RESUMO
The deficit of effective treatments for Chagas disease has led to searching for new substances with therapeutic potential. Natural products possess a wide variety of chemical structural motifs and are thus a valuable source of diverse lead compounds for the development of new drugs. Castanedia santamartensis is endemic to Colombia, and local indigenous communities often use it to treat skin sores from leishmaniasis; however, its mechanism of action against the infective form of Trypanosoma cruzi has not been determined. Thus, we performed chemical and biological studies of two alcoholic leaf extracts of C. santamartensis to identify their active fractions and relate them to a trypanocidal effect and evaluate their mechanism of action. Alcoholic extracts were obtained through cold maceration at room temperature and fractionated using classical column chromatography. Both ethanolic and methanolic extracts displayed activity against T. cruzi. Chemical studies revealed that kaurenoic acid was the major component of one fraction of the methanolic extract and two fractions of the ethanolic extract of C. santamartensis leaves. Moreover, caryophyllene oxide, kaurenol, taraxasterol acetate, pentadecanone, and methyl and ethyl esters of palmitate, as well as a group of phenolic compounds, including ferulic acid, caffeic acid, chlorogenic acid, myricetin, quercitrin, and cryptochlorogenic acid were identified in the most active fractions. Kaurenoic acid and the most active fractions CS400 and CS402 collapsed the mitochondrial membrane potential in trypomastigotes, demonstrating for the first time the likely mechanism against T. cruzi, probably due to interactions with other components of the fractions.
Assuntos
Asteraceae , Extratos Vegetais/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Diterpenos/química , Mitocôndrias/efeitos dos fármacos , Extratos Vegetais/química , Folhas de PlantaRESUMO
Introduction: Chronic Chagasic cardiomyopathy (CCC), caused by the protozoan Trypanosoma cruzi, is the most severe manifestation of Chagas disease.CCC is characterized by cardiac inflammation and fibrosis caused by a persistent inflammatory response. Following infection, macrophages secrete inflammatory mediators such as IL-1ß, IL-6, and TNF-α to control parasitemia. Although this response contains parasite infection, it causes damage to the heart tissue. Thus, the use of immunomodulators is a rational alternative to CCC. Rho-associated kinase (ROCK) 1 and 2 are RhoA-activated serine/threonine kinases that regulate the actomyosin cytoskeleton. Both ROCKs have been implicated in the polarization of macrophages towards an M1 (pro-inflammatory) phenotype. Statins are FDA-approved lipid-lowering drugs that reduce RhoA signaling by inhibiting geranylgeranyl pyrophosphate (GGPP) synthesis. This work aims to identify the effect of statins on U937 macrophage polarization and cardiac tissue inflammation and its relationship with ROCK activity during T. cruzi infection. Methods: PMA-induced, wild-type, GFP-, CA-ROCK1- and CA-ROCK2-expressing U937 macrophages were incubated with atorvastatin, or the inhibitors Y-27632, JSH-23, TAK-242, or C3 exoenzyme incubated with or without T. cruzi trypomastigotes for 30 min to evaluate the activity of ROCK and the M1 and M2 cytokine expression and secretion profiling. Also, ROCK activity was determined in T. cruzi-infected, BALB/c mice hearts. Results: In this study, we demonstrate for the first time in macrophages that incubation with T. cruzi leads to ROCK activation via the TLR4 pathway, which triggers NF-κB activation. Inhibition of ROCKs by Y-27632 prevents NF-κB activation and the expression and secretion of M1 markers, as does treatment with atorvastatin. Furthermore, we show that the effect of atorvastatin on the NF-kB pathway and cytokine secretion is mediated by ROCK. Finally, statin treatment decreased ROCK activation and expression, and the pro-inflammatory cytokine production, promoting anti-inflammatory cytokine expression in chronic chagasic mice hearts. Conclusion: These results suggest that the statin modulation of the inflammatory response due to ROCK inhibition is a potential pharmacological strategy to prevent cardiac inflammation in CCC.
Assuntos
Cardiomiopatias , Doença de Chagas , Inibidores de Hidroximetilglutaril-CoA Redutases , Trypanosoma cruzi , Humanos , Animais , Camundongos , Trypanosoma cruzi/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Quinases Associadas a rho/metabolismo , NF-kappa B/metabolismo , Atorvastatina/farmacologia , Células U937 , Macrófagos/metabolismo , Doença de Chagas/genética , Citocinas/metabolismo , Cardiomiopatias/metabolismo , Inflamação/metabolismoRESUMO
BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America and is widely distributed worldwide because of migration. In 30% of cases, after years of infection and in the absence of treatment, the disease progresses from an acute asymptomatic phase to a chronic inflammatory cardiomyopathy, leading to heart failure and death. An inadequate balance in the inflammatory response is involved in the progression of chronic Chagas cardiomyopathy. Current therapeutic strategies cannot prevent or reverse the heart damage caused by the parasite. Aspirin-triggered resolvin D1 (AT-RvD1) is a pro-resolving mediator of inflammation that acts through N-formyl peptide receptor 2 (FPR2). AT-RvD1 participates in the modification of cytokine production, inhibition of leukocyte recruitment and efferocytosis, macrophage switching to a nonphlogistic phenotype, and the promotion of healing, thus restoring organ function. In the present study, AT-RvD1 is proposed as a potential therapeutic agent to regulate the pro-inflammatory state during the early chronic phase of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 wild-type and FPR2 knock-out mice chronically infected with T. cruzi were treated for 20 days with 5 µg/kg/day AT-RvD1, 30 mg/kg/day benznidazole, or the combination of 5 µg/kg/day AT-RvD1 and 5 mg/kg/day benznidazole. At the end of treatment, changes in immune response, cardiac tissue damage, and parasite load were evaluated. The administration of AT-RvD1 in the early chronic phase of T. cruzi infection regulated the inflammatory response both at the systemic level and in the cardiac tissue, and it reduced cellular infiltrates, cardiomyocyte hypertrophy, fibrosis, and the parasite load in the heart tissue. CONCLUSIONS/SIGNIFICANCE: AT-RvD1 was shown to be an attractive therapeutic due to its regulatory effect on the inflammatory response at the cardiac level and its ability to reduce the parasite load during early chronic T. cruzi infection, thereby preventing the chronic cardiac damage induced by the parasite.
Assuntos
Cardiomiopatia Chagásica/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/administração & dosagem , Animais , Cardiomiopatia Chagásica/genética , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Doença Crônica/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Coração/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/imunologia , Nitroimidazóis/administração & dosagem , Carga Parasitária , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/imunologia , Trypanosoma cruzi/fisiologiaRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, endemic in Latin America but distributed worldwide because of migration. Without appropriate treatment, the disease progresses from an acute asymptomatic phase to a chronic, progressive inflammatory cardiomyopathy causing heart failure and death. Despite specific trypanocidal therapy, heart damage progression cannot be stopped or reversed. Statins, as part of their pleiotropic actions, can modulate chagasic myocarditis by inducing the production of 15-epi-lipoxin A4 (15-epi-LXA4), a proresolution lipid mediator in inflammation. Furthermore, several reports suggest that simvastatin activates the Notch pathway after stroke in cerebral endothelial cells, enhancing blood flow by promoting angiogenesis. Thus, statins are an attractive therapeutic strategy for modulating the Notch pathway to reverse the chronic heart damage induced by T. cruzi BALB/c mice chronically infected with T. cruzi were treated with 1 mg/kg/day simvastatin or 25 µg/kg/day 15-epi-LXA4 for 20 days. During the treatment period, cardiac function was evaluated by echocardiography. At 80 days postinfection, the heart tissues were assessed for Notch 1 activity. T. cruzi infection activated the Notch 1 pathway, and simvastatin (but not 15-epi-lipoxin A4) produced a further increase in that activity, correlating with improvement in the ejection fraction and histopathologic findings typical of T. cruzi infection, including improvements in inflammation and fibrosis. Moreover, simvastatin increased the number of isolectin B4-positive cells, suggesting active angiogenesis in the chronically infected hearts without alteration of the parasitic load. Simvastatin, probably acting through the Notch 1 pathway, decreases inflammation, improving cardiac function in mice chronically infected with T. cruzi.
Assuntos
Cardiomiopatia Chagásica , Doença de Chagas , Trypanosoma cruzi , Animais , Cardiomiopatia Chagásica/tratamento farmacológico , Células Endoteliais , Camundongos , Camundongos Endogâmicos BALB C , Sinvastatina/farmacologia , Sinvastatina/uso terapêuticoRESUMO
Since cancer cells have different mitochondrial bioenergetic requirements than non-cancerous cells, therapeutic inhibition of its mitochondrial functionality continues to be an important target for anticancer drug discovery. In this study, a series of acylhydroquinones with different acyl-chain length, and their chlorinated derivatives, in the aromatic ring, synthesized by Fries rearrangement under microwave irradiation, were evaluated for their anticancer activity in two leukemia cell lines. Findings from the primary and secondary screening of the 18 acylhydroquinones, tested at 5 µM on acute promyelocytic leukemia HL-60 and acute lymphoblastic leukemia CEM cells lines, identified an acylchlorohydroquinone (12) with a highly selective anti-proliferative effect toward HL-60 cells. This compound induced S-phase arrest in the cell cycle progression of HL-60 cells with insignificant toxicity on leukemic CEM cells and non-cancerous Hs27 cells. In HL-60 leukemic cells, 12 triggered increased mitochondrial NADH oxidation, increased respiration in presence of oligomycin (state 4o), mitochondrial depolarization, and ROS production, suggesting an uncoupling of OXPHOS. This provoked a metabolic adaptation dependent on AMPK/ACC/autophagy axis, having the mitochondrial ß-oxidation a pro-survival role since the combination of 12 and etomoxir, a carnitine palmitoyl-transferase (CPT) inhibitor promoted extensive HL-60 cell death. Finally, 12-induced metabolic stress sensitized to HL-60 cells to cell death by the FDA-approved anti-leukemic drug ABT-199, a BH3 mimetic. Therefore, our results suggest that acylchlorohydroquinone is a promising scaffold in anti-promyelocytic leukemia drug research.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Hidroquinonas/química , Fosforilação Oxidativa/efeitos dos fármacos , Sulfonamidas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Hidroquinonas/farmacologia , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, a parasite endemic to Latin America. Most infections occur in children by vector or congenital transmission. Trypanosoma cruzi establishes a complexity of specific molecular parasite-host cell interactions to invade the host. However, most studies have been mainly focused on the interaction between the parasite and different cell types, but not on the infection and invasion on a tissue level. During congenital transmission, T. cruzi must cross the placental barrier, composed of epithelial and connective tissues, in order to infect the developing fetus. Here we aimed to study the global changes of transcriptome in the placental tissue after a T. cruzi challenge. RESULTS: Strong changes in gene expression profiling were found in the different experimental conditions, involving the reprogramming of gene expression in genes involved in the innate immune response. CONCLUSIONS: Trypanosoma cruzi induces strong changes in genes involved in a wide range of pathways, especially those involved in immune response against infections.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/parasitologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Imunidade Inata/genética , Placenta/parasitologia , Doença de Chagas/imunologia , Doença de Chagas/transmissão , Criança , Regulação para Baixo , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/imunologia , Humanos , Placenta/citologia , Gravidez , Análise Serial de Tecidos , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/fisiologia , Regulação para CimaRESUMO
BACKGROUND: Congenital Chagas disease is caused by the protozoan parasite Trypanosoma cruzi that must cross the placental barrier during transmission. The trophoblast constitutes the first tissue in contact with the maternal-blood circulating parasite. Importantly, the congenital transmission rates are low, suggesting the presence of local placental defense mechanisms. On the other hand, the placenta is considered an immune regulatory organ since it acts as a modulator of fetal and maternal immune responses. We have previously proposed that local placental factors, such as the epithelial turnover of the trophoblast and the innate immune response initiated by Toll-like receptors (TLRs), might prevent parasite infection and congenital transmission. Here, we studied in an ex vivo infected human placental chorionic villi explant HPCVE model, the relationship between TLR-2 activation in response to T. cruzi trypomastigotes, the secreted profile of cytokines, the integrity of the placental barrier and the expression of trophoblast turnover markers. RESULTS: TLR-2 inhibition increases the parasite induced histopathological damage, prevents secretion of IL-6 and IL-10, decreases expression of PCNA (proliferation marker) and of ß-hCG (differentiation marker) while increasing caspase 3 activity (cell death marker). CONCLUSION: Our results suggest that TLR-2 is not only involved in the local secretion of cytokines but also regulates, at least partially, the trophoblast turnover.
Assuntos
Doença de Chagas/imunologia , Placenta/imunologia , Complicações Infecciosas na Gravidez/imunologia , Receptor 2 Toll-Like/fisiologia , Animais , Chlorocebus aethiops , Citocinas/metabolismo , Feminino , Humanos , Imunidade Inata , Placenta/metabolismo , Gravidez , Trypanosoma cruzi , Células VeroRESUMO
PROBLEM: Trypanosoma cruzi and Toxoplasma gondii present, respectively, low and high congenital transmission rates. The placenta as an immune regulatory organ expresses TLRs, leading to the secretion of cytokines. Both parasites are recognized by TLR-2, TLR-4, and TLR-9. Here, we studied if the parasites induce differences in TLR protein expression, cytokine profiles, and whether receptor inhibition is related to parasite infection. METHOD OF STUDY: Placental tissue explants were infected ex vivo with each parasite, TLRs protein expression, cytokine profile and parasite infection were determined by Western blotting, ELISA and qPCR. RESULTS: Trypanosoma cruzi and Toxoplasma gondii infection is related to TLR-2 and TLR-4/TLR-9, respectively. Trypanosoma cruzi elicits an increase in TNF-α, IL-1ß, IL-6, IL-8 and IL-10 cytokine secretion whereas T. gondii only increases the secretion of IL-8. CONCLUSION: The susceptibility of the placenta to each parasite is mediated partially by the innate immune response.
Assuntos
Vilosidades Coriônicas/imunologia , Vilosidades Coriônicas/parasitologia , Citocinas/imunologia , Receptores Toll-Like/imunologia , Toxoplasma , Trypanosoma cruzi , Animais , Doença de Chagas/imunologia , Chlorocebus aethiops , DNA de Protozoário , Feminino , Humanos , Carga Parasitária , Gravidez , Toxoplasma/genética , Toxoplasmose/imunologia , Trypanosoma cruzi/genética , Células VeroRESUMO
Congenital transmission of Trypanosoma cruzi (T. cruzi) is partially responsible for the progressive globalization of Chagas disease. During congenital transmission the parasite must cross the placental barrier where the trophoblast, a continuous renewing epithelium, is the first tissue in contact with the parasite. The trophoblast turnover implies cellular proliferation, differentiation and apoptotic cell death. The epithelial turnover is considered part of innate immunity. We previously demonstrated that T. cruzi induces cellular differentiation and apoptosis in this tissue. Here we demonstrate that T. cruzi induces cellular proliferation in a trophoblastic cell line. We analyzed the cellular proliferation in BeWo cells by determining DNA synthesis by BrdU incorporation assays, mitotic index, cell cycle analysis by flow cytometry, as well as quantification of nucleolus organizer regions by histochemistry and expression of the proliferation markers PCNA and Ki67 by Western blotting and/or immunofluorescence. Additionally, we determined the ERK1/2 MAPK pathway activation by the parasite by Western blotting.