The role of toll-like receptors in myocardial toxicity induced by doxorubicin

Journal Pre-proof The role of Toll-like receptors in myocardial toxicity induced by doxorubicin Cai Xinyong, Zeng Zhiyi, Hong Lang, Yan Peng, Wu Xiaocheng, Zhang Ping, Shao Liang

PII:

S0165-2478(19)30464-X

DOI:

https://doi.org/10.1016/j.imlet.2019.11.001

Reference:

IMLET 6391

To appear in:

Immunology Letters

Received Date:

8 September 2019

Revised Date:

21 October 2019

Accepted Date:

6 November 2019

Please cite this article as: Xinyong C, Zhiyi Z, Lang H, Peng Y, Xiaocheng W, Ping Z, Liang S, The role of Toll-like receptors in myocardial toxicity induced by doxorubicin, Immunology Letters (2019), doi: https://doi.org/10.1016/j.imlet.2019.11.001

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The role of Toll-like receptors in myocardial toxicity induced by doxorubicin

Cai Xinyong1#, Zeng Zhiyi 1#, Hong Lang1#, Yan Peng 1, Wu Xiaocheng1, Zhang Ping2, Shao Liang1*

1

Department of Cardiology, Jiangxi provincial People's Hospital Affiliated to Nanchang University, Nanchang

330006, Jiangxi, China 2

Department of Neurology, Jiangxi provincial People's Hospital Affiliated to Nanchang University, Nanchang

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330006, Jiangxi, China .

#, contribute equal to this article *Corresponding

authors: Shao Liang, E-mail: [email protected]

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Highlight

Doxorubicin may cause cardiac toxicity which limits its clinical application.

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Toll-like receptors (TLRs) play important roles in the cardiac toxicity induced by

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doxorubicin.

This review aimed to explore the role of TLRs in the cardiac toxicity induced by

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Abstract

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doxorubicin and provide possible solutions.

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Doxorubicin is an effective antitumor drug commonly used in the treatment of a wide variety of cancers. However, doxorubicin may cause cardiac toxicity, which can cause

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congestive heart failure in severe cases, and this seriously limits its clinical application. It is believed that doxorubicin promotes the formation of reactive oxygen species, inducing oxidative stress, and at the same time, reduces the content of antioxidant substances in cardiac tissues, causing adverse effects. Toll-like receptors (TLRs) are biomolecules expressed on the surfaces of macrophages, dendritic cells, and epithelial cells that can recognize various types of pathogen-related or damage-related molecular patterns. In recent years, a large number of studies have confirmed that TLRs play

important roles in the cardiac toxicity induced by doxorubicin. This review aimed to explore the role of TLRs in the cardiac toxicity induced by doxorubicin and provide possible solutions. Keywords: Doxorubicin; Cardiotoxicity; Toll-like receptors

Doxorubicin is an effective antitumor drug commonly used in the treatment of many different kinds of cancers. However, doxorubicin may cause cardiac toxicity, which can cause congestive heart failure in severe cases, and this seriously limits its

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clinical application. Recent studies suggested that doxorubicin promotes the formation of reactive oxygen species (ROS), resulting in oxidative stress, and at the same time causes adverse effects by reducing the content of antioxidant substances in cardiac tissues. Toll-like receptors (TLRs) are biomolecules expressed on the surfaces of

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macrophages, dendritic cells, and epithelial cells that can recognize various types of

pathogen-related (PAMPs) or damage-related molecular patterns (DAMPs). In recent

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years, a large number of studies have confirmed that TLRs plays important roles in the cardiac toxicity induced by doxorubicin. This review aimed to explore the role of TLRs

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in the cardiac toxicity induced by doxorubicin and provide possible solutions. 1. Mechanisms of the cardiac toxicity induced by doxorubicin

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Doxorubicin is widely used in the chemotherapy of lung cancer, breast cancer, solid tumors, and leukemia. Cardiotoxicity is one of the main adverse effects of this drug [1-2]. Retrospective analysis of doxorubicin’s clinical use in adults showed that the

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incidence of congestive heart failure increased from 3 to 5% at a dose of 400 mg•m-2, and from 18 to 48% at a dose of 700 mg•m-2. Moreover, the cardiac toxicity caused by

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doxorubicin has serious consequences, resulting in a poor prognosis and death for up to 61% of patients in which it develops [3]. It is generally believed that the cardiac toxicity induced by doxorubicin mainly involves the following mechanisms as Figure1 shown: 1) when metabolized in vivo, doxorubicin generates large amounts of ROS and reactive nitrogen species (RNS), including -OH, O2-, H2O2, ONOO-, etc., while the activity of peroxidase in cardiomyocytes is generally low, meaning these ROS can obviously cause cardiac toxicity [4]; (2) doxorubicin causes the peroxidation of cardiac

phospholipids and the uncoupling of the respiratory chain, which leads to cardiac mitochondrial metabolic disorder and cell apoptosis [5]; (3) doxorubicin can reduce the intracellular bcl-2/Bax ratio, and then release cytochrome c in the mitochondria, leading to apoptosis [6]; (4) the high levels of expression of topoisomerase 2 in cardiomyocytes in combination with doxorubicin lead to the formation of DNA complexes, resulting in double-strand breakage and cardiomyocyte death [7-9]; and (5) doxorubicin induces the expression of nitric oxide synthase in the body, and the resulting increased nitric oxide concentration can nitrify and inactivate functional enzymes in the myocardium, such as

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myofibril creatine kinase, resulting in myocardial damage [10]. Among the above mechanisms, studies on the myocardial toxicity of mitochondria in doxorubicin have attracted wide attention in recent years. Abdullah et al.

[11]

confirmed by autophagy flux measurement that REDOX induced autophagosome

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accumulation occurred due to the blocking of lysosomal degradation process. Using

GFP - LC3 and mRFP - GFP - LC3 transgenic mice (TG), confirmed in the body more

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supple than star induced autophagy body and autolytic enzyme body accumulation, acute and chronic more gentle than star related cardiomyopathy involves a multifocal

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disease process, its reason including autophagy and autophagy-lysosome, mitochondria, dynamics and the accumulation of oxidative phosphorylation to adjust the change of

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protein expression and mitochondrial respiratory dysfunction. Michael et al. [12] used H9c2 myocardial myoblasts expressing mitochondrial targeted fluorescent protein MitoDsRed or the new double fluorescent mitosis response reporter gene mt-rosella to

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test the possibility that unchecked mitochondrial mitosis and mitochondrial phagocytosis might damage the viability of myocardial cells and lead to cell death.

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Mice lacking motility associated protein 1(DRP1) were protected from doxorubicininduced cardiac injury, strongly supporting the role of DRP1 dependent mitochondrial fragments in doxorubicin cardiotoxicity.In addition, doxorubicin accelerates the mitochondrial autophagy flux, while DRP1 fragmentation weakens the autophagy flux, which was evaluated by the mitochondrial autophagy reporter mt-rosella, suggesting the necessity of mitochondrial fragmentation in doxorubicin-induced mitochondrial autophagy. It has been reported that doxorubin can inhibit cardiac mitochondrial

autophagy because it can isolate parkin and p53, thereby reducing mitochondrial translocation of parkin protein and mitochondrial integration in autophagy vacuoles [13]

.Lebrecht etc. [14] confirmed that the mtDNA damage induced by Adriamycin is a

significant start and could lead to mtDNA coding exclusive decreased, the activity of respiratory chain complexes followed by muscle mitochondrial Ca2+ accumulation, mitochondrial dysfunction and ahead of mitochondrial respiratory chain dysfunction secondary to precipitation of calcium deposition, is the forerunner of chronic toxicity of Adriamycin heart. Karin et al. found that transgenic mice lacking mitochondrial

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topoisomerase I were very sensitive to cardiac toxicity of Adriamycin, which provided support for cardiac toxicity caused by mitochondrial DNA damage. [15] 2. Cardiac toxicity of doxorubicin

The cardiac toxicity caused by doxorubicin can be classified into three types based

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on the timing of its occurrence: (1) acute or subacute, (2) chronic, and (3) late cardiac

toxicity [16]. Acute or subacute cardiotoxicity occurs during treatment and within a few

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days after administration of the drug, often presenting as intracardiac conduction disorder and arrhythmia, and in a few cases presenting as pericarditis and acute left

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heart failure. Acute cardiotoxicity due to doxorubicin has a very low incidence, mostly occurring only after the use of high doses of the drug. Chronic cardiotoxicity occurs

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within one year of chemotherapy, and is characterized by left ventricular dysfunction, which can lead to left heart failure. Late cardiac toxicity occurs 1 to 5 years after treatment, and is expressed as heart failure, cardiomyopathy, and arrhythmia. Children

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and adolescents are more sensitive to the cardiotoxicity induced by anthracene antitumor drugs than adults [17]. The cardiotoxicity caused by this drug does not include

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subclinical cardiovascular injury occurring early in the use of chemotherapy drugs, but rather mainly includes one or more of the following : (1) cardiomyopathy with a reduced left ventricular ejection fraction (LVEF), which presents as lower overall energy or significantly lower ventricular septal movement; (2) symptoms associated with congestive heart failure (CHF); (3) CHF-related signs, such as a third heartbeat, tachycardia, or both; (4) the LVEF being decreased by at least 5% from baseline to an absolute value < 55%, accompanied by CHF symptoms or signs; or (5) the LVEF being

decreased by at least 10% to an absolute value < 55%, but without being accompanied by symptoms or signs [18] 3. Toll-like receptors (TLRs) 3.1. Classification, structure, and ligands of TLRs TLRs are transmembrane receptors like those encoded by the Drosophila Toll gene (d Toll) discovered by Medzhidov et al.

[19]

in 1977 in the process of studying the

embryonic development of Drosophila. Homologous Toll-like proteins were later identified on the surfaces of human cells, and termed TLRs. The first human Toll-like

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protein to be identified, TLR4, is closely related to the immune inflammatory response. So far, 13 mammalian species of the TLR family have been found, among which at

least 11 species of human TLRs have been found [20]. Studies have found that TLRs are

mainly expressed in immune cells, such as monocytes, granulocytes, dendritic cells,

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etc., which act as recognition receptors for various pathogenic microorganisms to

initiate host defense responses. In addition, previous studies have shown that TLRs are

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also expressed in cardiomyocytes and cardiovascular endothelial cells [21]. For example, Boyd et al. [22] found that TLRs 2, 3, 4, 5, 7, and 9 are present in mouse cardiomyocytes.

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TLRs form a group of conservative transmembrane receptor proteins, including extracellular, transmembrane, and intracellular Toll interleukin receptor (TIR) proteins , which all belong to the type I transmembrane receptor group. The extracellular

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[23]

region is rich in leucine repeat regions, which are the sites at which ligands bind TLRs. TLRs in the intracellular region are responsible for recruiting downstream connective

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proteins, such as myeloid differential factor 88 (MyD88). Those in the transmembrane region are rich in mesocysteine. Based on the different locations at which TLRs are

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expressed, they are mainly divided into cell surface receptors and intracellular receptors. The TLRs that are recognized as cell surface receptors mainly include TLRs 1, 2, 4, 5, 6, and 10. The TLRs recognized as intracellular receptors include TLRs 3, 4, 7, 8, and 9 [24]. The ligands binding to TLRs are mostly viruses and nucleic acids, such as doublestranded DNA, single-stranded DNA, CpG DNA, lactoferrin, and so on. Of the above receptors, it is known that TLR4 can not only recognize lipopolysaccharides, respiratory syncytial virus proteins, and lipoteichoic acid, but also recognizes some

endogenous substances, thus initiating the innate immune response to resist invasions by pathogenic microorganisms and participating in the tissue damage repair process [2526]

. TLRs play important roles in Adriamycin induced pathological changes, and these

TLRs respond immediately to pathogenic and non-pathogenic ligands produced by damaged tissues. 3.2. TLR-mediated signal transduction pathways Signal transduction pathways involving TLRs are mainly divided into those that rely on the molecule MyD88 for ligand binding, and those that do not (as in Figure2).

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After ligand binding, TLRs induce the activation of Nuclear Factor-κB (NF-κB) in the cell nucleus, which leads to the secretion and release of a large number of bioactive substances that initiate the innate immune response, such as tumor necrosis factor alpha,

interleukins (IL-1, IL-3, and IL-6), intercellular adhesion molecule-1 (ICAM-1),

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monocyte chemo attractant protein 1 (McP-1), cluster of differentiation proteins (CD40 and CD80), etc. All of the aforementioned stimulating molecules can enhance the

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immune defense of body tissues and eliminate pathogenic microorganisms invading the bodies of human beings. Prior studies found that TLR4 does not directly recognize

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lipopolysaccharide (LPS), and LPS can only be recognized by it after its activity is enhanced by CD14, a cell-anchored molecule that binds to TLR4 and its satellite protein

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MD2. LPS bind to TLR4 on the cell surface to form a stable receptor-ligand complex to recruit the downstream adaptor protein molecule MyD88 [27]. Once activated, MyD88 combines with proteins in the IRAKs (interleukin receptor-associated kinases) family,

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and these IRAKs then reactivate tumor necrosis factor receptor-associated factor 6 (TRAF6). Then, TGF-3-activated kinase I (TAKI) is activated by transformed growth

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factor-P. After the induction of TAKI phosphorylation by IKK (inhibitor of kappa B kinase), IKK complexes are formed. The IKK complex phosphorylates the inhibitor of kappa B protein until degradation, and then NF-κB enters the human nucleus to induce the expression of inflammatory pre-cytokines; the aforementioned steps comprise the complete process of the TLR4-mediated MyD88 signal transduction pathway. All TLRs, except TLR3, mediate signal transduction via interactions with MyD88, so it can be seen that the MyD88 molecule is the joint molecule required in most TLR signal

transduction processes. In addition, there is a TLR-mediated signaling pathway independent of MyD88, in which TIPAP (TIP domain-containing adaptor protein), TRIF (TIR domain-containing adaptor inducing IFN-β), and TRAM (TRIF-related adaptor molecule), which all have similar effects to that of MyD88, interact with each other. Further initiation of interferon regulatory factor 3 (IRF3), NF-κB, and activator protein 1 (AP-1) will eventually lead to the activation of type I interferon and NF-κB and produce an inflammatory factor-mediated immune response to eliminate adverse stimuli.

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Ha et al. [28] transfected an adenovirus into the myocardium cells of rat hearts after abdominal aortic constriction to reduce MyD88 expression levels in them. Pathological

sections made after 21 days showed that the degree and extent of apoptosis and fibrosis

in myocardium cells transfected with the negative-dominant MyD88 adenovirus were

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significantly less than those in the control group. Further studies have found that TLR4

is involved in myocardial fibrosis and other ventricular remodeling processes by

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regulating the phosphorylation level of the NF-κB signal transduction pathway. In addition, studies have also shown that the expression of TLR4 in the cardiac tissues of

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patients with heart failure is significantly increased, and the phosphorylation levels of downstream signaling molecules in the IRAK and NF-κB pathways are increased, [29]

.

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resulting in the increased expression of these downstream signaling molecules

Bowie et al. [30] demonstrated that acute heart failure induced by myocardial infarction after ischemia reperfusion activated TLR4, which then translocated into the human

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nucleus to activate NF-κB and the transcription and replication of the related genes IL1 and TNF-3, activating a large number of inflammatory cells, which then migrated to

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and infiltrated the damaged and necrotic area around the infarction lesion. Shimamot et al. [31] found that in an ischemia-reperfusion mouse model in which the TLR4 gene was knocked out, the myocardial inflammatory response and response to tissue injury were significantly less strong than those in the control group. However, in the experimental group, the inflammatory response and injury response caused by ischemia reperfusion were significantly reduced after the administration of a TLR4 antagonist. All of the

above results indicate that TLR4 is involved in various cardiovascular events, and that it is also an important part of the pathogenesis of many cardiovascular diseases. 3.3 role of TLRs in myocardial diseases 3.3.1. Septic cardiomyopathy At least, there are four PRRs expressing in cardiomyocytes, including CD14 and TLR2, TLR3, TLR 4, and TLR 9 as shown in Table.1. TLR receptors and myocardial diseases were first studied in septic cardiomyopathy. Studies have shown that after LPS stimulation of the heart, TLR4 can up-regulate the expressions of TNF, IL-1, IL-6 and

[32-33]

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NOS2, while CD14 and TLR4 are involved in lps-induced left ventricular dysfunction .Thomas et al. demonstrated that IRAK1 (a downstream signaling molecule of

TLRs) -deficient mice were protected from LPS-induced death and cardiac dysfunction. In order to compare the function of the myocardial cells and white blood cell surface

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TLR4, Tavener [34], such as the use of LPS stimulation TLR4 positive white blood cells

in mice and TLR4 defects of myocardial cells, the results showed that LPS stimulation

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after cardiac function is reduced, and TLR4 defects white blood cells in mice and TLR4 positive myocardial cells of LPS is characterized by no reactivity, the surface of the

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leucocytes TLR4, instead of myocardial cell TLR4 may participate in the middle of the back dysfunction septic shock.TLR4 also activates the pro-inflammatory cell pathway

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of endothelial cells, which may directly lead to septic cardiomyopathy caused by endothelial dysfunction. Studies suggest that TLR2 is also involved in the occurrence and progression of septic cardiomyopathy. Studies have shown that TLR2, participated

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in the staphylococcus aureus sepsis induced cardiac TNF, IL-1 beta, and NO expression level raised, TLR2 also mediates the left ventricular dysfunction [35], compared with

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wild type mice, TLR2 gene knockout mice significantly higher survival rate, cardiac function improved, and further analysis found that the myocardial and cycle of inhibiting factor with decreased and neutrophil migration enhancements. 3.3.2 Myocardial ischemia-reperfusion injury Myocardial ischemia-reperfusion injury is directly related to the toxic reaction of reactive oxygen species during cardiac reperfusion. Studies have confirmed that ischemia/reperfusion directly leads to increased expression of inflammatory mediators

TNF, il-1 beta, il-6 and NO [36].This powerful inflammatory response causes many heart side effects, most notably left ventricular dysfunction. Recent experimental studies have shown that TLR2 and TLR4 signaling pathways mediate left ventricular dysfunction after myocardial ischemia reperfusion injury

[37-38]

.In vitro studies have

shown that oxidative stress induced by hydrogen peroxide is sufficient to induce increased expression of TLR2-mediated signaling pathway and can be inhibited by antiTLR2 antibodies [39]. It is also verified that the tlr2-tirap (TIR adaptor protein) signaling pathway is involved in the progression of left ventricular dysfunction induced by

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ischemia reperfusion in adults. [39] After ischemia reperfusion, cardiac systolic function was significantly impaired in wild-type mice, while left ventricular developed pressure (LVDP) in tlr2-deficient mice decreased. The above studies were mainly conducted in

isolated reperfusion hearts, suggesting that left ventricular dysfunction may be partly

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related to tlr2-mediated signaling pathways. Recent studies using tlr2-deficient mice

with bone marrow transplantation have found that the infarct size of wild-type mice

[40].

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with tlr2-deficient bone marrow transplantation is similar to that of tlr2-deficient mice In contrast, tlr2-deficient mice transplanted with bone marrow from wild-type mice

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were not protected from ischemia reperfusion injury, while mice treated with TLR2 inhibitors reduced infarct size and improved heart function. In addition, inflammation

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and apoptosis signals were reduced in antibody-treated mice compared with untreated mice, suggesting that tlr2-positive white blood cells may determine the extent of myocardial injury in this model [40].The study also reported that the infarct area of TLR4

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sudden change mice (C3HeJ) decreased after short blockage of left anterior descending artery, while the TLR4 defect also reduced myocardial neutrophil infiltration and

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complement deposition

[39]

. It is reported that tlr4-deficient mice had decreased

infarction area after ischemia reperfusion, while the inhibition of PI3K (phosphocreatine kinase 3) /Akt signaling pathway resulted in increased infarction area in tlr4-deficient mice. [37] However, initial treatment with the TLR4 antagonist eritoran has significantly reduced the size of myocardial infarction after myocardial ischemia and reperfusion [31].In addition, the study also found that inhibition of TLR signaling pathways downstream of molecules such as MyD88, nf-kappa B also can alleviate

myocardial ischemia-reperfusion injury, TLR/MyD88 nf-kappa B the activation of signaling pathways involved in the occurrence and progress of myocardial injury of ischemia reperfusion

[41-42],

thus further experiments should focus on whether by

inhibiting TLR2/4 signaling pathways and reducing ischemia-reperfusion injury. 3.3.3. Myocardial ischemia-preconditioning Myocardial ischemia-preconditioning refers to that repeated short ischemic attacks can protect the myocardium from subsequent long-term ischemia, thereby reducing myocardial cell damage caused by myocardial ischemia-reperfusion and increasing the

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tolerance of myocardial cells to ischemia and hypoxia. Recent studies suggest that TLR signaling pathway plays an important role in reducing tissue damage in many

myocardial ischemia reperfusion models. Izuishi et al. [43] showed that HMGB1 (highmobility group box protein 1, a TLR4 ligand) pre-treatment could have cell protective

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effect on liver ischemia reperfusion in wild mice, but had no effect on TLR4 mutant

mice. In addition, tlr2-specific ligand therapy significantly reduced the cerebral

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infarction area during focal ischemia reperfusion in mice. [44]. Dong et al. [45] found that this repeated injury to the heart was manifested as the transient occurrence of ischemia

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after reperfusion, which could produce cellular protection through tlr2-tirap-dependent signaling pathways. Ischemic preconditioning can significantly increase the percentage

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of LVDP recovery in wild-type mice after ischemia-reperfusion, but has no effect on the heart of tirap-deficient mice. Ischemic preconditioning also significantly increased cardiac function in mice with TLR4 but not TLR2.TLR2 agonist Pam3CSK4 reduced

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the effects of ischemic preconditioning in wild-type mice rather than tlr2-deficient mice. Mersmann et al.

[46]

also confirmed that Pam3CSK4 pretreatment mice could reduce

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myocardial infarction area after ischemia reperfusion, reduce trocitin T level and improve cardiac function, accompanied by decreased leukocyte infiltration in ischemic areas and decreased expression of CXCL10 (CXC chemo hemic ligand 10).These data suggest that the innate immune system may play a role in short-term myocardial protection by activating the tlr2/4-mediated cellular protection signaling pathway. 3.3.4. Heart failure and myocardial remodeling

The relationship between heart failure and inflammation was first reported by Levine et al. [47], who found that TNF expression levels were up-regulated in heart failure. Many studies have found that in addition to TNF, other pro-inflammatory cytokines and chemokines are also involved in the progression of heart failure 49]

[48-

.Recent clinical and experimental studies have suggested that TLRs plays an

important role in the occurrence and development of heart failure. Frantz et al. [50] first reported TLR4 expression in human and rodent hearts, and both ischemic cardiomyopathy and experimental heart failure in rodents found that TLR4 expression [51]

also reported that TLR4

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in myocardial tissue was up-regulated. Timmers et al.

mutant rats had less left ventricular remodeling after myocardial infarction, so as to

protect left ventricular function, and non-infarcted interstitial fibrosis and myocardial hypertrophy were also less common. The mutant mice produced decreased

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inflammatory cytokines and increased collagen density in the infarcted area, suggesting that tlr4-mediated LPS signal transduction had a significant effect on left ventricular

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systolic function in mice. However, Shishido et al. [52] found that the mortality rate and the incidence of left ventricular dysfunction were significantly reduced in tlr2-deficient

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mice after coronary artery ligation and modeling. Compared with wild-type mice, tlr2deficient mice had less ventricular remodeling under similar infarction area conditions.

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Histological examination revealed that TLR2 defects of infarction area of myocardial fibrosis in mice significantly reduced, further analysis found that these may be related to TLR2 heart defect mice TGF - beta 1 and type I collagen expression reduced, in

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addition, the oxidative stress plays an important role in heart failure, can be triggered by TLR2 nf-kappa B.These studies suggest that TLR2 and TLR4 may be new targets

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for the treatment of ischemic heart failure. Irks et al. [53] reported that both TLR4 and il1 receptors were up-regulated in the myocardium of patients with deteriorating cardiac function who needed to be equipped with left ventricular assist devices. The expression of TLR4 mRNA is higher in patients with dilated cardiomyopathy than in patients with ischemic heart disease. Recently, Mann et al. [54] studied the activation of innate immune genes in patients with heart failure caused by dilated cardiomyopathy, ischemic cardiomyopathy and viral cardiomyopathy, and found that the expression of TLR2/4 in

transplanted hearts of patients with three types of cardiomyopathy was decreased. However, the downstream factor mediators TIRAP and irak-1 were increased. These data suggest that adaptive innate immune responses may be involved in the development of heart failure through the continuous expression of preinflammatory cytokines.TLR2 /4 mediates a series of signal transmission through the recognition of various pathogen-related molecular patterns (PAMP), which eventually activates nfkappa B, induces the production of inflammatory cytokines and adhesion molecules by monocytes/macrophages, leading to further decline of cardiac function.

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In conclusion, the TLR2/4 signaling pathway plays an important role in maintaining cardiovascular system homeostasis. However, this short-term beneficial effect may be offset by down regulation of expression (e.g., septic cardiomyopathy). Therefore, the

resulting in myocardial toxic side effects.

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application of doxorubicin will inevitably affect TLRs and then cardiomyocytes,

4. The role of TLRs in cardiac toxicity induced by doxorubicin

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4.1. The effect of TLR4 on cardiac toxicity induced by doxorubicin

TLR4 was the first TLR protein discovered in humans. It is the specific pattern

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recognition receptor of lipopolysaccharides (LPS) and belongs to the group of type I transmembrane receptors. The TLR4-mediated signal transduction processes in humans

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include MyD88-dependent and MyD88-independent pathways. A large number of studies on myocardial TLRs have shown that almost all kinds of cardiovascular diseases involve TLRs. For example, in cultured neonatal rat myocardial cells, TLR2 was found

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to activate the NF-κB-mediated apoptosis induced by hydrogen peroxide, while TLR4 activation could instead inhibit apoptosis of myocardial cells [55]. In TLR4-deficient

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mice with myocardial ischemia reperfusion (I/R), the severity of inflammation and tissue damage was also significantly reduced. Eritoran (E 5564), a TLR4 antagonist, can reduce the inflammatory stress in I/R patients [55]. In 1999, Frantz et al. [56] reported increased TLR4 expression in human heart failure and ischemic rat hearts. Subsequently, numerous studies have shown that tlr4-mediated signaling pathways are involved in myocardial I/R injury. Compared with wild-type (WT) I/R mice, tlr4deficient mice had a lower incidence of myocardial infarction after I/R [57].Interestingly,

giving mice a specific TLR4 antagonist heritage oran significantly reduced the size of myocardial infarction compared to untreated I/R mice [58].Both TLR4 deficiency and TLR4 antagonist decrease will weaken I/R, increase NF-k B binding activity in myocardium, and reduce the production of inflammatory cytokines after I/R in myocardium

[57-59]

.Cha et al.

[60]

found a significant improvement in cardiac function

after myocardial I/R compared to WT mice in hearts isolated from tlr4-deficient mice. Since there were no circulating cells throughout I/R in this isolated heart model, the results suggest that TLR4 expression by cardiomyocytes may cause cardiac dysfunction [61]

recently reported that in myocardial I/R

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during I/R.In line with this, Fallach et al.

and sepsis models, cardiac cells expressing TLR4 play a key role in myocardial

dysfunction. These authors observed that cardiac function in tlr4-deficient mice was

significantly improved after myocardial I/R or endotoxin treatment compared to WT

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I/R mice. There is a growing amount of evidence that, even in the absence of infection, endogenous ligands, including signals from damaged and stressed cells, can induce a

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pro-inflammatory response by the natural immune system via TLRs, triggering an immune response known as a ‘risk mode’ immune response. Recent studies have shown

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that heat shock proteins (HSPs) may be important endogenous ligands that induce natural and acquired immune responses and promote the generation of an autoimmune

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response [62-63]. Thus, we speculate that the activation of TLR4 by doxorubicin might be caused by oxidative stress, which leads to myocardial cell damage by triggering cell membrane lipid peroxidation. This changes the structure of the membrane, eventually

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leading to the integrity of the membrane being comprised, damaging the cell membrane structure and leading to the formation of a large number of endogenous ligands and

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overflow thereof, with the result that TLR4 is activated and subsequent immune responses are initiated. We present it in Figure3. During Adriamycin treatment, TLR4 induced apoptosis, oxidative stress and cardiac inflammation, and increased endothelin1 caused left ventricular dysfunction. The down-regulation of TLR4 reduces the formation of myocardial reactive oxygen species and prevents the down-regulation of transcription factor gat-4.Through the above mechanism, TLR4 participates in

extracellular degradation by inducing viscous circulation [64].TLR4 also increases levels of tumor necrosis factor (tnf-alpha) [65] 4.2. The effect of TLR2 on cardiac toxicity induced by doxorubicin TLR2, the most widely distributed TLR in mammals, is found mainly in the lungs, heart, brain, and muscle tissues. TLR2 is widely distributed in the body’s immune system and serves as its first line of defense. Indeed, while T cells are TLR2– (i.e. lack TLR2), B cells, monocytes, macrophages, neutrophils, and most other immune cells are all TLR2+. TLR2 is also expressed at high levels in the spleen and peripheral blood

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leukocytes. Recent research confirmed that TLR2 is also expression on the surfaces of dendritic cells, which directly or indirectly promotes the synthesis and release of

inflammatory factors, and also has significant antibacterial and antiviral activities. For

example, Ueland [66] found that TLR2 was involved in the development of myocardial

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inflammation and heart failure after myocardial infarction, and further that doxorubicin may make the TLR2 on the myocardial cell membrane enhanced the transduction of

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harmful signal proteins, which leads to myocardial damage. During the metabolism of doxorubicin, many ROS and RNS, including - OH, O2-, H2O2, ONOO-, etc., are

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produced in the body, and in vitro studies have shown that the oxidative stress induced in the heart muscle cells by hydrogen peroxide was mediated by TLR2-mediated

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signaling pathways, and its expression could thus be increased or decreased by antibodies targeting TLR2 [67]. Shao [68] and other studies showed that TLR2 expression was increased during the doxorubicin treatment of patients with impaired cardiac

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function, and a large number of studies have also shown that TLR2 activation can aggravate myocardial reperfusion injuries

[69]

. In TLR2-knockout mice, this treatment

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can reduce the growth of the infarction area caused by myocardial ischemia reperfusion injury, neutrophil infiltration, production of oxygen free radicals, and release of cytokines

[22]

. TLR2 can also recognize lipoproteins and peptidoglycans, causing the

activation of the NF-κB pathway, and impairing the systolic functioning of myocardial cells [22, 70]. Nozaki et al. [71] also found that doxorubicin decreased myocardial injury in TLR2 knockout mice, and they induced myocardial dysfunction in wild-type (WT) mice and tlr-2 knockout (KO) mice with a single dose of 20 mg/kg IP, 5 days after

Adriamycin injection, left ventricular dimension at end-diastole was smaller and fractional shortening was higher in KO mice compared with WT mice (P<0.01). Nuclear factor- B activation and production of proinflammatory cytokines after Dox were suppressed in KO mice compared with WT mice (P<0.01). The numbers of TUNEL-positive nuclei and Dox-induced caspase-3 activation were less in KO mice than in WT mice (P<0.01). Survival rate was significantly higher in KO mice than in WT mice 10 days after Dox injection (46% vs 11%, P<0.05).These findings suggest that TLR-2 may play a role in the regulation of inflammatory and apoptotic mediators

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in the heart after Dox administration. It has been reported that TLR2 deficiency can reduce Adriamycin induced cardiac dysfunction, reduce local inflammatory response, inhibit myocardial cell apoptosis and improve animal survival rate, suggesting that

TLR2 mediates Adriamycin induced cardiac dysfunction. Adriamycin induced

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cardiomyopathy has an increase in oxidative stress, which is associated with an increase in TLR2, which induces nuclear factor kappa B (nf-kappa B) and ultimately leads to

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apoptosis [67,72].

4.2. Effects of other TLRs on the cardiac toxicity induced by doxorubicin

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TLR3 is composed of 904 amino acids, and its relative molecular weight is about 125 kDa. Doxorubicin exposure can lead to ROS generation, mitochondrial damage,

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and the expression and release of apoptosis proteins in myocardial cells. One study found that TLR3 activation can increase the production of ROS, impair vasodilation, impair endothelial exchange, and aggravate endothelial cells [73-76]. At the same time,

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TLR3 can also recognize nucleic acid components in viruses, bacteria, and infected cells, and then induce the production of pro-inflammatory cytokines. The up regulated

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expression of human TLR3 on smooth muscle cells of atherosclerotic tissue can increase the expression of many chemokines and inflammatory cytokines [77-78]. The nuclear transcription factor NF-κB is an inducible and ubiquitous transcription factor that plays a central regulatory role in the transcription of a large number of genes, and thus plays extensive and important roles in immunity, inflammation, cell survival, proliferation, differentiation, and apoptosis. Through the induction and regulation of tumor necrosis factor, intercytokines (IL-1, IL-6, and IL-8), and other genes, the

inflammatory response is induced and stimulated by this transcription factor

[79-82]

. In

addition, doxorubicin can be metabolized in the body to generate many ROS and RNS, which induces the expression of nitric oxide synthase in the body. The higher the concentration of nitric oxide is relative to those of the myocardial enzymes (such as myofibril creatine kinase) that function in the inactivation of nitrification, the lower the content of TLR3 will be causing myocardial damage. This was shown by the results of research by Shao [40], in which the doxorubicin treatment of patients with impaired cardiac function increased the expression of TLR3 and reduced that of TLR2 as shown

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in Figure4. This suggests that TLR2-TLR3 imbalance has a role in doxorubicin-induced heart failure, and indirectly confirms the role of TLR3 in doxorubicin-induced myocardial toxicity.TLR3 has the ability to stimulate type 1 interferon (IFN-1) based on the efficacy of Adriamycin and produces the IFN stimulating gene. During

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Adriamycin treatment, dead tumor cells release their own RNA.This self-rna further triggers TLR3 signaling [83].

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The roles of other TLRS have not been determined, and further studies are needed to confirm what functions these have in the body, including in the heart

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5. Conclusion

In the treatment of tumors with doxorubicin, some TLRs are up regulated, while

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some are down regulated, which cannot be clearly explained by any known single mechanism. The interactions of these different TLRs with other molecules over the course of disease progression and resistance are certainly not the same. Therapies

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targeting TLRs have become a new way to treat many diseases, but more in vivo studies are needed to provide clearer evidence of their effects. How doxorubicin interferes with

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the TLRs of cardiac myocytes and the mechanisms by which doxorubicin acts on various TLRs are not fully understood. Similarly, there is considerable debate as to whether increasing or decreasing the activities of certain TLRs is the primary cause of doxorubicin cardiotoxicity. Further understanding of the mechanisms of the cardiac toxicity caused by doxorubicin will be helpful to prevent and treat the cardiomyopathy caused by doxorubicin treatment in the future.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or

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comparable ethical standards. Informed consent

Informed consent was obtained from all individual participants included in the

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study.

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Table1.The role of TLRs on cardiovascular system TLRs

Pathways

Effects

Enhance the signal transduction of harmful proteins, recognize lipoprotein and peptidoglycan, and cause nf-kb activity

TLR2

It is involved in the formation of heart failure after myocardial inflammation and myocardial infarction [66], aggravating myocardial reperfusion injury [69], and impaired myocardial cell systolic function [22, 70].

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Recognize nucleic acid components in viruses, bacteria, and infected cells and induce the production of pro-inflammatory cytokines Activation of nf-kb induces a pro-inflammatory response of the natural immune system and triggers an immune response

TLR3

Causes apoptosis, oxidative stress and cardiac inflammation, and causes left ventricular dysfunction

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TLR4

Induced increase inflammation myocardial damage

[79-82]

[55]

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It is closely related to Recognition of cytosine cardiovascular diseases such as phosphate - guanine sequence (CpG hypertension [84] ischemic heart DNA), by mtDNA action, into the disease [85-87] and heart failure activation of p38 mitogenic [88-89]. Insufficient capillary activation of egg white kinase density of myocardium after (p38MAPK) and nuclear factor myocardial infarction can affect the kappab (nf-kappab) signaling repair and remodeling of pathway, inducing the innate myocardium after myocardial immune response of cells infarction [90]

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TLR9

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Figure1.Molecular mechanism of doxorubicin toxicity: It mainly includes :(1) DNA destruction (2)

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free radical production (3) other mechanisms

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Figure2. TLRs signaling pathway. Mainly rely on the MyD88 after TLRs and its ligand binding and

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not rely on the MyD88 two signal transduction pathways

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Figure3. Mechanism of TLR4 in myocardial injury of doxorubicin: Doxorubicin activates oxidative stress, which in turn activates TLR4, which causes myocardial injury through the Myd88/TRIF

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pathways

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Figure4.Mechanism of TLR2/3 in myocardial injury of doxorubicin: Large amounts of ROS and RNS produced by doxorubicin during metabolism in vivo cause up-regulation of TLR2 and downregulation of TLR3, respectively, leading to myocardial damage