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Protective effects of dimethyl itaconate in mice acute cardiotoxicity induced by doxorubicin
Biochemical and Biophysical Research Communications 517 (2019) 538e544
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Protective effects of dimethyl itaconate in mice acute cardiotoxicity induced by doxorubicin Qing Shan a, b, 1, Xiaoyu Li c, 1, Mei Zheng d, 1, Xi Lin e, Guotao Lu e, f, Dongming Su g, h, *, Xiang Lu a, ** a
Department of Geriatrics, The Second Affilicated Hospital, Nanjing Medical University, Nanjing, 211166, People's Republic of China Department of Geriatrics, Affilicated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225000, People's Republic of China Department of Pathophysiology, Nanjing Medical University, Nanjing, 211166, People's Republic of China d Department of Cardiology, Beijing Jishuitan Hospital & the 4th Medical College of Peking University, Peking University, No. 31 Xinjiekou East Street, XiCheng District, Beijing, 100035, People's Republic of China e Department of Gastroenterology, Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225000, People's Republic of China f Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou, 225001, People's Republic of China g Center for Clinical Pathology and Laboratory, Affiliated Hospital of Yifu, Nanjing Medical University, Nanjing, 211166, People's Republic of China h Department of Pathoology, Nanjing Medical University, Nanjing, 211166, People's Republic of China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2019 Accepted 17 July 2019 Available online 31 July 2019
Doxorubicin (DOX) is an antitumor drug widely used in hematological tumors and various solid tumors. However, the cardiotoxicity elicited by DOX severely limits its clinical treatment. Dimethyl itaconate (DI), a common form of itaconate, is found many potential targets for prevent heart injury. Here we employed wild type and Nrf2 knockout mice and induced a cardiotoxicity model by administration of DOX to clarify the effects of DI. After treatment with DI, we found that it could effectively alleviate the cardiotoxicity by analyzing morphology, LDH levels and heart weight/body weight ratio changes. Meanwhile we demonstrated that RIP3, a key protein of necrosis, was significantly decreased in DI treated group. Further we observed that treatment with DI could suppress oxidative stress by altering Nrf2/HO-1. Compared with vehicle group, DI could increase the tissue SOD and GSH, and reduce MDA levels, then DHE staining revealed that the level of ROS in DI group reduced by half. Finally, transmission electron microscope (TEM) data showed that treatment with DI obviously decreased the mitochondrial damage. While Nrf2 was ablated in mice, the protective effects of DI were vanished and SOD, GSH, MDA became unchanged related to vehicle group. This report provides the evidence for the protective effects of DI treatment in cardiotoxicity induced by DOX. On mechanisms, DI could reduce the oxidative stress by altering Nrf2/HO-1 pathway and prevent mitochondrial from damage. Taken together, these findings of this paper will afford the new therapeutic targets in DOX related cardiotoxicity. © 2019 Elsevier Inc. All rights reserved.
Keywords: Dimethyl itaconate Acute cardiotoxicity Doxorubicin Oxidative stress
1. Introduction
* Corresponding author. Department of Pathology, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing, 211166. People's Republic of China. ** Corresponding author. Department of Geriatrics, The Second Affiliated Hospital, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing, 211166, People's Republic of China. E-mail addresses: [email protected] (D. Su), [email protected] (X. Lu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.07.046 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Doxorubicin (DOX) is an anthracycline non-specific antitumor drug targeted to cell cycle, which inhibits tumor cell proliferation in different growth phase. Due to its highly effective and broadspectrum anti-tumor effects, DOX has been widely used in clinical chemotherapy for hematological tumors and various solid tumors since the 1960s [1]. However, the cardiotoxicity of DOX severely restrains its application. In long-term clinical treatment with DOX, patients are often suffered from delayed and irreversible myocardial damage and the toxicity often shows dosage-dependent DOX accumulation [2]. At a therapeutic dosage of DOX, the main clinical
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manifestations of early cardiotoxicity are arrhythmia and cardiac dysfunction [3e5], while long-term medication is prone to induce dilated cardiomyopathy, congestive, heart failure and even lifethreatening [6e9], thus The US FDA's Black Box Warning requirement for DOX is also based on its cardiotoxicity [10]. Currently, there are absent of effective treatments and prevention for its unanticipated toxicity. It has been shown that mechanisms of DOX induced cardiotoxicity involve many factors, such as apoptosis [11], autophagy [12], vasoactive amine release [13], oxidative stress [14e16] and mitochondrial damage [17e20]. Specially, the oxidative stress pathway involved in mitochondrial damage has gradually become the main issue in injury process. To our interesting, it has been proved that inhibition of oxidative stress can effectively alleviate acute myocardial damage induced by DOX [21e23]. Further, finding effective drugs for anti-oxidative stress might be an effective strategy to prevent myocardial damage induced by DOX in clinical therapeutic. Itaconic acid is an unsaturated dibasic organic acid, which contains unsaturated double bonds with active chemical properties. It can carry out various addition reactions, esterification reactions and polymerization reactions. Therefore, itaconic acid is an important raw material for chemical synthesis industry and chemical production. Recently, itaconate has been found to play an important role in cellular immune metabolism and antimicrobial defense [24,25]. However, whether it has a protective effect on acute cardiotoxicity induced by DOX has not been reported yet. In this paper, dimethyl itaconate (DI), a common form of itaconate, was used to investigate its effects.
2. Materials and methods 2.1. Animal and reagents Male C57BL/6 and Nrf2 KO mice were obtained from the Model Animal Center of Nanjing University. 8 weeks old mice were used for experiment and animal feeding. Mice were exposed to 12/12 h light and dark cycles with freedom for eating and drinking. All animal protocols were performed in accordance with the Principles of Laboratory Animal Management (NIH Publication 85Y23, revised in 1996). All animal experiments were performed in accordance with the ethical requirements of animal experiments at Nanjing Medical University. DOX was purchased from Medchem Express (MCE Co. Ltd., Shanghai, China) and DI was purchased from Aladdin (Aladdin Bio-Chem Technology Company, Shanghai, China). The LDH assay kit was purchased from Nanjing Jiancheng (Nanjing Jiancheng Corp., Nanjing, China). Antibodies against-RIP3, Nrf2 and HO-1 were purchased from Abcam (Cambridge, UK). MDA, GSH, SOD commercial kits were purchased from Nanjing Key-Gen (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China). Dihydroethidium (DHE) staining solution and electron microscope fixative were purchased from Servicebio (Wuhan Servicebio Technology Co. Ltd., Wuhan, China).
2.2. Mouse model preparation and administration of DI According to previous literatures [26,27], we used intermittent administration of DOX to induce acute myocardial injury. DOX was dissolved in PBS for further protocol. Acute myocardial injury was elicited by DOX (total dosage: 200 mg/kg) intraperitoneally injected at 1st and 3rd day. Itaconic acid was dissolved in PBS and administered at first 4 days with 100 mg/kg per day since DOX intraperitoneal injection.
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2.3. Sample collection and preparation Mice were sacrificed on the 5th day after the first DOX dose. All mice were sacrificed after anesthesia, then systemic circulation perfusion was performed. The heart tissue was collected for morphological study with 4% paraformaldehyde fixation and to other protocols with storing in liquid nitrogen. 2.4. HE stains After tissue dehydration, waxing, and embedding, 5 mm slices were made and baked at 60 C. The tissue sections were stained with hematoxylin and eosin for 3 and 2 min respectively. The gradient alcohol is used to dehydrate and the xylene is used to transparentize. Finally, the slices were observed under a microscope for morphologic study. 2.5. Immunohistochemistry (IHC) Briefly, 5 mm thickness sections were dewaxed, gradient alcohol hydration, heating for antigen retrieval and 3% H2O2 removal of endogenous peroxidase activity. Then added to RIP3 (1:200) primary antibody 4 C overnight, after washing, secondary antibody was incubated for 1 h at room temperature. Finally, hematoxylin stained. 2.6. Western bolt Heart tissue proteins were extracted and the protein concentration was determined by the BCA assay. PVDF membrane with constant flow 200 mA was used to transfer membrane. After membrane was blocked with 5% milk for 1 h the primary antibody was incubated overnight at 4 C. The primary antibody dilution ratios were: Nrf2 (1: 1000 dilution), HO-1 (1: 1000 dilution), Lamin B1 (1: 1000 dilution), and b-actin (1: 2000 dilution). The second antibody was incubated for 1 h at room temperature. b-actin or Lamin B1 was used as a loading control. 2.7. Analysis oxidative stress Heart tissue was added to PBS with a ratio of 1:9, homogenized to prepare a suspension, and centrifuged to obtain a supernatant. SOD, MDA, GSH in the supernatant were detected according to the kits’ instruction to present oxidative stress level. 2.8. Cell ROS production detection ROS fluorescent probe-Dihydroethidium (DHE) was employed to detect cellular ROS production. DHE can enter the cell through the living cell membrane freely, and is oxidized by ROS to form ethylene oxidein the cell. Ethylene oxide can be incorporated in the chromosomal DNA then red fluorescence is detectable to represent ROS. Fresh heart tissue was embedded in OCT and 7 mm sections were performed. The slices were incubated with DHE staining solution at 37 C in the dark for half an hour after washed with PBS for 3 times, then subjected to DAPI staining. Finally, photographs were taken under an immunofluorescence microscope. 2.9. Transmission electron microscopy Fresh tissue was quickly placed in 2.5% glutaraldehyde for 24 h at 4 C and then using 1% citric acid$0.1 M phosphate buffer PB (pH 7.4) fixed tissue at room temperature (20 C) for 2 h. Alcohol and acetone were used to dehydrate. Acetone: 812 embedding agents ¼ 1:1 for 2e4h, acetone:812 embedding agent ¼ 2:1
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permeate overnight, pure 812 embedding agent 5e8h. The pure 812 embedding agent was poured into the embedding plate, and the sample was inserted into the embedding plate with at 37 C overnight. The ultra-thin slices (60e80 nm) was made by JEOL1010. Double staining of Uranium-Plumbum (2% uranyl acetate saturated alcohol solution, plumbum citrate, stained for 15 min respectively), and the sections were dried overnight. Observed under a transmission electron microscope, image analysis was performed. 2.10. statistical analysis GraphPad Prism 7.0 software was used for data analysis. Data are shown as mean ± SEM. Statistical significance was determined by performing t tests (two-tailed), one-way ANOVA. Image J software was used to analyze WB and morphologic photos. The optical density of the immunoreactive bands target proteins relative to b-
actin for the cytoplasm extraction or Lamin B1 for the nuclear extraction represented the protein contents level in WB. p value with 0.05 was considered to be statistically significant. 3. Results 3.1. DI effectively alleviated the acute damage of cardiomyocytes induced by DOX To investigate the role of DI in cardiotoxicity, DOX induced acute myocardial injury model were applicated in our study. The flow chart of experiments shown in Fig. 1A. As shown in Fig. 1B, the cardiomyocytes of the control group were arranged neatly and densely. DOX significantly caused the arrangement disorder of myocardial cells vacuolization of the cytoplasm, blurred transverse lines, and widening of the intercellular space in the murine model, accompanied by a high serum LDH level (Fig. 1C) and enhanced
Fig. 1. DI alleviated the acute damage of cardiomyocytes induced by DOX in mice. (A) The experimental protocol with DI in DOX-induced cardiomyocytes induced model. (B) Representative HE staining and histological scores of heart. (CeD) Changes in HW/BW and serum LDH levels in mice. (E) Representative immunohistochemistry images for RIP3 expression in the heart. **p < 0.01, and ***p < 0.001.
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heart weight/body weight (HW/BW) ratio (Fig. 1D). These suggested that DOX caused severe acute myocardial damage in mice. Compared with DOX treated group, additional DI administration group exhibited a reduction in cardiac pathological damage, a repression in plasma LDH levels, and less heart weight/body weight ratio, which suggest DI significantly attenuate DOX-induced cardiomyocyte injury. As a critical regulator of cell necrosis, RIP3 is significantly up-regulated during DOX-induced myocardial injury. IHC staining in Fig. 1E confirmed that DI could significantly downregulate RIP3 expression in cardiomyocytes, which further suggested that DI might have an obvious protective effect against the myocardial injury.
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3.3. Nrf2 deficiency offset the protective effect of DI on DOXinduced cardiomyocyte injury Finally, in order to further clarify the mechanism of action of DI to protect DOX-induced cardiomyocyte injury, Nrf2 KO mice were involved in our experiments. Firstly, we observed that Nrf2
3.2. DI effectively alleviated DOX-induced mitochondrial damage and altered Nrf2/HO-1 oxidative stress pathway Oxidative stress and mitochondrial damage play an important role in DOX-induced myocardial injury. Moreover, previous studies have pointed out that DI could activate Nrf2/HO-1 pathway to alleviate inflammatory responses in macrophage [28]. We further observed the protective effect of DI on myocardial mitochondrial damage. By detection of myocardial oxidative stress products and Nrf2/HO-1 protein level, we found that DI could significantly activate the Nrf2/HO-1 pathway, elevate the tissue levels of SOD and GSH and reduce MDA levels (Fig. 2AeC). TEM analysis confirmed that DOX treatment led to mitochondrial swelling, lysis and rupture in cardiomyocyte. Nevertheless, decreased mitochondrial damage was discovered after administration of DI, as shown in Fig. 3A. In addition, DHE staining showed a robust increase (6 times up) in ROS after DOX administration, while DI could reduce the level of ROS by half (Fig. 3B and C).
Fig. 3. DI alleviated DOX-induced mitochondrial damage and reduced ROS production. (A) Representative transmission electron microscopy images for cardiac tissue.(B) Representative immunofluorescence image of DHE. Stained sections of cardiac tissue in magnification 200. (C) Densitometric analysis of DHE fluorescence.
Fig. 2. DI reduced the oxidative stress response of cardiac tissue in mice. (AeB) Protein levels of nuclear Nrf2 and HO-1 in the cardiac tissues were analyzed by Western blotting. Lamin B1 and b-actin were used as a control for protein loading. (C) Levels of oxidative stress products (MDA, SOD and GSH) of cardiac tissues. *p < 0.05, **p < 0.01, and ***p < 0.001.
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deficiency could exacerbate DOX-induced myocardial cell damage (date not shown), which is consistent to Sy Li's research [29]. Intriguingly, the protective effect of DI was vanished in Nrf2 KO mice. We employed HE staining, serum LDH concentration, heart weight/body weight ratio and myocardial tissue oxidative stress products (MDA, GSH, and SOD) to assess the myocardial injury. It's evident that protective effects of DI on DOX-induced cardiomyocyte necrosis is abolished by Nrf2 deficiency (Fig. 4). 4. Discussion In current study, we demonstrated that DI, an antiinflammatory metabolite, could alleviate acute cardiotoxicity induce by DOX involving mitochondrial damage and oxidative stress pathway. Mitochondria are the major toxic targets of DOX [17e20]. The mitochondrial inner membrane is rich in cardiolipin, which has high affinity with DOX. That leads to a large accumulation of ROS in
cardiomyocytes, disrupting mitochondrial membrane permeability and affecting the maintenance of mitochondrial membrane potential. Additionally, a large number of free radicals are also produced in metabolic process of DOX, which cause mitochondrial oxidative damage. These factors cause mitochondrial dysfunction and induce cardiotoxicity together. In this report, we illustrated that DI could effectively protect against acute injury induced by DOX in cardiomyocytes for the first time. Dexrazoxane is the unique cardioprotective agent approved by FDA for anthracycline induced cardiotoxicity, however, its protection is not sufficient. Therefore, more studies of new cardioprotective agents are required. Itaconic acid is derived from the cellular mitochondrial matrix and has a clear anti-inflammatory and immune-modulatory function [30e32]. Studies have shown that itaconic acid can reduce the release of lipopolysaccharidestimulated bone marrow derived macrophage inflammatory factors, and has a definite protective effect on inflammatory diseases such as ischemia-reperfusion and sepsis in mice [28]. Besides the
Fig. 4. Nrf2 deficiency offset the protective effect of DI on DOX -induced cardiomyocyte injury. (A) Representative HE staining of cardiac tissue. (BeC) Changes in HW/BW and serum LDH levels in mice. (C) Levels of oxidative stress products (MDA, SOD and GSH) of cardiac tissues.
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various immunoregulatory effects in myeloid cells, Recently, Daniels et al. find that neuronal itaconate restricts Zika virus replication by reprogramming neuronal metabolism [33]. However, whether it has a protective effect on DOX-induced cardiomyocyte injury has not been reported. Through animal experiments, we found that after administration of itaconic acid, the acute injury of cardiomyocytes induced by DOX was alleviated, the expression of RIP3 protein was down-regulated, the mitochondrial damage and ROS production of myocardial cells were decreased. These phenomena suggested the protective effect of itaconic acid on DOX-induced acute injury of cardiomyocytes. Studies also have shown that Nrf2/HO-1 mediated oxidative stress product plays an important role in DOX-induced cardiomyocyte injury [14e16,34]. Myocardial cell injury aggravated in Nrf2-deficient mice, while activation of Nrf2/HO-1 pathway can effectively protect myocardial cell injury. Researches of Mills EL et al. have shown that itaconic acid exerts an anti-inflammatory pathway by activating the Nrf2/HO-1 pathway. Firstly, we detected that itaconic acid can activate the Nrf2/HO-1 pathway in myocardial tissue with a change in oxidative stress products. Subsequently, Nrf2 deficiency can counteract the protective effect of itaconic acid. These results directly suggested that itaconic acid as a therapeutic drug to protect the myocardial injury elicited by DOX through the Nrf2/HO-1 pathway. 5. Conclusion Our study demonstrates that the protective effect of itaconic acid on DOX-induced acute myocardial injury was acted through activating the Nrf2/HO-1 oxidative stress pathway. In clinic application, itaconic acid may be used as a therapeutic drug to prevent DOX-induced myocardial injury.
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All authors of this paper have no conflict of interests to disclose. Acknowledgements This work was supported by Yangzhou Science, Education and Health Special Fund [LJRC201818], Jiangsu Province Education Office of the major basic research projects [15KJA310001], National Natural Science Foundation of China [81670263].
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Protective effects of dimethyl itaconate in mice acute cardiotoxicity induced by doxorubicin Qing Shan a, b, 1, Xiaoyu Li c, 1, Mei Zheng d, 1, Xi Lin e, Guotao Lu e, f, Dongming Su g, h, *, Xiang Lu a, ** a
Department of Geriatrics, The Second Affilicated Hospital, Nanjing Medical University, Nanjing, 211166, People's Republic of China Department of Geriatrics, Affilicated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225000, People's Republic of China Department of Pathophysiology, Nanjing Medical University, Nanjing, 211166, People's Republic of China d Department of Cardiology, Beijing Jishuitan Hospital & the 4th Medical College of Peking University, Peking University, No. 31 Xinjiekou East Street, XiCheng District, Beijing, 100035, People's Republic of China e Department of Gastroenterology, Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, 225000, People's Republic of China f Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou, 225001, People's Republic of China g Center for Clinical Pathology and Laboratory, Affiliated Hospital of Yifu, Nanjing Medical University, Nanjing, 211166, People's Republic of China h Department of Pathoology, Nanjing Medical University, Nanjing, 211166, People's Republic of China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 July 2019 Accepted 17 July 2019 Available online 31 July 2019
Doxorubicin (DOX) is an antitumor drug widely used in hematological tumors and various solid tumors. However, the cardiotoxicity elicited by DOX severely limits its clinical treatment. Dimethyl itaconate (DI), a common form of itaconate, is found many potential targets for prevent heart injury. Here we employed wild type and Nrf2 knockout mice and induced a cardiotoxicity model by administration of DOX to clarify the effects of DI. After treatment with DI, we found that it could effectively alleviate the cardiotoxicity by analyzing morphology, LDH levels and heart weight/body weight ratio changes. Meanwhile we demonstrated that RIP3, a key protein of necrosis, was significantly decreased in DI treated group. Further we observed that treatment with DI could suppress oxidative stress by altering Nrf2/HO-1. Compared with vehicle group, DI could increase the tissue SOD and GSH, and reduce MDA levels, then DHE staining revealed that the level of ROS in DI group reduced by half. Finally, transmission electron microscope (TEM) data showed that treatment with DI obviously decreased the mitochondrial damage. While Nrf2 was ablated in mice, the protective effects of DI were vanished and SOD, GSH, MDA became unchanged related to vehicle group. This report provides the evidence for the protective effects of DI treatment in cardiotoxicity induced by DOX. On mechanisms, DI could reduce the oxidative stress by altering Nrf2/HO-1 pathway and prevent mitochondrial from damage. Taken together, these findings of this paper will afford the new therapeutic targets in DOX related cardiotoxicity. © 2019 Elsevier Inc. All rights reserved.
Keywords: Dimethyl itaconate Acute cardiotoxicity Doxorubicin Oxidative stress
1. Introduction
* Corresponding author. Department of Pathology, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing, 211166. People's Republic of China. ** Corresponding author. Department of Geriatrics, The Second Affiliated Hospital, Nanjing Medical University, 101 Longmian Avenue, Jiangning District, Nanjing, 211166, People's Republic of China. E-mail addresses: [email protected] (D. Su), [email protected] (X. Lu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.07.046 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Doxorubicin (DOX) is an anthracycline non-specific antitumor drug targeted to cell cycle, which inhibits tumor cell proliferation in different growth phase. Due to its highly effective and broadspectrum anti-tumor effects, DOX has been widely used in clinical chemotherapy for hematological tumors and various solid tumors since the 1960s [1]. However, the cardiotoxicity of DOX severely restrains its application. In long-term clinical treatment with DOX, patients are often suffered from delayed and irreversible myocardial damage and the toxicity often shows dosage-dependent DOX accumulation [2]. At a therapeutic dosage of DOX, the main clinical
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manifestations of early cardiotoxicity are arrhythmia and cardiac dysfunction [3e5], while long-term medication is prone to induce dilated cardiomyopathy, congestive, heart failure and even lifethreatening [6e9], thus The US FDA's Black Box Warning requirement for DOX is also based on its cardiotoxicity [10]. Currently, there are absent of effective treatments and prevention for its unanticipated toxicity. It has been shown that mechanisms of DOX induced cardiotoxicity involve many factors, such as apoptosis [11], autophagy [12], vasoactive amine release [13], oxidative stress [14e16] and mitochondrial damage [17e20]. Specially, the oxidative stress pathway involved in mitochondrial damage has gradually become the main issue in injury process. To our interesting, it has been proved that inhibition of oxidative stress can effectively alleviate acute myocardial damage induced by DOX [21e23]. Further, finding effective drugs for anti-oxidative stress might be an effective strategy to prevent myocardial damage induced by DOX in clinical therapeutic. Itaconic acid is an unsaturated dibasic organic acid, which contains unsaturated double bonds with active chemical properties. It can carry out various addition reactions, esterification reactions and polymerization reactions. Therefore, itaconic acid is an important raw material for chemical synthesis industry and chemical production. Recently, itaconate has been found to play an important role in cellular immune metabolism and antimicrobial defense [24,25]. However, whether it has a protective effect on acute cardiotoxicity induced by DOX has not been reported yet. In this paper, dimethyl itaconate (DI), a common form of itaconate, was used to investigate its effects.
2. Materials and methods 2.1. Animal and reagents Male C57BL/6 and Nrf2 KO mice were obtained from the Model Animal Center of Nanjing University. 8 weeks old mice were used for experiment and animal feeding. Mice were exposed to 12/12 h light and dark cycles with freedom for eating and drinking. All animal protocols were performed in accordance with the Principles of Laboratory Animal Management (NIH Publication 85Y23, revised in 1996). All animal experiments were performed in accordance with the ethical requirements of animal experiments at Nanjing Medical University. DOX was purchased from Medchem Express (MCE Co. Ltd., Shanghai, China) and DI was purchased from Aladdin (Aladdin Bio-Chem Technology Company, Shanghai, China). The LDH assay kit was purchased from Nanjing Jiancheng (Nanjing Jiancheng Corp., Nanjing, China). Antibodies against-RIP3, Nrf2 and HO-1 were purchased from Abcam (Cambridge, UK). MDA, GSH, SOD commercial kits were purchased from Nanjing Key-Gen (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China). Dihydroethidium (DHE) staining solution and electron microscope fixative were purchased from Servicebio (Wuhan Servicebio Technology Co. Ltd., Wuhan, China).
2.2. Mouse model preparation and administration of DI According to previous literatures [26,27], we used intermittent administration of DOX to induce acute myocardial injury. DOX was dissolved in PBS for further protocol. Acute myocardial injury was elicited by DOX (total dosage: 200 mg/kg) intraperitoneally injected at 1st and 3rd day. Itaconic acid was dissolved in PBS and administered at first 4 days with 100 mg/kg per day since DOX intraperitoneal injection.
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2.3. Sample collection and preparation Mice were sacrificed on the 5th day after the first DOX dose. All mice were sacrificed after anesthesia, then systemic circulation perfusion was performed. The heart tissue was collected for morphological study with 4% paraformaldehyde fixation and to other protocols with storing in liquid nitrogen. 2.4. HE stains After tissue dehydration, waxing, and embedding, 5 mm slices were made and baked at 60 C. The tissue sections were stained with hematoxylin and eosin for 3 and 2 min respectively. The gradient alcohol is used to dehydrate and the xylene is used to transparentize. Finally, the slices were observed under a microscope for morphologic study. 2.5. Immunohistochemistry (IHC) Briefly, 5 mm thickness sections were dewaxed, gradient alcohol hydration, heating for antigen retrieval and 3% H2O2 removal of endogenous peroxidase activity. Then added to RIP3 (1:200) primary antibody 4 C overnight, after washing, secondary antibody was incubated for 1 h at room temperature. Finally, hematoxylin stained. 2.6. Western bolt Heart tissue proteins were extracted and the protein concentration was determined by the BCA assay. PVDF membrane with constant flow 200 mA was used to transfer membrane. After membrane was blocked with 5% milk for 1 h the primary antibody was incubated overnight at 4 C. The primary antibody dilution ratios were: Nrf2 (1: 1000 dilution), HO-1 (1: 1000 dilution), Lamin B1 (1: 1000 dilution), and b-actin (1: 2000 dilution). The second antibody was incubated for 1 h at room temperature. b-actin or Lamin B1 was used as a loading control. 2.7. Analysis oxidative stress Heart tissue was added to PBS with a ratio of 1:9, homogenized to prepare a suspension, and centrifuged to obtain a supernatant. SOD, MDA, GSH in the supernatant were detected according to the kits’ instruction to present oxidative stress level. 2.8. Cell ROS production detection ROS fluorescent probe-Dihydroethidium (DHE) was employed to detect cellular ROS production. DHE can enter the cell through the living cell membrane freely, and is oxidized by ROS to form ethylene oxidein the cell. Ethylene oxide can be incorporated in the chromosomal DNA then red fluorescence is detectable to represent ROS. Fresh heart tissue was embedded in OCT and 7 mm sections were performed. The slices were incubated with DHE staining solution at 37 C in the dark for half an hour after washed with PBS for 3 times, then subjected to DAPI staining. Finally, photographs were taken under an immunofluorescence microscope. 2.9. Transmission electron microscopy Fresh tissue was quickly placed in 2.5% glutaraldehyde for 24 h at 4 C and then using 1% citric acid$0.1 M phosphate buffer PB (pH 7.4) fixed tissue at room temperature (20 C) for 2 h. Alcohol and acetone were used to dehydrate. Acetone: 812 embedding agents ¼ 1:1 for 2e4h, acetone:812 embedding agent ¼ 2:1
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permeate overnight, pure 812 embedding agent 5e8h. The pure 812 embedding agent was poured into the embedding plate, and the sample was inserted into the embedding plate with at 37 C overnight. The ultra-thin slices (60e80 nm) was made by JEOL1010. Double staining of Uranium-Plumbum (2% uranyl acetate saturated alcohol solution, plumbum citrate, stained for 15 min respectively), and the sections were dried overnight. Observed under a transmission electron microscope, image analysis was performed. 2.10. statistical analysis GraphPad Prism 7.0 software was used for data analysis. Data are shown as mean ± SEM. Statistical significance was determined by performing t tests (two-tailed), one-way ANOVA. Image J software was used to analyze WB and morphologic photos. The optical density of the immunoreactive bands target proteins relative to b-
actin for the cytoplasm extraction or Lamin B1 for the nuclear extraction represented the protein contents level in WB. p value with 0.05 was considered to be statistically significant. 3. Results 3.1. DI effectively alleviated the acute damage of cardiomyocytes induced by DOX To investigate the role of DI in cardiotoxicity, DOX induced acute myocardial injury model were applicated in our study. The flow chart of experiments shown in Fig. 1A. As shown in Fig. 1B, the cardiomyocytes of the control group were arranged neatly and densely. DOX significantly caused the arrangement disorder of myocardial cells vacuolization of the cytoplasm, blurred transverse lines, and widening of the intercellular space in the murine model, accompanied by a high serum LDH level (Fig. 1C) and enhanced
Fig. 1. DI alleviated the acute damage of cardiomyocytes induced by DOX in mice. (A) The experimental protocol with DI in DOX-induced cardiomyocytes induced model. (B) Representative HE staining and histological scores of heart. (CeD) Changes in HW/BW and serum LDH levels in mice. (E) Representative immunohistochemistry images for RIP3 expression in the heart. **p < 0.01, and ***p < 0.001.
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heart weight/body weight (HW/BW) ratio (Fig. 1D). These suggested that DOX caused severe acute myocardial damage in mice. Compared with DOX treated group, additional DI administration group exhibited a reduction in cardiac pathological damage, a repression in plasma LDH levels, and less heart weight/body weight ratio, which suggest DI significantly attenuate DOX-induced cardiomyocyte injury. As a critical regulator of cell necrosis, RIP3 is significantly up-regulated during DOX-induced myocardial injury. IHC staining in Fig. 1E confirmed that DI could significantly downregulate RIP3 expression in cardiomyocytes, which further suggested that DI might have an obvious protective effect against the myocardial injury.
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3.3. Nrf2 deficiency offset the protective effect of DI on DOXinduced cardiomyocyte injury Finally, in order to further clarify the mechanism of action of DI to protect DOX-induced cardiomyocyte injury, Nrf2 KO mice were involved in our experiments. Firstly, we observed that Nrf2
3.2. DI effectively alleviated DOX-induced mitochondrial damage and altered Nrf2/HO-1 oxidative stress pathway Oxidative stress and mitochondrial damage play an important role in DOX-induced myocardial injury. Moreover, previous studies have pointed out that DI could activate Nrf2/HO-1 pathway to alleviate inflammatory responses in macrophage [28]. We further observed the protective effect of DI on myocardial mitochondrial damage. By detection of myocardial oxidative stress products and Nrf2/HO-1 protein level, we found that DI could significantly activate the Nrf2/HO-1 pathway, elevate the tissue levels of SOD and GSH and reduce MDA levels (Fig. 2AeC). TEM analysis confirmed that DOX treatment led to mitochondrial swelling, lysis and rupture in cardiomyocyte. Nevertheless, decreased mitochondrial damage was discovered after administration of DI, as shown in Fig. 3A. In addition, DHE staining showed a robust increase (6 times up) in ROS after DOX administration, while DI could reduce the level of ROS by half (Fig. 3B and C).
Fig. 3. DI alleviated DOX-induced mitochondrial damage and reduced ROS production. (A) Representative transmission electron microscopy images for cardiac tissue.(B) Representative immunofluorescence image of DHE. Stained sections of cardiac tissue in magnification 200. (C) Densitometric analysis of DHE fluorescence.
Fig. 2. DI reduced the oxidative stress response of cardiac tissue in mice. (AeB) Protein levels of nuclear Nrf2 and HO-1 in the cardiac tissues were analyzed by Western blotting. Lamin B1 and b-actin were used as a control for protein loading. (C) Levels of oxidative stress products (MDA, SOD and GSH) of cardiac tissues. *p < 0.05, **p < 0.01, and ***p < 0.001.
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deficiency could exacerbate DOX-induced myocardial cell damage (date not shown), which is consistent to Sy Li's research [29]. Intriguingly, the protective effect of DI was vanished in Nrf2 KO mice. We employed HE staining, serum LDH concentration, heart weight/body weight ratio and myocardial tissue oxidative stress products (MDA, GSH, and SOD) to assess the myocardial injury. It's evident that protective effects of DI on DOX-induced cardiomyocyte necrosis is abolished by Nrf2 deficiency (Fig. 4). 4. Discussion In current study, we demonstrated that DI, an antiinflammatory metabolite, could alleviate acute cardiotoxicity induce by DOX involving mitochondrial damage and oxidative stress pathway. Mitochondria are the major toxic targets of DOX [17e20]. The mitochondrial inner membrane is rich in cardiolipin, which has high affinity with DOX. That leads to a large accumulation of ROS in
cardiomyocytes, disrupting mitochondrial membrane permeability and affecting the maintenance of mitochondrial membrane potential. Additionally, a large number of free radicals are also produced in metabolic process of DOX, which cause mitochondrial oxidative damage. These factors cause mitochondrial dysfunction and induce cardiotoxicity together. In this report, we illustrated that DI could effectively protect against acute injury induced by DOX in cardiomyocytes for the first time. Dexrazoxane is the unique cardioprotective agent approved by FDA for anthracycline induced cardiotoxicity, however, its protection is not sufficient. Therefore, more studies of new cardioprotective agents are required. Itaconic acid is derived from the cellular mitochondrial matrix and has a clear anti-inflammatory and immune-modulatory function [30e32]. Studies have shown that itaconic acid can reduce the release of lipopolysaccharidestimulated bone marrow derived macrophage inflammatory factors, and has a definite protective effect on inflammatory diseases such as ischemia-reperfusion and sepsis in mice [28]. Besides the
Fig. 4. Nrf2 deficiency offset the protective effect of DI on DOX -induced cardiomyocyte injury. (A) Representative HE staining of cardiac tissue. (BeC) Changes in HW/BW and serum LDH levels in mice. (C) Levels of oxidative stress products (MDA, SOD and GSH) of cardiac tissues.
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various immunoregulatory effects in myeloid cells, Recently, Daniels et al. find that neuronal itaconate restricts Zika virus replication by reprogramming neuronal metabolism [33]. However, whether it has a protective effect on DOX-induced cardiomyocyte injury has not been reported. Through animal experiments, we found that after administration of itaconic acid, the acute injury of cardiomyocytes induced by DOX was alleviated, the expression of RIP3 protein was down-regulated, the mitochondrial damage and ROS production of myocardial cells were decreased. These phenomena suggested the protective effect of itaconic acid on DOX-induced acute injury of cardiomyocytes. Studies also have shown that Nrf2/HO-1 mediated oxidative stress product plays an important role in DOX-induced cardiomyocyte injury [14e16,34]. Myocardial cell injury aggravated in Nrf2-deficient mice, while activation of Nrf2/HO-1 pathway can effectively protect myocardial cell injury. Researches of Mills EL et al. have shown that itaconic acid exerts an anti-inflammatory pathway by activating the Nrf2/HO-1 pathway. Firstly, we detected that itaconic acid can activate the Nrf2/HO-1 pathway in myocardial tissue with a change in oxidative stress products. Subsequently, Nrf2 deficiency can counteract the protective effect of itaconic acid. These results directly suggested that itaconic acid as a therapeutic drug to protect the myocardial injury elicited by DOX through the Nrf2/HO-1 pathway. 5. Conclusion Our study demonstrates that the protective effect of itaconic acid on DOX-induced acute myocardial injury was acted through activating the Nrf2/HO-1 oxidative stress pathway. In clinic application, itaconic acid may be used as a therapeutic drug to prevent DOX-induced myocardial injury.
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All authors of this paper have no conflict of interests to disclose. Acknowledgements This work was supported by Yangzhou Science, Education and Health Special Fund [LJRC201818], Jiangsu Province Education Office of the major basic research projects [15KJA310001], National Natural Science Foundation of China [81670263].
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