Sesquiterpene Lactone Potentiates the Immunomodulatory, Antiparasitic and Cardioprotective Effects on Anti-Trypanosoma Cruzi Specific Chemotherapy T
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International Immunopharmacology 77 (2019) 105961 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Sesquiterpene lactone potentiates the immunomodulatory, antiparasitic and cardioprotective effects on anti-Trypanosoma cruzi specific chemotherapy T Elda Gonçalves-Santosa, Diego F. Vilas-Boasa, Lívia F. Diniza, Marcia P. Velosob, Ana L. Mazzetia, Maria R. Rodriguesb, Carla M. Oliveirab, Victor Hugo C. Fernandesd, Rômulo D. Novaesc, ⁎ Daniela A. Chagas-Paulad, Ivo S. Caldasa, a Institute of Biomedical Sciences, Department of Pathology and Parasitology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil b Pharmaceutical Sciences Institute, Federal University of Alfenas, Alfenas 37130-001, MG, Brazil c Institute of Biomedical Sciences, Department of Structural Biology, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil d Chemistry Institute, Federal University of Alfenas, 37130-001 Alfenas, MG, Brazil ARTICLE INFO ABSTRACT Keywords: We investigated the immunomodulatory, antiparasitic and cardioprotective effects of a sesquiterpene lactone Chagas’s disease (SL) administered alone or combined with benznidazole (Bz), in a murine model of Chagas’ disease by in vitro Benznidazole and in vivo assays. Antiparasitic and cytotoxic potential of tagitinin C (SL) and Bz were tested in vitro against T. Infectious myocarditis cruzi epimastigotes and cardiomyocytes. Swiss mice challenged with T. cruzi were also treated for 20 days with Sesquiterpene lactone tagitinin C (10 mg/kg) alone and combined with Bz (100 mg/kg). Tagitinin C exhibited a higher antiparasitic Tagitinin C (IC50: 1.15 µM) and cytotoxic (CC50 at 6.54 µM) potential than Bz (IC50: 35.81 µM and CC50: 713.5 µM, re- spectively). When combined, these drugs presented an addictive interaction, determining complete suppression of parasitemia and parasitological cure in all infected mice (100%) compared to those receiving Bz alone (70%). Anti-T. cruzi immunoglobulin G, and pro-inflammatory cytokines IFN-γ and TNF-α levels were reduced in ani- mals treated with tagitinin C combined with Bz, while IL-10 production was unaffected. Heart inflammation was undetectable in 90% of the animals receiving this combination, while only 50% of the animals receiving Bz alone showed no evidence of myocarditis. Together, our findings indicated that the combination of tagitinin C and Bz exerts potent antiparasitic, immunomodulatory and cardioprotective effects. Due to the remarkable suppression of parasitemia and high parasitological cure, this combination was superior to Bz monotherapy, indicating a high potential for the treatment of Chagas’s disease. 1. Introduction Thus, eliminating the parasite and adjusting the immune response are the two pillars of Chagas’s disease treatment [7,8]. Currently, this Chagas’s disease is a neglected infection linked to poverty and treatment is limited to benznidazole and nifurtimox, whose anti- caused by the parasite protozoan Trypanosoma cruzi [1]. About 6 mil- parasitic and immunomodulatory potential are more effective in acute lion people are infected by this parasite and at least 50,000 cases of infections [9,10]. However, these drugs do not guarantee cure even in annual deaths are reported worldwide, especially in Latin American the acute phase of infections [11–14], and the therapeutic failure is countries, where the disease is endemic [2,3]. Most of the fatal cases are even more frequent in the chronic phase of Chagas’s disease [15].In consequence of a severe infectious cardiomyopathy associated with addition, both references drugs require prolonged administration [16]. intense and unbalanced Th1 immunological polarization, as well as, Because of this, they induce dose-dependent systemic toxicity and ser- immunomediated (i.e., autoimmunity) and reactive myocardial da- ious side effects (i.e., vomiting, anorexia, hepatotoxicity, bone marrow mage; which courses with cardiomyocytolysis, myonecrosis, heart fi- depression, dermatitis, polyneuropathy), which determined about 60% brosis, electromechanical dysfunction and cardiac failure [4,5]. of treatment discontinuation [17,18]. Thus, the limited pharmacolo- It is well established that the direct destruction of the host cells by gical efficiency, drug toxicity and low adherence to the chemotherapy the parasite, and the secondary immunomediated lesions of the target protocol are recognized as direct causes of therapeutic failure for both organs are the most serious pathological events of Chagas’s disease [6]. and unique available references drugs [15,19]. ⁎ Corresponding author at: Department of Parasitology and Pathology, Federal University of Alfenas, Alfenas, Minas Gerais 37130-001, Brazil. E-mail address: [email protected] (I.S. Caldas). https://doi.org/10.1016/j.intimp.2019.105961 Received 18 September 2019; Received in revised form 1 October 2019; Accepted 2 October 2019 Available online 01 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved. E. Gonçalves-Santos, et al. International Immunopharmacology 77 (2019) 105961 The high prevalence, morbidity, mortality, increasing of infection in both was applied: 5:0, 4:1, 3:2, 2:3, 1:4 and 0:5. A volume of 150 μL/ non-endemic countries, and money spent with the prevention of well of each dilution was added to a 96-well sterile polystyrene plate. Chagas’s disease worldwide, indicate the need and urgency for new and Negative controls (containing LIT medium), positive controls (con- effective therapeutic strategies against T. cruzi infection [20,21]. In this taining the LIT medium and the parasite) and controls containing the sense, active molecules derived from natural products with potent im- medium and drug in the absence of the parasite were added to evaluate munomodulatory and anti-T. cruzi activities has been reported in vitro the potential of the compound to reduce the dye. After incubation for [22]. However, their biotechnological, antiparasitic and im- 72 h in a BOD oven, at 20 °C, 20 μL of resazurin was added; after further munobiological potential in vivo remains poor explored for the treat- incubation for 12 h, the reaction was read on a spectrophotometer at ment of the Chagas’s disease. 570 nm and 600 nm. From the percent inhibition of each concentration Sesquiterpene lactones (SL) are a chemical group of molecules with of the substances. The IC50 and IC50 FICs were calculated with the aid of marked pharmacological potential due to a broad spectrum of bioactive the CalcuSyn program, and dose-response curves were constructed in effects, especially potent anti-inflammatory, analgesic, anti-microbial, Graph Pad Prism 5.0. anti-diabetic, and anti-parasitic activities [23,24]. In vitro evidence suggests that the trypanocidal effect of SL is potentially mediated by the 2.3.2. Cytotoxicity assay inhibition of trypanothione reductase, an enzyme essential to protect T. Cells from the H9c2 line (American Type Culture Collection, ATCC: cruzi against molecular oxidative damage and death [25]. Sesqui- CRL 1446) from neonatal rat cardiomyoblasts were cultured at 37 °C terpene lactones also exerts potent anti-inflammatory effects [26], de- and 5% CO2 in DMEM medium (NaHCO3, HEPES and Penicillin/ sirable properties in anti-T. cruzi drugs, since the sustained pro-in- Streptomycin) supplemented with 10% FBS. The concentration of 1x103 flammatory response is a major cause of excessive or uncontrolled cells/mL was added in duplicate in a 96-well plate and after incubation inflammation, heart damage and death in patients with Chagas’s dis- for 24 h at 37 °C, 5% CO2, the medium was replaced with 200 μL/well ease [27,28]. of a new medium containing seven different concentrations of the drug As SL combine antiparasitic and anti-inflammatory potential, we Bz and substance TC (1:2 dilution starting at the concentration of used and in vitro and in vivo integrated approach to investigate the 200 μg/mL). Negative controls (medium) and positive controls immunomodulatory, antiparasitic, cytotoxic and cardioprotective po- (medium + cells) were used in each plate. After 72 h of incubation, tential of the SL, tagitinin C (TC), administered alone or combined with 20 μL/well of resazurin was added and after 12 h the reading was car- benznidazole (Bz), a pro-oxidant drug used as a first-line treatment for ried out using a spectrophotometer at an absorbance of 570 nm and Chagas’s disease. 600 nm. After calculating the percent inhibition of each drug con- centration and of the substances tested, the Graph Pad Prism program 2. Materials and methods 5.0 was used to calculate CC50 (concentration of substances and drug reducing 50% of cell viability). 2.1. Parasite strain and benznidazole 2.4. T. cruzi infection and chemotherapy strategy in vivo The T. cruzi Y strain (DTU II), which is well characterized as par- tially sensitive to experimental chemotherapy with Bz [29], was used in 2.4.1. Animals and infection this study. The drug benznidazole (Bz) (compound N-benzyl-2- (2-ni- Five groups of 10 female Swiss mice were used, weighing approxi- troimidazole) acetamide) was supplied by the Pharmaceutical Labora- mately 37 g, from the Central Biotherm of the Federal University of tory of Pernambuco – LAFEPE (Recife, Pernambuco, Brazil). Alfenas (UNIFAL-MG). Four groups of animals were inoculated in- traperitoneally with 1000 blood trypomastigotes obtained from pre- 2.2. Plant material and tagitinin C viously infected animals. Parasitemia and parasitic load were used and primary markers of infection [32].