Oleanolic acid modulates the immune-inflammatory response in mice with experimental autoimmune myocarditis and protects from cardiac injury. Therapeutic implications for the human disease

Highlights

• OA ameliorates developing and established experimental autoimmune myocarditis (EAM).

• OA reduces auto-antibodies and harmful proinflammatory/profibrotic cytokines levels.

• OA increases the number of Treg cells in EAM.

• OA enhances immunoregulatory molecules and promotes an antiinflammatory environment.

• OA exerts a direct and pleiotropic protective role in cardiac cells.

Abstract

Myocarditis and dilated cardiomyopathy (DCM) are inflammatory diseases of the myocardium, for which appropriate treatment remains a major clinical challenge. Oleanolic acid (OA), a natural triterpene widely distributed in food and medicinal plants, possesses a large range of biological effects with beneficial properties for health and disease prevention. Several experimental approaches have shown its cardioprotective actions, and OA has recently been proven effective for treating Th1 cell-mediated inflammatory diseases; however, its effect on inflammatory heart disorders, including myocarditis, has not yet been addressed. Therefore, the present study was undertaken to determine the effectiveness of OA in prevention and treatment of experimental autoimmune myocarditis (EAM). The utility of OA was evaluated in vivo through their administration to cardiac α-myosin (MyHc-α_614–629)-immunized BALB/c mice from day 0 or day 21 post-immunization to the end of the experiment, and in vitro through their addition to stimulated-cardiac cells. Prophylactic and therapeutic administration of OA dramatically decreased disease severity: the heart weight/body weight ratio as well as plasma levels of brain natriuretic peptide and myosin-specific autoantibodies production were significantly reduced in OA-treated EAM animals, compared with untreated ones. Histological heart analysis showed that OA-treatment diminished cell infiltration, fibrosis and dystrophic calcifications. OA also decreased proliferation of cardiac fibroblast in vitro and attenuated calcium and collagen deposition induced by relevant cytokines of active myocarditis. Furthermore, in OA-treated EAM mice the number of Treg cells and the production of IL-10 and IL-35 were markedly increased, while proinflammatory and profibrotic cytokines were significantly reduced. We demonstrate that OA ameliorates both developing and established EAM by promoting an antiinflammatory cytokine profile and by interfering with the generation of cardiac-specific autoantibodies, as well as through direct protective effects on cardiac cells. Therefore, we envision this natural product as novel helpful tool for intervention in inflammatory cardiomyopathies including myocarditis.

Introduction

Myocarditis, an inflammatory disease of the heart, is a precursor of dilated cardiomyopathy (DCM) and represents the most common cause of chronic heart failure, or even sudden death, in people younger than 40 years of age [1], [2], [3]. The pathogenesis of myocarditis is not fully understood but there is substantial evidence suggesting that the autoimmune response to heart antigens, particularly cardiac myosin, after viral infection may contribute to the disease process [4], [5]. Interestingly, patients with heart-specific autoantibodies but no evidence of viral genome persistence show improvement in their cardiac function on immunosuppressive therapy, thus pointing to autoimmunity as a key factor in the pathogenesis of postinflammatory cardiomyopathy [6], [7].

Experimental autoimmune myocarditis (EAM) is an animal model for human fulminant myocarditis in the acute phase, which develops into postmyocarditic DCM in the chronic phase. EAM has been produced in susceptible rodent animals by immunization with cardiac specific peptides [8], [9]. In the myosin-induced myocarditis model, pathogenic roles for macrophages, CD4⁺ T cells and autoantibodies have been described, and proinflammatory cytokines and chemokines are upregulated during the course of the autoimmune response [10].

At present, therapeutic strategies in myocarditis have focussed on the different stages of the disease, targeting the elimination of the infecting agent, inhibition of the heart-specific autoimmune response, as well as reduction of inflammation and inflammatory-associated tissue remodeling. Toxicity, limited efficacy and expense, however, are serious disadvantages of the new anti-inflammatory and/or immunosuppressive agents. Hence, the identification of novel molecules with low toxicity is a very useful challenge for the treatment of myocarditis and DCM.

Along these lines, there is considerable interest in bioactive compounds present in edible plants and thus in “letting your food be your medicine”, as recommended by Hippocrates. Triterpenoids stand out amongst these bioactive plant constituents that are being actively studied for their beneficial effects on human health. Oleanolic acid (OA) is a naturally occurring triterpene that possesses numerous biological activities and displays many pharmacological properties with therapeutic potential [11]. OA is widely present in edible and medicinal plants, being an integral part of the human diet, and is extensively used in Asian medicine, usually in the form of herbal extracts [12]. Among other plant species, OA can be extracted from olive leaves and roots (Olea Europaea), a plant that has been widely utilized in folk medicine of African and European Mediterranean countries. In addition, it is present in grapes and olives, a substantial component of the Mediterranean diet, and particularly considerable amounts are found in grape- and olive pomace. So, to fully characterize their health-related properties, it could be both interesting for drug development and clinical studies, as well as for nutraceutical studies. At present, OA, as well as herbal extracts containing OA, are well known for their anti-inflammatory, immunomodulatory and antioxidative properties, and its effectiveness in the treatment of some pathological conditions has been extensively documented [12], [13], [14], [15], [16], [17], [18]. However, the potential beneficial actions of OA on cardiac damage, in the context of inflammatory heart disease, remain speculative and have not been addressed. In the present study, we tested the hypothesis that OA can act as a potent anti-inflammatory/immunoregulatory drug in myocarditis.