort loss, ality wa and s dec 90% reased of them  to 40% had  by 15 died   by days. The mice in both the CON and AOS groups were all alive over the entire observation period  eight days after DOX (20 mg/kg) treatment (Figure 2A). However, these signs were attenuated in (15 days). The heart‐to‐body weight ratio (Figure 2B) on the fifth day after DOX injection (15 mg/kg)  the mice pre-treated with AOS (200 mg/kg/day, seven days) and mortality was decreased to 40% was obviously decreased compared with the CON group, but the AOS pretreatment (200 mg/kg/day,  by 15 days. The mice in both the CON and AOS groups were all alive over the entire observation seven days) significantly improved the loss of the heart‐to‐body weight ratio compared with the CON  period (15 days). The heart-to-body weight ratio (Figure 2B) on the fifth day after DOX injection + DOX group. Similarly, cTnI—a specific biomarker of cardiac injury—was significantly increased in  (15 mg/kg) was obviously decreased compared with the CON group, but the AOS pretreatment the CON + DOX group compared with the CON and AOS groups. However, the cTnI level was  (200 mg/kg/day, seven days) significantly improved the loss of the heart-to-body weight ratio significantly decreased in the AOS + DOX group compared with the CON + DOX group (Figure 2C).  compared with the CON + DOX group. Similarly, cTnI—a specific biomarker of cardiac injury—was significantly increased in the CON + DOX group compared with the CON and AOS groups. However, the cTnI level was significantly decreased in the AOS + DOX group compared with the CON + DOX group (Figure 2C). Mar. Drugs 2016, 14, 231 4 of 13 Mar. Drugs 2016, 14, 231  4 of 13  Mar. Drugs 2016, 14, 231  4 of 13  Figure 2. AOS pretreatment attenuates acute DOX cardiotoxicity in mice. (A) Kaplan‐Meier survival  Figure 2. AOS pretreatment attenuates acute DOX cardiotoxicity in mice. (A) Kaplan-Meier survival curve analysis of mice after DOX injection with or without AOS pretreatment (200 mg/kg/day, 7 days).  curve analysis of mice after DOX injection with or without AOS pretreatment (200 mg/kg/day, 7 days). n = 10 in each group; (B) Graphs showing the heart weight to body weight ratio. HW: heart weight;  n = 10Figure in each  2. AO group S pretreatment ; (B) Graphs  attenuates showing acute the DOX heart cardiot weight oxito city body  in miweight ce. (A) Kaplan ratio.‐HW Meie:r heart surviva weight; l  BW: body weight. n = 6 in each group; (C) Evaluation of plasma cTnI levels. n = 6 in each group. * p <  curve analysis of mice after DOX injection with or without AOS pretreatment (200 mg/kg/day, 7 days).  BW: body 0.05 vs weight. . the CONn group; = 6 in   each p < 0.05 gr vs oup; . the( C CON ) Evaluation  + DOX group. of plasma   cTnI levels. n = 6 in each group. n = 10 in each group; (B) Graphs showing the heart weight to body weight ratio. HW: heart weight;  * p < 0.05 vs. the CON group; p < 0.05 vs. the CON + DOX group. BW: body weight. n = 6 in each group; (C) Evaluation of plasma cTnI levels. n = 6 in each group. * p <  2.3. AOS Pretreatment Protects DOX‐Induced Myocardial Histological Alterations and Apoptosis in Mice  0.05 vs. the CON group;   p < 0.05 vs. the CON + DOX group.  2.3. AOS Pretreatment Protects DOX-Induced Myocardial Histological Alterations and Apoptosis in Mice To examine whether AOS pretreatment (200 mg/kg/day, seven days) attenuates DOX‐induced  cardiac injury, we analyzed the heart sections with hematoxylin and eosin (HE) staining through  2.3. AOS Pretreatment Protects DOX‐Induced Myocardial Histological Alterations and Apoptosis in Mice  To examine whether AOS pretreatment (200 mg/kg/day, seven days) attenuates DOX-induced electron  microscopy.  As  shown  in  Figure  3,  AOS  by  itself  had  no  effect  on  cardiac  morphology.  cardiac injury, we analyzed the heart sections with hematoxylin and eosin (HE) staining through To examine whether AOS pretreatment (200 mg/kg/day, seven days) attenuates DOX‐induced  Consistent  with  a  previous  study  [15],  DOX‐treated  mice  exhibited  extensive  focal  cytoplasmic  electron card micr iac injur oscopy y, we . As  anashown lyzed the in heart Figur sections e 3, AOS  withby  hematoxylin itself had and no eosin effect (HE) on car stain diac ing through morphology   . vacuolization, a specific change of DOX‐induced cardiac injury, whereas this effect was significantly  electron  microscopy.  As  shown  in  Figure  3,  AOS  by  itself  had  no  effect  on  cardiac  morphology.  Consistent with a previous study [15], DOX-treated mice exhibited extensive focal cytoplasmic reduced  by  AOS  pretreatment.  Terminal  deoxynucleotidyl  transferase‐mediated  dUTP  nick  end  Consistent  with  a  previous  study  [15],  DOX‐treated  mice  exhibited  extensive  focal  cytoplasmic  vacuolization, labeling (TU a specific NEL) assa change ys wereof perform DOX-induced ed to determine cardiac  the injury effect of , wher  AOS eas on ap this opto efsis fect  in DOX was‐significantly treated  vacuolization, a specific change of DOX‐induced cardiac injury, whereas this effect was significantly  hearts. As shown in Figure 4, TUNEL‐positive cardiomyocytes were rarely observed in the heart  reduced by AOS pretreatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick end reduced  by  AOS  pretreatment.  Terminal  deoxynucleotidyl  transferase‐mediated  dUTP  nick  end  sections of mice in the CON and AOS groups. Conversely, a significantly larger number of TUNEL‐ labeling (TUNEL) assays were performed to determine the effect of AOS on apoptosis in DOX-treated labeling (TUNEL) assays were performed to determine the effect of AOS on apoptosis in DOX‐treated  positive cardiomyocytes (16.6%) were detected in the CON + DOX group, whereas this change was  hearts. As shown in Figure 4, TUNEL-positive cardiomyocytes were rarely observed in the heart hearts. As shown in Figure 4, TUNEL‐positive cardiomyocytes were rarely observed in the heart  significantly mitigated by AOS pretreatment (9.6%).  sections of mice in the CON and AOS groups. Conversely, a significantly larger number of sections of mice in the CON and AOS groups. Conversely, a significantly larger number of TUNEL‐ TUNEL-positive cardiomyocytes (16.6%) were detected in the CON + DOX group, whereas this positive cardiomyocytes (16.6%) were detected in the CON + DOX group, whereas this change was  change sign was ificant significantly ly mitigated mitigated  by AOS pretrea by AOS tment pr etr (9.6% eatment ).  (9.6%). Figure  3.  Effect  of  AOS  pretreatment  on  DOX‐induced  myocardial  histological  alterations.  Representative  histopathological  findings  at  400×  magnification  of  mouse  hearts  stained  with  hematoxylin and eosin (HE). Black arrows indicate extensive cytoplasmic vacuolization and nuclear  Figure  3.  Effect  of  AOS  pretreatment  on  DOX‐induced  myocardial  histological  alterations.  condensation or dissolution, n = 6 in each group.  Figure 3. Effect of AOS pretreatment on DOX-induced myocardial histological alterations. Representative  histopathological  findings  at  400×  magnification  of  mouse  hearts  stained  with  Representative histopathological findings at 400 magnification of mouse hearts stained with hematoxylin and eosin (HE). Black arrows indicate extensive cytoplasmic vacuolization and nuclear  hematoxylin and eosin (HE). Black arrows indicate extensive cytoplasmic vacuolization and nuclear condensation or dissolution, n = 6 in each group.  condensation or dissolution, n = 6 in each group. Mar. Drugs 2016, 14, 231 5 of 13 Mar. Drugs 2016, 14, 231  5 of 13  Figure  4.  AOS  pretreatment  decreases  DOX‐induced  myocardial  apoptosis.  (A)  Representative  Figure 4. AOS pretreatment decreases DOX-induced myocardial apoptosis. (A) Representative photomicrographs  at  400×  magnification  of  mouse  hearts  stained  with  TUNEL,  apoptotic  photomicrographs at 400 magnification of mouse hearts stained with TUNEL, apoptotic cardiomyocyte nuclei appear brown‐stained, whereas normal nuclei appear blue; (B) Quantitative  cardiomyocyte nuclei appear brown-stained, whereas normal nuclei appear blue; (B) Quantitative analysis of the percentage of TUNEL‐positive cells. * p < 0.05 vs. the CON group;   p < 0.05 vs. the  analysis of the percentage of TUNEL-positive cells. * p < 0.05 vs. the CON group; p < 0.05 vs. the CON + DOX group, n = 6 in each group.  CON + DOX group, n = 6 in each group. 2.4. AOS Pretreatment Reduces DOX‐Induced Oxidative Stress in Mice  2.4. AOS Pretreatment Reduces DOX-Induced Oxidative Stress in Mice Nicotinamide‐adenine  dinucleotide  phosphate  (NADPH)  oxidases  are  involved  in  DOX‐ induced  oxidative  stress,  and  DOX‐stimulated  NADPH‐dependent  superoxide  production  in  the  Nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are involved in DOX-induced heart is critically dependent on gp91 (phox) [8]; therefore, we measured the protein expression of  oxidative stress, and DOX-stimulated NADPH-dependent superoxide production in the heart is gp91(phox) in the hearts. As shown in Figure 5A, AOS alone did not have an obvious effect on the  critically dependent on gp91 (phox) [8]; therefore, we measured the protein expression of gp91(phox) expression of gp91(phox). However, DOX treatment significantly increased the expression of gp91  in the hearts. As shown in Figure 5A, AOS alone did not have an obvious effect on the expression (phox) by 3.0‐fold compared with the CON group, and this effect was decreased to 2.1‐fold by AOS  of gp91(phox). However, DOX treatment significantly increased the expression of gp91 (phox) by pretreatment (200 mg/kg/day, seven days). Similar results were found in assessing the expression of  3.0-fold compared with the CON group, and this effect was decreased to 2.1-fold by AOS pretreatment lipid peroxide 4‐HNE (Figure 5B). AOS by itself had no marked effect on lipid peroxidation compared  (200 mg/kg/day, seven days). Similar results were found in assessing the expression of lipid peroxide with the CON group but obviously reduced the accumulation of 4‐HNE induced by DOX. The levels  4-HNE of(Figur  the lipi ed 5 B). peroxide AOS by  4‐HN itself E in had  the no CO marked N + DOX effect  grou on p and lipid AOS per oxidation + DOX gro compar up were ed fo with und to the be CON   groupincre butaobviously sed  by  3.3‐ rand educed   1.7‐fol the d, accumulation respectively,  comp of 4-HNE ared  to  induced that  in  the by  CON DOX.   grou The p.  levels Representa of the tivelipid   western blot images are shown in Figure 5C.  peroxide 4-HNE in the CON + DOX group and AOS + DOX group were found to be increased by 3.3- and 1.7-fold, respectively, compared to that in the CON group. Representative western blot images are shown in Figure 5C. Mar. Drugs 2016, 14, 231 6 of 13 Mar. Drugs 2016, 14, 231  6 of 13  Mar. Drugs 2016, 14, 231  6 of 13  Figure 5. AOS pretreatment inhibits oxidative stress in the heart treated with DOX. (A,B) Quantitative  Figure 5. AOS pretreatment inhibits oxidative stress in the heart treated with DOX. (A,B) Quantitative analyses of gp91 phox and 4‐HNE protein expression; (C) Representative blots of gp91 phox, 4‐HNE  analyses of gp91 phox and 4-HNE protein expression; (C) Representative blots of gp91 phox, 4-HNE Figure 5. AOS pretreatment inhibits oxidative stress in the heart treated with DOX. (A,B) Quantitative  and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). * p < 0.05 vs. the CON group;   p < 0.05  and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * p < 0.05 vs. the CON group; p < 0.05 vs. analyses of gp91 phox and 4‐HNE protein expression; (C) Representative blots of gp91 phox, 4‐HNE  vs. the CON + DOX group, n = 6 in each group.  the CON + DOX group, n = 6 in each group. # and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). * p < 0.05 vs. the CON group;   p < 0.05  vs. the CON + DOX group, n = 6 in each group.  2.5. AOS Pretreatment Ameliorates DOX‐Induced Endoplasmic Reticulum‐Mediated Apoptosis in Mice  2.5. AOS Pretreatment Ameliorates DOX-Induced Endoplasmic Reticulum-Mediated Apoptosis in Mice CHOP and Caspase‐12 are known to be involved in endoplasmic reticulum‐mediated apoptosis  2.5. AOS Pretreatment Ameliorates DOX‐Induced Endoplasmic Reticulum‐Mediated Apoptosis in Mice  CHOP and Caspase-12 are known to be involved in endoplasmic reticulum-mediated apoptosis in in the context of DOX cardiotoxicity [16,17]. Our results demonstrated that AOS by itself had no  CHOP and Caspase‐12 are known to be involved in endoplasmic reticulum‐mediated apoptosis  the context of DOX cardiotoxicity [16,17]. Our results demonstrated that AOS by itself had no significant significant effect on the expressions of CHOP and Caspase‐12 compared to the CON group (Figure  in the context of DOX cardiotoxicity [16,17]. Our results demonstrated that AOS by itself had no  6A,B). CHOP and Caspase‐12 protein expressions in the CON + DOX group were significantly higher  effect on the expressions of CHOP and Caspase-12 compared to the CON group (Figure 6A,B). CHOP significant effect on the expressions of CHOP and Caspase‐12 compared to the CON group (Figure  than that of the CON group, and this increase was markedly abolished by AOS pretreatment (200  and Caspase-12 protein expressions in the CON + DOX group were significantly higher than that of 6A,B). CHOP and Caspase‐12 protein expressions in the CON + DOX group were significantly higher  mg/kg/day,  seven  days).  DOX  administration  significantly  inhibited  the  expression  of  Bcl‐2  the CON group, and this increase was markedly abolished by AOS pretreatment (200 mg/kg/day, than that of the CON group, and this increase was markedly abolished by AOS pretreatment (200  compared  to  the  CON  group,  whereas  AOS  pretreatment  restored  the  expression  of  Bcl‐2  to  the  seven days). DOX administration significantly inhibited the expression of Bcl-2 compared to the mg/kg/day,  seven  days).  DOX  administration  significantly  inhibited  the  expression  of  Bcl‐2  normal level (Figure 6D). Bax protein expression in the CON + DOX group was significantly higher  CON group, whereas AOS pretreatment restored the expression of Bcl-2 to the normal level (Figure 6D). compared  to  the  CON  group,  whereas  AOS  pretreatment  restored  the  expression  of  Bcl‐2  to  the  than  that  of  the  CON  group,  whereas  AOS  pretreatment  significantly  decreased  the  expression  Bax protein expression in the CON + DOX group was significantly higher than that of the CON group, normal level (Figure 6D). Bax protein expression in the CON + DOX group was significantly higher  (Figure 6E). Representative western blot images are shown in Figure 6C,F.  whertha easn AOS that  pr of etr the eatment   CON  gro significantly up,  whereadecr s  AOS eased   pretrea the expr tment ession   signif(Figur icantly e  decre 6E). Raepr sedesentative   the  expression western   (Figure 6E). Representative western blot images are shown in Figure 6C,F.  blot images are shown in Figure 6C,F. Figure  6.  AOS  pretreatment  inhibits  endoplasmic  reticulum‐mediated  apoptosis  in  hearts  treated  with  DOX.  (A,B)  Quantitative  analyses  of  CHOP  and  Caspase‐12  protein  expression;  (C)  Figure 6. AOS pretreatment inhibits endoplasmic reticulum-mediated apoptosis in hearts treated with Figure  6.  AOS  pretreatment  inhibits  endoplasmic  reticulum‐mediated  apoptosis  in  hearts  treated  Representative blots of CHOP, Caspase‐12 and GAPDH; (D,E) Quantitative analyses of Bcl‐2 and Bax  DOX. (A,B) Quantitative analyses of CHOP and Caspase-12 protein expression; (C) Representative blots with  DOX.  (A,B)  Quantitative  analyses  of  CHOP  and  Caspase‐12  protein  expression;  (C)  protein expression; (F) Representative blots of Bcl‐2, Bax and GAPDH. * p < 0.05 vs. the CON group;  of CHOP Representative , Caspase-12  blots and  of CHOP, GAPDH;  Caspase (D,E‐)12 Quantitative  and GAPDH;analyses  (D,E) Quantitative of Bcl-2 and  analyses Bax pr ofotein  Bcl‐2 expr and Bax ession;    p < 0.05 vs. the CON + DOX group, n = 6 in each group.  protein expression; (F) Representative blots of Bcl‐2, Bax and GAPDH. * p < 0.05 vs. the CON group;  (F) Representative blots of Bcl-2, Bax and GAPDH. * p < 0.05 vs. the CON group; p < 0.05 vs. the  p < 0.05 vs. the CON + DOX group, n = 6 in each group.  CON + DOX group, n = 6 in each group. Mar. Drugs 2016, 14, 231 7 of 13 3. Discussion Treatment progression has improved the survival rate of patients with cancers. However, the progressive, irreversible chemotherapy-induced cardiotoxicity leaves the survivors at a higher risk of death than the general population [18,19]. DOX, one of the most effective chemotherapy agents, is broadly used as one component of treatment protocols, yet its administration is greatly compromised by its cardiotoxicity. Hence, it is urgent for cardio-oncologists to identify strategies that can protect against DOX-induced cardiotoxicity without affecting its anticancer activities. The current study demonstrated that AOS was cardioprotective in the face of acute DOX cardiotoxicity. We found that inhibitions of oxidative stress and endoplasmic reticulum-mediated apoptosis were associated with the cardioprotection. This present study was the first to demonstrate that mice pre-treated with AOS were well protected against acute DOX cardiotoxicity, as evidenced by a significantly improved survival rate, heart weight to body weight ratio, cardiac contractile dysfunction, histological alterations, and decreased the myocardial apoptosis. Plasma cTnI monitoring is broadly used in clinical practice and animal experiments as a specific biomarker of heart injury [20,21]. The plasma level of cTnI was significantly elevated in the DOX-treated mice, whereas AOS pretreatment down-regulated this increase, indicating the strong cardioprotective efficacy against acute DOX cardiotoxicity. AOS has also been shown to provide protection against many diseases including Alzheimer ’s disease and asthma [22,23]. By using neuron-like PC12 cells, Tusi et al. previously showed that AOS decreased H2O2-induced oxidative stress and endoplasmic reticulum-mediated apoptosis [12]. In contrast, AOS was also found to have the capacity to trigger an oxidative burst and induce resistance against infection in a few members of brown algae [24], which reminds us that AOS maybe exhibit different bioactivity in face of different injuries. Available laboratory evidence suggests that increased oxidative stress is well known as the causative factor in acute DOX cardiotoxicity [5,6,8]. Previous studies have confirmed that DOX induces the overproduction of a cascade of reactive oxygen species (ROS) such as hydroxyl radicals, oxyanions and hydrogen peroxide, which are involved in DOX-induced cardiotoxicity [25,26]. NADPH oxidases, with the primary function of ROS generation, are activated by various stimuli that are important in cardiac remodeling and are also capable of modulating other ROS sources [27]. Using rat embryonic myocyte H9C2 cell cultures, Gilleron et al. reported that the activity of NADPH oxidases increased more than two-fold after one hour of DOX incubation [28]. As a multi-component enzyme, NADPH oxidases are the primary source of oxidative stress in acute DOX cardiotoxicity. Once stimulated, the cytosolic complex migrates to the membrane and assembles with other subunits to form an active oxidase with the ability to produce superoxide anions [29]. The previous study demonstrated that DOX-induced NADPH-dependent superoxide generation was critically dependent on NOX2 (also named gp91 (phox)). Knockout of NOX2 protected the mice against heart failure, cardiomyocyte atrophy, and apoptosis after DOX treatment [8]. Overproduction of oxidants and electrophiles during DOX treatment activates the oxidative response of membrane lipids, leading to the accumulation of 4-HNE, which has been shown to affect mitochondrial function, modify protein functions and be a specific biomarker of DOX-induced oxidative stress [30,31]. Consistent with these studies, the expression of gp91 (phox), the pivotal NADPH oxidase subunit, was up-regulated after DOX administration. Similarly, the expression of the lipid peroxide 4-HNE was also up-regulated. However, pretreatment with AOS inhibited the oxidative stress process by down-regulating the expressions of gp91 (phox) and 4-HNE in the heart. Moreover, our results showed that AOS pretreatment had no obvious effect on the oxidative stress in the heart under physiological conditions. Endoplasmic reticulum-mediated apoptotic signaling has been studied in the development of heart failure [32,33]. It is also believed that increased oxidative stress can directly induce endoplasmic reticulum stress [34,35]. Chua et al. found that DOX could activate the endoplasmic reticulum-initiated apoptotic response and further augment endoplasmic reticulum stress in mouse hearts [36]. Signal transduction pathways such as the Caspase-12 and C/EBP homologous protein (CHOP) pathways can mediate and augment endoplasmic reticulum-mediated apoptosis [37]. Mar. Drugs 2016, 14, 231 8 of 13 Mar. Drugs 2016, 14, 231  8 of 13  In the context of endoplasmic reticulum stress, Caspase-12, serving as an endoplasmic reticulum membrane-resident pro-apoptotic molecule, activates the caspase cascades which mainly induce cell resident pro‐apoptotic molecule, activates the caspase cascades which mainly induce cell death [38].  death [38]. Consistent with previous research that demonstrates that the inhibition of Caspase-12 Consistent with previous research that demonstrates that the inhibition of Caspase‐12 cleavage is  cleavage is linked with the recovery of heart function [39], our data suggested that AOS pretreatment linked with the recovery of heart function [39], our data suggested that AOS pretreatment attenuated  attenuated Caspase-12 cleavage in the face of DOX insult, leading to decreased myocardial apoptosis Caspase‐12  cleavage  in  the  face  of  DOX  insult,  leading  to  decreased  myocardial  apoptosis  and  and improved cardiac dysfunction. Many studies have demonstrated that CHOP, a marker of improved cardiac dysfunction. Many studies have demonstrated that CHOP, a marker of endoplasmic  endoplasmic reticulum-mediated apoptosis [40], can promote apoptosis by inhibiting expression reticulum‐mediated  apoptosis  [40],  can  promote  apoptosis  by  inhibiting  expression  of  the  anti‐ of the anti-apoptotic factor Bcl-2 and activating expression of the pro-apoptotic factor Bax [41]. apoptotic factor Bcl‐2 and activating expression of the pro‐apoptotic factor Bax [41]. Over‐expression of  Over-expression of CHOP decreased the expression of Bcl-2 protein and facilitated translocation CHOP decreased the expression of Bcl‐2 protein and facilitated translocation of the Bax protein from  of the Bax protein from the cytosol to the mitochondria [42]. Our data also supported this hypothesis. the cytosol to the mitochondria [42]. Our data also supported this hypothesis. Administration of DOX  Administration of DOX significantly activated the CHOP signaling pathway-mediated myocardial significantly activated the CHOP signaling pathway‐mediated myocardial apoptosis by regulating  apoptosis by regulating the expressions of Bcl-2 and Bax. However, the detrimental effect was the  expressions  of  Bcl‐2  and  Bax.  However,  the  detrimental  effect  was  ameliorated  by  AOS  ameliorated by AOS pretreatment. Importantly, our results showed that AOS pretreatment had pretreatment. Importantly, our results showed that AOS pretreatment had no marked effects on the  no marked effects on the endoplasmic reticulum mediated-apoptotic signaling pathway under endoplasmic reticulum mediated‐apoptotic signaling pathway under physiological conditions.  physiological conditions. 4. Materials and Methods  4. Materials and Methods 4.1. 4.1. Preparatio Preparationn of of AOS AOS  AOS, AOS, aa gift gift fr from om Qingdao Qingdao BZ BZ Oligo Oligo Biotech Biotech Co. Co.  Ltd Ltd (Qingdao, (Qingdao,  China), China),  was was  pr produced oduced  by by the the  method method of of enzymatic enzymatic degradation degradation as as pr previously eviously described described [4 [433] ].. Br Briefly iefly,, alginate alginate (5 (5 g) g) derived derived fr from om  Laminaria Laminaria japonica japonica with with an an M/G M/G ratio ratio of of 1.86/1 1.86/1 [4 [44 4]] wa was s d dissolved issolved in in 50 5000 mL mL of of 50 50 mmol/L mmol/L Tris Tris-HCL ‐HCL  buf buffe ferr (pH (pH 7.0), 7.0), which which was was added added to to 200 200 units units of of alginate alginate lyase lyase purified purified fr from om Pseudomonas Pseudomonas sp. sp. HZJ HZJ  216. 216. The The en enzymatic zymatic rre eaction action was was performed performed at at 30 30 °C C for for 6 6 h, h, and and th then en termi terminated nated by by boiling boiling in in water water  for for 55 min. min. The The AOS AOS was was initially initially sep separat arated ed by by adjusting adjusting the the pH pH of of the the supernatan supernatant t to to 2.85. 2.85. Next, Next, the the  methods methods of ofanion-exchange  anion‐exchange chr  chrom omatography atography and and desalting  desalting wer were e used used to further  to furt purify her purify the AOS.  the  Then, AOS.  the Then, methods   the  methods of High  of Performanc   High  Per eformance Gel Permeation   Gel  Pe Chr rmeation omatography   Chroma (HPGPC) tography (Dionex, (HPGPC Sunnyvale, )  (Dionex,  CA, Sunnyv USA) ale, and   CA, Electr   USA) ospray   and Ionization   ElectrMass ospraSpec y  Ionization troscopy (ESI-MS) Mass  Spectros (Agilentcopy Technologies,   (ESI‐MS) Santa   (Agil Clara, ent  CA, Technologies, USA) wer eSa used nta C to lar determine a, CA, USA the ) were relative  used molecular  to determine mass the and relative degree molecula of polymerization r mass and (DP) degre ofe  the of polymeriza AOS. The rtion elative  (DPmolecular ) of the AOS. weight  The of relative AOS ismolecula approximately r weight1.2  of kDa AOS(Figur  is app erS1), oxim and atelthe y 1.2 DP kD ofa  AOS (Figure mainly  S1),  ranges and the fr DP om  2ofto AOS 6 (Figur  mainly es S2 range and S3, s from Tables  2 to S1 6and  (Fig S2). ures Lastly  S2 and , the S3 1H , Tables Nuclear  S1Magnetic  and S2).  Resonance Lastly,  the (1H-NMR) 1H  Nuclear spectr   Magn oscopy etic  Resonance method was  (1 performed H‐NMR)  spectr to determine oscopy the method M/G  ratio was  of performed AOS which   to  is determine 1/2.6 (Figur  the eM/G S4). rat The io chemical of AOS wh strich uctur  is 1/ e2. of6 AOS (Figur acquir e S4).ed  The thr che ough mical enzymatic  structure degeneration  of AOS acquwas ired  shown through in en Figur zymatic e 7.  degeneration was shown in Figure 7.  Figure  7.  Schematic  representation  of  the  molecular  structure  of  alginate  oligosaccharide  (AOS)  Figure 7. Schematic representation of the molecular structure of alginate oligosaccharide (AOS) prepared by enzymatic degradation.  prepared by enzymatic degradation. 4.2. Animals and Drug Administration  4.2. Animals and Drug Administration Adult  male  C57BL/6  mice  (22–25  g;  Department  of  Laboratory  Animal  Science,  Fudan  Adult male C57BL/6 mice (22–25 g; Department of Laboratory Animal Science, Fudan University, University, (Shanghai, China) were maintained under specific pathogen‐free conditions in an animal  (Shanghai, China) were maintained under specific pathogen-free conditions in an animal room on a room  on  a  12/12  h  light/dark  cycle  with  free  access  to  water  and  food.  All  experiments  were  performed in strict accordance with the guidelines of the China Council on Animal Management. The  protocols  were  approved  by  the  Committee  on  the  Ethics  of  Animal  Experiments  of  Fudan  University.  The  model  of acute DOX  cardiotoxicity was  induced  by intraperitoneal  injection  of a  Mar. Drugs 2016, 14, 231 9 of 13 12/12 h light/dark cycle with free access to water and food. All experiments were performed in strict accordance with the guidelines of the China Council on Animal Management. The protocols were approved by the Committee on the Ethics of Animal Experiments of Fudan University. The model of acute DOX cardiotoxicity was induced by intraperitoneal injection of a single dose (15 mg/kg) of DOX (Sigma-Aldrich, St. Louis, MO, USA) [15]. The AOS was dissolved in normal saline with a concentration of 2.0% w/v. In part one, the mice were randomly divided into the following four groups according to treatment: vehicle control group (CON, n = 6), DOX group (CON + DOX, n = 6), AOS treatment group (AOS-T, n = 6) and AOS treatment + DOX group (AOS-T + DOX, n = 6). The mice were treated with AOS (200 mg/kg/day) or an equal volume of normal saline by gavage for 5 consecutive days from the day of DOX injection to the end of the study based on a previous study [21]. In part two, the mice were randomly divided into the following four groups according to treatment: vehicle control group (CON, n = 6), DOX group (CON + DOX, n = 6), AOS pretreatment group (AOS, n = 6), and AOS pretreatment + DOX group (AOS + DOX, n = 6). The mice were pretreated with AOS (200 mg/kg/day) or an equal volume of normal saline by gavage for 7 consecutive days, and on the eighth day, the mice were acutely injected intraperitoneally with DOX or an equal volume of normal saline. Next, the mice received the same daily dose of AOS (200 mg/kg/day) and normal saline by gavage for another five days. At the end of the study, cardiac function was measured. Following anesthesia, body weights were recorded, blood samples were collected, and the hearts were rapidly removed, weighed, and then snap frozen in liquid nitrogen for further examinations. For survival observation, the mice were treated with a single high dose (20 mg/kg) of DOX, and the mortality was monitored (n = 10 in each group) for 15 days after DOX injection. A total of 112 animals were used in the experiments. 4.3. Determination of Cardiac Troponin-I Level On the fifth day after DOX injection, blood samples were collected from the carotid artery with EDTA-containing syringes prior to heart harvest. The plasma was prepared by centrifugation at 4000 rpm for 30 min and 4 C immediately frozen and stored at 80 C. The plasma was thawed only once for cardiac troponin-I (cTnI) measurement. The level of plasma cTnI was quantified with a Mouse Cardiac troponin-I for plasma kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assay was implemented according to the protocol provided by the manufacturer. 4.4. Hemodynamic Measurements Transthoracic echocardiography was performed by using an animal specific instrument (Vevo707B, Visual Sonics Inc., Toronto, ON, Canada) on the fifth day after DOX and normal saline injection. The mice were anesthetized with 1%–2% isoflurane, and the heart rates were stably maintained between 450 and 500 beats per min. After the B- and M-mode images were acquired, the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured as previously described [45]. All measurements were performed by three experienced technicians who were blinded to the animal groups. 4.5. Histopathological Analysis The heart tissue was immediately placed in 10% neutral buffered formalin for 24 h. Then, the specimen was embedded in paraffin, and serial sections were cut at 4 m thickness. The sections were stained with hematoxylin and eosin (HE). Eight microscopic fields (400) from each section were captured from each slide and imaged with a color digital camera. 4.6. Detection of Myocardial Apoptosis Myocardial apoptosis was examined by using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Paraffin-embedded sections of heart tissues were stained using an In Situ Cell Apoptosis Detection Kit (POD; Roche Diagnostics Corp, Indianapolis, IN, Mar. Drugs 2016, 14, 231 10 of 13 USA) according to the manufacturer ’s protocol. Eight microscopic fields (400) from each section were analyzed by counting brown nuclei. The percentage of TUNEL-positive nuclei (brown nuclei) was calculated. 4.7. Western Blot Analyses Hearts were quickly removed and frozen in liquid nitrogen. Total protein was extracted from the homogenized heart tissues using RIPA lysis buffer (Beyotime Biotechnology, Nanjing, China) and a complete protease inhibitor cocktail. Subsequently, extracted proteins (50 g) were loaded in a 10% or 12% polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. The protein expression was detected by immunoblotting with antibodies against gp91 (phox), 4-HEN, Caspase-12 (Abcam), CHOP, Bax, and Bcl-2 (Cell Signaling Technology). After three washes, the blots were incubated with radish peroxidase-conjugated rabbit secondary antibody immunoglobulin G (Kangchen Biotechnology, Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. The protein bands were detected by chemiluminescence and quantified using a Bio-Rad equipped with image software basic Quantity One (Bio-Rad, Hercules, CA, USA). 4.8. Statistical Analysis Data were expressed as the mean  standard deviation and compared by one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test using Graph Pad Prism 6 software (San Diego, CA, SA). Survival curves were determined by the Kaplan-Meier estimator and compared by a log-rank test. A p < 0.05 was considered statistically significant. 5. Conclusions The current study is the first to demonstrate that AOS successfully prevents acute DOX cardiotoxicity in mice, at least in part, by suppression of oxidative stress and endoplasmic reticulum-mediated apoptosis. Our data indicate that AOS may clinically serve as a novel preventive strategy against acute DOX cardiotoxicity. However, we should not ignore the question of whether AOS administration interferes with the anticancer activity of DOX, which was not illuminated in the present study. Moreover, the pharmacokinetics of AOS were also not investigated. The challenge for future studies is to focus on answering these important questions. Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/14/12/231/s1, Figure S1: The relative molecular mass of AOS analyzed by High Performance Gel Permeation Chromatography (HPGPC). The relative molecular mass of AOS is 1.2 kDa, which was determined by the different molecular weights of dextran standards from the National Institutes for Food and Drug Control, Figure S2: The degree of polymerization (DP) of AOS analyzed by Electrospray Ionization Mass Spectroscopy (ESI-MS), Figure S3: The different degree of polymerization (DP) of AOS analyzed by High Performance Gel Permeation Chromatography (HPGPC), Figure S4: The M/G ratio of AOS analyzed by 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy. The M/G ratio of AOS was approximately 1/2.6 by calculating the integral areas of the M and G residues, Figure S5: Effects of AOS administered after DOX injection on cardiac dysfunction induced by DOX insult. (A) The cardiac function of mice receiving DOX injection with or without AOS (200 mg/kg, 5 days) treatment was measured after 5 days, and representative echocardiographic images were acquired; (B–E) Data of cardiac function were collected. LVEDD: left ventricular end-diastolic dimension; LVESD: left ventricular end-systolic dimension; EF: left ventricular ejection fraction; and FS: left ventricular fractional shortening. * p < 0.05 vs. the CON group; n = 6 in each group, Table S1: Electrospray Ionization Mass Spectroscopy (ESI-MS) data analysis of AOS. The DP of AOS mainly ranged from 2 to 6, Table S2: The relative percentages of the different degrees of polymerization of AOS. The AOS mainly contains DP2~DP6; the relative contents of different DP are DP2 26.27%, DP3 32.50%, DP4 18.55%, DP5 13.98%, DP6 3.69%, and >DP7 5.01%, according to the peak area normalization method. 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Published: Dec 20, 2016

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