Natural products with anti-inflammatory and immunomodulatory activities against autoimmune myocarditis

Accepted Manuscript Title: Natural products with anti-inflammatory and immunomodulatory activities against autoimmune myocarditis Authors: Behjat Javadi, Amirhossein Sahebkar PII: DOI: Reference:

S1043-6618(17)30888-5 http://dx.doi.org/doi:10.1016/j.phrs.2017.07.022 YPHRS 3653

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Pharmacological Research

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17-7-2017 26-7-2017

Please cite this article as: Javadi Behjat, Sahebkar Amirhossein.Natural products with anti-inflammatory and immunomodulatory activities against autoimmune myocarditis.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.07.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Natural products with anti-inflammatory and immunomodulatory activities against autoimmune myocarditis

Behjat Javadi1*, Amirhossein Sahebkar,2,3*

1

Department of Traditional Pharmacy, School of Pharmacy, Mashhad University of Medical

Sciences, 2

Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

3

School of Medicine, University of Western Australia, Perth, Australia

Correspondence: Behjat Javadi (E-mail: [email protected]) Amirhossein Sahebkar (E-mail: [email protected]; [email protected])

1

Graphical abstract

Abstract Myocarditis is an inflammatory disease of the myocardium associated with immune dysfunction which may frequently lead to the development of dilated cardiomyopathy. Experimental autoimmune myocarditis is an animal model which mimics myocarditis in order to allow assessment of the therapeutic effects of different molecules on this disease. We aimed to review the inflammatory and immunological mechanisms involved in the pathogenesis of the myocarditis and finding natural products and phytochemicals with anti-myocarditis activities based on studies of cardiac myosin-induced experimental autoimmune myocarditis in rodents. A number of natural molecules (e.g. apigenin, berberine and quercetin) along with some plant extracts were found to be effective in alleviating experimental autoimmune myocarditis. Upregulation of Th1-type cytokines and elevation of the Th2type cytokines (IL-4 and IL-10), mitigation of oxidative stress, modulation of mitogen-activated protein kinase signaling pathways and increasing Sarco-endoplasmic reticulum Ca2+-ATPase levels are among the most important anti-myocarditis mechanisms for the retrieved molecules and extracts. Interestingly, there are structural similarities between the anti-EAM compounds, suggesting the presence of similar pharmacophore and enzymatic targets for these molecules. Naturally occurring molecules discussed in the present article are potential anti-myocarditis drugs and future additional animal studies and clinical trials would shed more light on their effectiveness in the treatment of myocarditis and prevention of dilated cardiomyopathy.

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Abbreviations: DCM = dilated cardiomyopathy; EAM = experimental autoimmune myocarditis; CM = cardiac myosin; CHF = heart failure; LV = left ventricular; ECM = extracellular matrix; IL = interleukin ; TNF- α = tumor necrosis factor-α; IFN-γ = interferon-γ; STAT = signal transducer and activator of transcription; DM = diabetes mellitus; GSH-Px= glutathione peroxidase; MDA = malondialdehyde ; SOD = superoxide dismutase; ICAM-1= intercellular adhesion molecule-1; anti-MyHCα = myosin-specific autoantibodies; Treg cells =T regulatory cells; IR = ischemia reperfusion; ER = endoplasmic reticulum; MAPK= mitogen-activated protein kinases; HW/TL = heart weight/tibial length; SIRT1= silent mating type information regulation 2 homolog 1; RAGE = receptor for advanced glycation endproducts; SERCA2= sarco-endoplasmic reticulum Ca2+ATPase; OPN = osteopontin; EF= ejection fraction; FS = fractional shortening; VEGF = vascular endothelial growth factor; VCAM-1= vascular cell adhesion molecule-1; MPO = myeloperoxidase; DHE = dihydroethidium

Keywords: Myocarditis; Dilated cardiomyopathy; Experimental autoimmune myocarditis; Natural products; Anti-inflammatory; Immunomodulatory.

Introduction Myocarditis is an inflammatory disease of the myocardium associated with immune dysfunction which may frequently lead to the development of dilated cardiomyopathy (DCM) [1]. Myocarditis is diagnosed by endomyocardial biopsy using established immunological, histological and immunohistochemical criteria [2]. The incidence of myocarditis has been difficult to determine because clinical presentations of the disease vary widely. However, autopsy reports have revealed varying estimates ranging from 0.12% to 12%, according to the population studied [3]. Approximately 21% of acute myocarditis patients develop DCM [4]. Lymphocytes and mononuclear cells infiltration, enhanced pro-inflammatory chemokines, cytokines and circulating autoantibodies expression are frequently observed in myocarditis and DCM [5]. Some patients with myocarditis develop a fulminant course followed by death from intractable cardiogenic shock. Unfortunately, no effective treatment strategy for myocarditis has still been introduced [6]. Experimental autoimmune myocarditis (EAM) is a CD4+ T-cell-mediated disorder involving a Th1/Th2 imbalance. It is an animal model of myocarditis induced by immunizing them with cardiac myosin together with complete Freund’s adjuvant [7, 8]. Cardiac tissue obtained from EAM animals demonstrates enlargement of the heart, dilatation of ventricles, severe myocardial injuries and large areas of myocyte fibrosis similar to those observed in human myocarditis [5, 8, 9]. This model has been largely used to mimic moycaritis in order to investigate the effects of different molecules on this disease. Medicinal plants and natural products have long been used to manage various diseases. 3

Traditional Medical systems such as Persian Medicine, Chinese Traditional Medicine and Ayurvedic Medicine have used plants to alleviate and cure a wide range of cardiovascular problems [10, 11]. Many studies investigated the role of medicinal plants and phytochemicals in alleviating EAM severity. The aim of this article is to review natural products and phytochemicals with antimyocarditis activities based on studies of cardiac myosin (CM)-induced experimental autoimmune myocarditis (EAM) in rodents. The inflammatory and immunological mechanisms involved in the pathogenesis of the myocarditis are also discussed. Myocarditis: pathophysiological aspects According to the current WHO classification of cardiomyopathies, myocarditis and DCM represent the acute and chronic phases of an inflammatory disease of the myocardium originating from various etiologies including idiopathic, familial/genetic, viral, primarily organ-specific autoimmune or postinfectious immune causes [12, 13]. Myocarditis is usually self-limited but approximately half of the acute myocarditis patients with progressive autoimmune myocardial injury demonstrate significant left ventricular (LV) dysfunction and symptoms of heart failure (CHF), arrhythmias, and sudden cardiac death [4, 14]. Increasing evidence suggest that inflammation and autoimmunity play crucial roles in the pathogenesis of myocarditis and DCM [12]. It is evident that dysregulation of immune response against cardiac tissue can facilitate programmed death of myocytes and is responsible for the ongoing myocytolytic process [15]. In myocardial inflammation, both cellular and humoral immune responses participate in cardiac remodeling by affecting processes such as extracellular matrix (ECM) degradation, collagen deposition, cardiomyocyte hypertrophy and/or apoptosis leading to vascular injury and cardiomyocyte ischemia. These processes can be mediated by pro-inflammatory cytokines, oxidative stress and mitochondrial dysfunction, alterations of micro- and macrocirculation, metabolic alterations, endothelial dysfunction, NO production, derangement of catecholaminergic stimulation and autonomic dysfunction. Myocardial inflammation can also directly affect cardiomyocyte contractility through disruption of Ca2+ homeostasis [4]. However, Most of the mechanisms underlying immune-mediated injury leading to cardiac dysfunction and heart failure remain unknown. Experimental autoimmune myocarditis (EAM) is a rodent model resembling human giant cell myocarditis, which can lead to DCM [16]. EAM provides insights into the role of the immune responses in the development of DCM and CHF [17]. Histological examination of EAM hearts shows the infiltration of inflammatory cells along with myocardial damage two weeks after cardiac myosin immunization. Thereafter, myocarditis reaches the peak around the third week, and then gradually diminishes during the fourth week. In the later stage, the sixth week, myocarditis progresses to DCM. EAM in rats and mice is a T cell-mediated autoimmune disease [18]. T cells are lymphocytes that play a central role in cellmediated immunity [19]. Activated T cells (CD4+ and CD8+) secrete chemokines and cytokines which in turn activate other inflammatory cells, such as mast cells, macrophages, and neutrophils [18]. Mast cells are granulocytes normally present in a variety of organ tissues and have been shown to be involved in the pathogenesis of inflammatory diseases including cardiac inflammation and fibrosis. Mast cells produce several cytokines, including interleukin (IL)-1, IL-3, IL-4, IL-5, IL-6, tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), and, other central mediators that develop inflammatory reactions [20]. On the other hand, the anti-inflammatory cytokines present a series of immunoregulatory molecules that 4

control the proinflammatory cytokine response [21]. IL-10 is a multifunctional cytokine with the ability of modulating extracellular matrix biosynthesis. The principal function of IL-10 seems to be to limit and terminate inflammatory responses. [18, 22]. IL-10 can bind to specific IL-10 receptors located on mast cells to prevent the release of inflammatory mediators [18]. In addition, it has been shown that IL-10-Igcontaining medium, significantly inhibits IL-17 gene expression in IL-1-stimulated spleen cells to restore excessive increase of Th17/Th1 responses in EAM rats [23]. Signal transducer and activator of transcription (STAT) proteins are intracellular transcription factors that are capable of transmitting cytokine signals from the plasma to the nucleus, where they modulate the expression of a variety of target genes through binding to sequence-specific DNA elements [15]. In EAM, modulating the activities of STATs can lead to further suppression of Th17 and Th1 cell differentiation [1].

Natural products with the EAC attenuating activities A number of natural products and medicinal plants have been shown to prevent and alleviate myocarditis using EAM model. Chemical structures of natural products with anti-EAC activities are illustrated in Figure 1. Apigenin is a flavon which is widely distributed in vegetables and fruits, especially in celery. Numerous pharmacological studies revealed cardioprotective activities of apigenin [24, 25]. Zhang et al. reported that apigenin significantly upregulated serum levels of the Th1-type cytokines (IL-2, IFN-γ, and TNF-α) and elevated the Th2-type cytokines (IL-4 and IL-10) and attenuated the severity of EAM in myocarditis mice. Blood pressure and heart rate were not affected in the treatment groups, indicating that beneficial effects of apigenin on EAM might be independent of blood pressure lowering effects [1]. Moreover, apigenin treatment significantly improved hearts dysfunction, and attenuated cardiac fibrosis induced by type 2 diabetes mellitus (DM) in mice with experimental diabetic cardiomyopathy. The immunohistochemistry showed that DM significantly induced the over-accumulation of 4hydroxynonenal (a by-product of lipid peroxidation) in the cardiac tissue, which could be downregulated by apigenin treatment accompanying with decreasing of glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and superoxide dismutase (SOD). Apigenin also suppressed the enhanced activity of caspase3 and NF-κB/P65. Besides, the apoptotic nuclei in apigenin treated group were much less than that in cardiomyopathy control group. In addition, apigenin could inhibit the translocation of NF-κB and down-regulate Bax gene and cleaved-caspase 3 expression, in vitro [26]. Berberine, a isoquinoline alkaloid extracted from many plants of the general Berberis and Coptis, has been found to possess several health benefits [27-30] including ameliorating effects in congestive heart failure as well as immunomodulatory effects against immune-mediated disorders [1, 31]. Liu et al. investigated the protective activities of berberine against EAM and the possible underlying molecular mechanisms involved. The results revealed that berberine could significantly attenuate the impaired LV dysfunction and the pathophysiological severity and lower levels of anti-cardiac myosin antibody of EAM rats. Moreover, the excessive increase of Th17/Th1 responses induced in EAM rats was restored by berberine. Berberine also notably attenuated the excessive expression of phosphorylated (p)-STAT1, 5

STAT3 and STAT4. Therefore, berberine could ameliorate EAM through differentially modulating the activities of p-STAT1, p-STAT3 and p-STAT4 to further suppress Th17 and Th1 cell differentiation [1]. Curcumin is a natural polyphenolic compound abundant in the rhizome of the herbaceous plant turmeric, Curcuma longa L. (Zingiberaceae) which is commonly used as a spice and coloring agent in Asian cuisine and Traditional Medicine. Numerous pharmacological activities have been reported [3242]. It has been found that curcumin exerts protective effect in the acute phase of EAM rats. Curcumin can reduce heart weight-to-body weight ratio (HW/BW; an index of myocardial hypertrophy) and inflammatory infiltration. Hemodynamic and echocardiographic measurements revealed improvement of both systolic and diastolic heart function of the myocardium of curcumin-treated EAM rats. Moreover, curcumin reduced the area of inflammatory lesions and the myocardial protein level of NF-κ B, IL-1b, TNF-α and GATA-4 [14]. Chlorogenic acid, an ester of caffeic acid and quinic acid, is an important phenolic compound found abundantly in coffee and tea [43]. Chlorogenic acid has been found to significantly suppress the intercellular adhesion molecule-1 (ICAM-1) expression in the EAM mice hearts. ICAM-1 plays an important role in cell-cell adhesion which is required in immune responses [44]. Furthermore, ICAM-1 suppression can lead to immune response modulation. The suppressed ICAM-1 (as a result of chlorogenic acid treatment) can lead to reducing myocardial fibrosis compared to control EAM hearts [45]. Emodin is an anthraquinone derivative isolated from a variety of plant species including Rheum palmatum L. Many studies have reported anti-inflammatory and immunosuppressive effect of emodin [46]. Emodin also has been shown to possess cardioprotective activities via anti-apoptotic mechanisms [47]. Song et al. revealed that emodin treatment significantly improved LV function and reduced the severity of myocarditis in EAM rats, as evidenced by echocardiographic and histopathological studies. Emodin treatment decreased the serum levels of proinflammatory cytokines TNF-α and IL-1β. Moreover, NF-κBp65, a rapid-response transcription factor that regulates proinflammatory cytokines, in the myocardial tissue was also suppressed in emodin treated rats [48]. Oleanolic acid is a naturally occurring triterpene which is widely distributed in plants such as olive oil. Oleanolic acid possesses numerous cardiovascular activities including anti-arrhythmic, immunomodulatory, antihyperlipidemic, vasodilatory, antiinflammatory, and antioxidant activities [4952]. Martín et al investigated the effects of prophylactic and therapeutic administration of oleanolic acid in EAM mice. Oleanolic acid could dramatically decrease disease severity: HW/BW ratios as well as plasma levels of brain natriuretic peptide and myosin-specific autoantibodies (anti-MyHCα) production were significantly reduced in oleanolic acid-treated EAM mice, compared with untreated ones. Histological heart analysis showed that oleanolic acid-treatment decreased cellular infiltration, fibrosis and myocardial calcifications. Oleanolic acid could also diminish proliferation of cardiac fibroblast in vitro and reduced cytokine-induced calcium and collagen deposits in active EAM. Furthermore, treatment of EAM animals with oleanolic acid was associated with a significantly elevated number of T regulatory cells (Treg cells), increased IL-10 and IL-35 production, and reduced secretion of proinflammatory and profibrotic cytokines. Therefore, enhancement of an antiinflammatory cytokine 6

response and inhibition of anti-MyHCα production are cardioprotective actions whereby oleanolic acid can improve EAM [53]. Quercetin is a bioactive flavonoids that occurs in several edible plants, and has several cardioprotective properties including anti-oxidant, antihypertensive, anti-inflammatory, and anti-coagulant activities together with protective action against myocardial ischemia/reperfusion (IR) injury. [54-56]. Quercetin (20 mg/kg) has been shown to significantly decrease the incidence of autoimmune myocarditis, HW/BW and macroscopic and microscopic scores of hearts in EAM rats. Further, levels of TNF-α and IL-17 were significantly lower in EAM rats treated with quercetin, while the level of IL-10 in serum and also in supernatants of lymph node cells was significantly higher in EAM rats treated with quercetin compared with untreated control rats [57]. Arumugam et al. performed an experiment to study the protective effects of quercetin (10 mg/kg; p.o.) in the progression of EAM to DCM in EAM rats. The results showed the protective effect of quercetin against cardiac remodeling and elevated endoplasmic reticulum (ER). Moreover, quercetin was found to preserve myocardial dimensions and cardiac function in rats. Quercetin treatment in rats was also associated with the modulation of MAPK signaling pathway as shown by down-regulation of myocardial endothelin-1 and mitogen-activated protein kinases (MAPK) [16]. Resveratrol is a natural stilbenoid found in grapes and wine and is reported to have cardioprotective and immunomodulatory effects [58-60]. Yoshida et al. examined the effect of pretreatment of myosin immunized rats with resveratrol (50mg/kg per day) on EAM in rats. Resveratrol could preserve cardiac function of rats according to echocardiographic analysis. Resveratrol attenuated the increased heart weight/tibial length (HW/TL) ratio which was observed in control rats (1.8-fold higher than unimmunized rats). Resveratrol significantly decreased cellular infiltration, fibrosis, and expression of inflammatory cytokines in the myocardium. Resveratrol decreased the elevated expression of antioxidant genes (MnSOD and Cu/Zn-SOD) and attenuated myocarditis. Silent mating type information regulation 2 homolog 1 (SIRT1) protein, a potential effector of resveratrol, was increased in the myocardium of control EAM rats but not in resveratrol treated rats. The SIRT1 was localized mainly in infiltrating mononuclear cells. Thus, resveratrol possibly limits lymphocyte proliferation by modulating the activity of SIRT1 in immune cells [61]. Morus alba L. commonly known as white mulberry is a small to medium-sized tree belonging to Moraceae. Mulberry leaves are commonly used to feed silkworm larvae [62]. Mulberry leaves have been reported to exert a wide spectrum of pharmacological activities including antioxidant, antidiabetic, anti-inflammatory, analgesic, antipyretic arterial pressure restoration, anti-atherosclerosis and hypolipidemic activities [62-65]. Arumugam et al. reported that EAM rats receiving a diet supplemented with 5% w/w of mulberry leaves powder exhibited a significant attenuation in expression of the myocardial p22phox (an essential subunit of the oxidative stress mediating enzyme NADPH-oxidase) level indicating modulation of oxidative stress caused by NADPH oxidase [6]. Advanced glycation end products (AGEs) are produced by oxidation of cellular lipids and proteins and can be a factor of aging and development of degenerative disease. The receptor for advanced glycation endproducts (RAGE) can bind to AGEs resulting in age-related chronic inflammatory diseases as evidenced by enhanced level of RAGE ligands in these disorders [66]. The expression of RAGE was significantly suppressed in the 7

mulberry leaves treated rats suggesting that mulberry leaves can attenuate cardiac damage induced by oxidative stress following EAM [6]. The expression of phospho-p38 MAPK and phospho-JNK (enzymes responsive to stress stimuli) which were significantly increased in the EAM rats were decreased in mulberry leaves treated rats indicating a protection from the cardiac damage induced by oxidative stress [6]. Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA2) is a Ca2+ ATPase which resides within myocytes and transfers Ca2+ from the cytosol of the cells to the lumen of the sarcoplasmic reticulum. The myocardial level of SERCA2 has been shown to decrease in EAM animals. SERCA2 and osteopontin (OPN; a member of the matricellular protein family whose expression increases in myocardial dysfunction [67]) levels were also found to be normalized in the mulberry leaves treated rats suggesting that calcium movement in the cardiac cells can be handled properly to maintain normal cardiac function. The mulberry leaves also significantly decreased cellular infiltration, myocardial fibrotic remodeling, mast cell density, phospho-c-Jun NH2-terminal protein kinase, glucose regulated protein and caspase levels in EAM rats [6]. It improved the LV ejection fraction (EF) and fractional shortening (FS) as evidenced by echocardiographic studies. The myocardial levels of endothelin-1, activated members of MAPK pathway, and vascular endothelial growth factor (VEGF) were also attenuated by mulberry leaves [68]. These results may suggest that mulberry leaves can preserve the cardiac function in EAM by modulating MAPK activation induced by oxidative stress and further protects against ER stress mediated apoptosis as well as cardiac hypertrophy [6]. Phytochemical analysis has reported the presence of large amounts of quercetin in mulberry leaves [69]. This suggests the observed anti-myocarditis activities of mulberry leaves are possibly attributed to its quercetin content. Cacao beans (Theobroma cacao L.) are fruits of an evergreen tree from Sterculiaceae, native to the tropical regions of Central and South America. Today, cocoa products are consumed worldwide and their polyphenolic constituents especially flavan-3-ols or catechins ((−)-epicatechin, (+)-catechin, (+)gallocatechin, and (−)-epigallocatechin) which are abundant in green tea, cocoa and many other plants have been found to possess antioxidant and antiradical properties. Moreover, catechins have many cardioprotective activities; including anti-inflammatory, anti-oxidative, anti-atherosclerosis, anti-obesity, anti-hypercholesterolemia and antihyperglycemia [9, 70, 71]. Chocolate and cocoa products also have been shown to lower the risk of coronary heart disease mortality, decrease insulin resistance and exhibit blood pressure-lowering, antihyperdipidemic, anti-inflammatory and anti-platelet aggregation effects in humans [72]. Zempo et al. reported that cocoa/chocolate polyphenols extract significantly suppressed the elevation of HW/BW and fibrotic area ratios and decreased cardiac cell infiltration in EAM mice. Reverse transcriptase-PCR revealed that mRNA expressions of IL-1β, IL-6, E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and collagen type 1 were lower in the cocoa group compared with the control group. Moreover, cacao treatment significantly decreased cardiac H2O2 concentrations. Cardiac myeloperoxidase (MPO) activity, the intensity of dihydroethidium (DHE) staining and the levels of phosphorylated NF-κB p65 were also lower in the cacao group compared with the control group [73]. In addition, Suzuki et al. showed that tea catechins significantly improved cardiac function in EAM rats compared to control. Pathologically, tea catechins-treated EAM hearts showed significantly less myocardial cell infiltration, fibrosis area and ventricular remodeling. Immunohistochemistry revealed that enhanced expression of NF-κB and ICAM-1 was suppressed by catechin administration. Further, 8

mRNA levels of TNF-α were decreased and Th2 cytokines were markedly enhanced in tea catechinstreated rats compared with the control group [74]. Conventional and investigational treatment options of myocarditis There is still no approved medicine for the treatment of myocarditis. However, many forms of myocarditis are treated symptomatically. The main principles of treatment are management of arrhythmia and heart failure and aetiology-targeted therapy [75]. Specific types of autoimmune myocarditis e.g. giant cell myocarditis are treated with a combination of immunosuppressant and other agents (cyclosporine and corticosteroids with or without azathioprine or muronomab-CDs). This therapeutic strategy may offer a median survival time of 12 months compared with 3 months for untreated patients. However, mechanical circulatory support or heart transplantation may be required in some patients within 1 year. Moreover, Withdrawal of immunosuppression can lead to the recurrence of symptoms and even fatal giant cell myocarditis [76]. Favorable response to immunosuppressive agents (cyclosporine, prednisolone and azathioprine) is reported mainly in virusnegative myocarditis, giant cell myocarditis, and autoimmun myocarditis [75]. Currently, several agents are under investigation to treat autoimmune myocarditis. Various anticardiac antibodies have been detected in myocarditis patients. Immunoadsorption can be employed to neutralize these autoantibodies. In a large cohort of unselected patients, 48% of inflammatory DCM patients undergoing immunoadsorption therapy positively responded to the treatment, and diseaserelated clinical and echocardiographic parameters were significantly improved. However, this treatment option is invasive (17.2% of patients experienced complications) and expensive. Moreover, further studies are needed to identify target myocarditis patients who might best benefit from this treatment [77]. Owing to the paucity of available treatment options for autoimmune myocarditis, naturally occurring agents could serve as interesting candidates should the results of clinical trials are supportive. However, the efficacy of discussed natural products has not yet been directly compared with Western investigational drugs mentioned above. Such comparative studies would shed light on the place of natural products in the management of autoimmune myocarditis with respect to efficacy, safety and cost-effectiveness. Discussion Based on the cardiac myosin-induced EAM studies in rodents, a number of plant extracts and phytochemicals have been found to serve as possible candidates for future studies on myocarditis (Figure 2). These compounds could suppress the infiltration of inflammatory cells and protect from myocardial damage and remodeling by a number of anti-inflammatory and immunomodulatory mechanisms. Upregulation of serum levels of the Th1-type cytokines and elevation of the Th2-type cytokines (IL-4 and IL-10) [1], decreasing serum levels of proinflammatory cytokines TNF-α, IL-1β and IL6, suppressing NF-κBp65 which regulates proinflammatory cytokines [48], restoring the excessive expression of phosphorylated (p)-STAT1, STAT3 and STAT4 and further suppressing Th17/Th1 responses 9

[1], suppressing ICAM-1, VCAM-1, E-selectin, and collagen type 1 expression [45, 73], reducing antiMyHCα, increasing the number of Treg cells [53], modulation of MAPK signaling pathway [16] and the activity of SIRT1 [61], alleviating oxidative stress caused by NADPH oxidase, suppressing the expression of RAGE, phospho-p38 MAPK and phospho-JNK, osteopontin and increasing SERCA2 levels are among the most important mechanisms against myocardial damage and remodeling [6]. In our study, polyphenols especially flavonoids and catechins, were found to be the dominating compounds with EAM-alleviating activities. These compounds (quercetin, apigenin and (−)-epicatechin) are well-known for their anti-oxidative stress, anti-inflammation and anti-apoptotic activities [54, 78]. Flavonoids are aromatic compounds containing two phenyl rings (A and B) and a heterocyclic ring (C) (Figure 3). The flavonoids are hydrophobic compounds and increased level of hydroxylation can enhance their water-solubility. The solubility of the flavonoids affects their free radical scavenging ability due to phase partitioning [79]. As mentioned before, recent evidence suggests that oxidative stress and myocardial apoptosis play important roles in the progression of EAM [6]. Therefore, it appears that the more hydroxyl group a flavonoid possesses, the stronger its antioxidant activity will be. Flavonoids have also been shown to possess cardioprotective effects in experimental myocardial ischemic injury through a variety of mechanisms including protecting mitochondrial function, improving the action of PPARγ receptor signaling, repressing inflammatory cascade and inhibiting apoptosis and PI3K/Akt pathway [54, 78, 80]. In fact, all anti-EAM compounds studied in the present article were found to be effective in alleviating and preventing experimental myocardial injury in animals suggesting their multidimensional cardioprotective properties [52, 81-85]. Catechins are polyphenolic compounds with numerous important cardiovascular activities such as regulation of lipid metabolism, protection against vascular endothelium and attenuation of hypertension [86]. These phytochemicals which are major compounds of abundantly consumed beverages, tea and cacao have been found to improve cardiac function in EAM animals [74]. Catechins contain two or more aromatic rings (A and B) connected with a carbon bridge, and a dihydropyran heterocycle (C ring) with a hydroxyl group on carbon 3. Aromatic rings A and B possess multiple aromatic hydroxyl groups which increase their hydrophilicity (Figure 4) [87]. It seems that flavonoid and catechin backbones bearing hydroxyl groups are crucial for their multiple effects on myocarditis. Interestingly, other compounds with high degrees of similarity to catechins and flavonoids like berberine, resveratrol and emodin also exhibited remarkable anti-EAM activities. Even curcumin with lower degrees of similarity with these compounds has been shown to exert satisfactory anti-myocarditis properties. This suggests the presence of similar enzymatic targets for these molecules which should be elucidated by future studies delving deeper into the structure-activity relationship along with molecular modeling for these compounds. Future studies are also warranted to examine anti-EAM properties of molecules similar to the mentioned compounds in order to discover and develop new bioactive pharmaceuticals for the treatment of myocarditis. Conclusions Considering the natural molecules discussed in the present article in future confirmatory animal studies and clinical trials would shed more light on their effectiveness and safety in the treatment of myocarditis 10

and preventing DCM. Additionally, the presence of structural similarity between these compounds makes it incumbent to screen the anti-EAM effects of other similar phytochemicals or synthetic derivatives designed based on the naturally occurring prototype structure.

Conflict of interest The author confirms that this article content has no conflict of interest.

Acknowledgements The author is thankful to the Mashhad University of Medical Sciences Research Council for providing access to the bibliographic databases and full texts of articles used in this review.

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Figure legends Fig. 1. Chemical structures of natural products with anti-EAC activities. Fig. 2. Pharmacological mechanisms for the therapeutic effects of natural products in autoimmune myocarditis. Activated T cells (CD4+ and CD8+) secrete chemokines and cytokines which in turn activate other inflammatory cells, such as mast cells, macrophages, and neutrophils. Cardiac inflammation and fibrosis take place as a result of the proinflammatory cytokines secretion from these cells. Inflammatory responses can be limited and terminated by IL-10. IL-10 binds to specific IL-10 receptors located on mast cells to prevent the release of inflammatory mediators. IL-10-Ig-containing medium inhibits IL-17 gene expression in IL-1-stimulated spleen cells to restore excessive increase of Th17/Th1 responses in EAM. Signal transducer and activator of transcription (STAT) proteins modulate the expression of a variety of target genes through binding to sequence-specific DNA elements. In EAM, modulating the activities of STATs can lead to further suppression of Th17 and Th1 cell differentiation. Natural products can alleviate EAM mainly through modulating the mentioned pathways. Fig. 3. Chemical structure of flavonoids backbone. Fig. 4. Chemical structure of catechins backbone.

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