RESUMO
The Oceanimonas sp. BPMS22-derived protein protease inhibitor (PPI) has been proven to shift macrophages towards an inflammatory state and reduce Leishmania donovani infection in vitro and in vivo. The current study explored and validated the mechanistic aspects of the PPI and Toll-like receptor (TLR) interaction. The PPI exhibited the upregulation of TLR2, TLR4, and TLR6 during treatment which was proven to orchestrate parasite clearance effectively. An in silico study confirmed the high interaction with TLR4 and PPI. Immune blotting confirmed the significant upregulation of TLR4 in macrophages irrespective of L. donovani infection. Pharmacological inhibition and immune blot study confirmed the involvement of the PPI in TLR4-mediated phosphorylation of p38 MAPK and dephosphorylation of ERK1/2, repolarizing to pro-inflammatory macrophage state against experimental visceral leishmaniasis. In addition, in TLR4 knockdown condition, PPI treatment failed to diminish M2 phenotypical markers (CD68, Fizz1, Ym1, CD206, and MSR-2) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-ß). Simultaneously, the PPI failed to upregulate the M1 phenotypical markers and pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, and IFN-γ) (p < 0.001) during the TLR4 knockdown condition. In the absence of TLR4, the PPI also failed to reduce the parasite load and T-cell proliferation and impaired the delayed-type hypersensitivity response. The absence of pro-inflammatory cytokines was observed during a co-culture study with PPI-treated macrophages (in the TLR4 knockdown condition) with day 10 T-cell obtained from L. donovani-infected mice. This study supports the immunotherapeutic potential of the PPI as it interacted with TLR4 and promoted macrophage repolarization (M2-M1) to restrict the L. donovani parasite burden and helps in the mounting immune response against experimental visceral leishmaniasis.
Assuntos
Anti-Infecciosos , Leishmania donovani , Leishmaniose Visceral , Leishmaniose , Animais , Camundongos , Receptor 4 Toll-Like/metabolismo , Inibidores de Proteases/metabolismo , Macrófagos , Citocinas/metabolismo , Leishmaniose/metabolismo , Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , Anti-Infecciosos/metabolismoRESUMO
Arachis hypogea L. protein fraction-2 (AHP-F2) from the Peanut shell was extracted and characterized and its potent immunomodulatory and anti-leishmanial role was determined in this present study. AHP-F2 was found to be a glycoprotein as the presence of carbohydrates were confirmed by the analysis of high-performance liquid chromatography (HPLC) yielded glucose, galactose, mannose, and xylose. AHP-F2 molecular mass was found to be â¼28 kDa as indicated in MALDI-TOF and peptide mass fingerprinting analysis followed by Mascot search. The peptide matches revealed the similarity of the mannose/glucose binding lectin with 71.07% in the BLAST analysis. After that, the 3D structure of the AHP-F2 model was designed and validated by the Ramachandran plot. The immunomodulatory role of AHP-F2 was established in murine peritoneal macrophages as induction of nitric oxide (NO), and stimulation of proinflammatory cytokines (IL-12 and IFN-γ) in a dose-dependent manner was observed. Interestingly, it was also found that AHP-F2 has interacted with the innate immune receptor, toll-like receptors (TLRs) as established in molecular docking as well as mRNA expression. The anti-leishmanial potential of AHP-F2 was revealed with a prominent inhibition of amastigote growth within the murine macrophages with prompt induction of nitrite release. Altogether, the isolated AHP-F2 from Arachis hypogea L. has strong immunomodulatory and anti-leishmanial potential which may disclose a new path to treat leishmaniasis.
Assuntos
Arachis , Leishmania donovani , Animais , Camundongos , Manose , Ativação de Macrófagos , Simulação de Acoplamento Molecular , Glicoproteínas , Glucose , Leishmania donovani/metabolismo , Óxido Nítrico/metabolismo , Camundongos Endogâmicos BALB CRESUMO
The present study aimed to validate the potential of a novel serine protein protease inhibitor (PPI), purified from marine Oceanimonas sp. BPMS22, induced M2 to M1 repolarization of the macrophages to treat visceral leishmaniasis (VL). Peptide mass fingerprint of the purified trypsin digested PPI peptide was obtained using matrix-assisted laser desorption ionization-time of flight combined with tandem mass spectrometry (MALDI-TOF MS/MS) and the sequence was used to construct a 3D protein model by homology modelling. The IC50 of PPI were 25.28 ± 1.675 µg/mL and 0.415 ± 0.015 µg/mL against promastigotes and intracellular amastigotes, respectively, indicating the host-directed therapy using PPI. The PPI enhanced the effector molecule i.e., nitric oxide (NO), and dampened the arginase activity in a dose-dependent manner. In vitro studies revealed that the BPMS22-derived PPI significantly (p < 0.05) decreased the mRNA expressions of M2 markers (FIZZ-1, YM-1, CD206, Arg-1) and increased the mRNA expressions of M1 markers (iNOS, IL-1ß, IL-12) in rIL-4 + rIL-10 induced M2 macrophages. Interestingly, the BPMS22-derived PPI also significantly (p < 0.05) decreased the FIZZ-1, YM-1, CD206, and Arg-1; significantly (p < 0.05) increased iNOS, IL-12, and IFN-γ mRNA expression in L. donovani -infected murine macrophages, alongside the decreased parasite load in it. Hence, PPI has the potential to repolarize the cytokines (rIL-4 + rIL-10) pre-stimulated and L. donovani-infected M2 macrophages to M1 phenotype in vitro. A decrease in parasite burden after treatment with PPI indicated the acceleration of the parasite killing by enhancing the macrophage effector functions. Further, in vivo PPI treatment reduced hepatic and splenic Leishman donovan units (LDU) up to 93.34 % and 87.63 %, respectively. This was followed by a surge in pro-inflammatory cytokines and dampening anti-inflammatory cytokines (p < 0.01), which exhibited anti-VL immunity. These observations might open new perspectives on PPI in macrophage repolarization to treat VL.
Assuntos
Leishmania donovani , Leishmaniose Visceral , Camundongos , Animais , Óxido Nítrico/metabolismo , Arginase/metabolismo , Inibidores de Proteases/metabolismo , Tripsina/metabolismo , Espectrometria de Massas em Tandem , Macrófagos , Citocinas/metabolismo , Interleucina-12/metabolismo , Imunidade , Serina/metabolismo , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: Leukemia is one of the severe cancer types all around the globe. Even though some chemotherapeutic drugs are available for treating leukemia, they have various side effects. As an alternative approach, herbal drugs are focused on current research to overcome leukemia. The present work was conducted to investigate the antileukemic mechanism of active phytochemical vitexin, which was isolated from ethno-medicine (Prosopis cineraria leaf) used by traditional healers of West Bengal, India. METHODS: Antiproliferative mechanisms of selected phyto-compound against K-562 cells were evaluated using cellular uptake, morphological changes, DNA fragmentation, mitochondrial membrane potential and signaling pathways analysis. KEY FINDINGS: Vitexin exhibited cytotoxicity by reducing mitochondrial membrane potential (32.40%) and causing DNA fragmentation (84.15%). The western blotting study indicated inhibition of cell survival proteins (BCR, ABL, H-RAS, N-RAS, K-RAS and RAF) and expression of apoptotic proteins (p38, BAX and caspase-9) in leukemia cells upon treatment with vitexin. CONCLUSIONS: Based on the results, presently investigated phyto-compound vitexin could be considered for developing safe and natural drugs to treat leukemia after conducting suitable preclinical and clinical trials.
Assuntos
Apigenina/farmacologia , Proteínas Oncogênicas v-abl/metabolismo , Prosopis , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Immune metabolic adaptation in macrophages by intracellular parasites is recognized to play a crucial role during Leishmania infection. However, there is little accessible information about changes in a metabolic switch in L. donovani infected macrophages. In previous studies, we have reported on the anti-leishmanial synergic effect of eugenol oleate with amphotericin B. In the present study, we demonstrated that glycolytic enzymes were highly expressed in infected macrophages during combinatorial treatment of eugenol oleate (2.5 µM) and amphotericin B (0.3125 µM). Additionally, we found that the biphasic role in arachidonic acid metabolite, PGE2, and LTB4, is released during this treatment. In vitro data showed that COX-2 mediated PGE2 synthesis increased significantly (p<0.01) in infected macrophages. Not only was the level of prostaglandin synthesis decreased 4.38 fold in infected macrophages after treatment with eugenol oleate with amphotericin B. The mRNA expression of PTGES, MPGES, and PTGER4 were also moderately expressed in infected macrophages, and found to be decreased in combinatorial treatment. In addition, NOS2 expression was activated by the phosphorylation of p38MAPK when combination-treated macrophages were promoted to kill intracellular parasites. The findings of the present study indicate that the synergism between eugenol oleate and amphotericin B could play an important role in immune metabolism adaptation with a concomitant increase in host immune response against the intracellular pathogen, L. donovani.
Assuntos
Leishmania donovani , Leishmaniose , Anfotericina B/farmacologia , Animais , Eugenol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ácido OleicoRESUMO
Conventional therapy of visceral leishmaniasis (VL) remains challenging with the pitfall of toxicity, drug resistance, and expensive. Hence, urgent need for an alternative approach is essential. In this study, we evaluated the potential of combination therapy with eugenol oleate and miltefosine in Leishmania donovani infected macrophages and in the BALB/c mouse model. The interactions between eugenol oleate and miltefosine were found to be additive against promastigotes and amastigotes with xΣFIC 1.13 and 0.68, respectively. Significantly (p < 0.001) decreased arginase activity, increased nitrite generation, improved pro-inflammatory cytokines, and phosphorylated p38MAPK were observed after combination therapy with eugenol oleate and miltefosine. >80% parasite clearance in splenic and hepatic tissue with concomitant nitrite generation, and anti-VL cytokines productions were observed after orally administered miltefosine (5 mg/kg body weight) and eugenol oleate (15 mg/kg body weight) in L. donovani-infected BALB/c mice. Altogether, this study suggested the possibility of an oral combination of miltefosine with eugenol oleate against visceral leishmaniasis.
Assuntos
Citocinas/metabolismo , Eugenol/uso terapêutico , Imunidade , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Óxido Nítrico/biossíntese , Fosforilcolina/análogos & derivados , Administração Oral , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/biossíntese , Interações Medicamentosas , Quimioterapia Combinada , Eugenol/administração & dosagem , Eugenol/farmacologia , Feminino , Imunidade/efeitos dos fármacos , Concentração Inibidora 50 , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Leishmania donovani/ultraestrutura , Leishmaniose Visceral/parasitologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/parasitologia , Macrófagos/ultraestrutura , Masculino , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , Parasitos/efeitos dos fármacos , Parasitos/crescimento & desenvolvimento , Parasitos/imunologia , Parasitos/ultraestrutura , Fosforilação/efeitos dos fármacos , Fosforilcolina/administração & dosagem , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Visceral leishmaniasis (VL) is an endemic fatal infectious disease in tropical and subtropical nations. The limited treatment options, long treatment regimens, invasive mode of administration of drugs, and lack of effective vaccination are the main reasons for the search of new alternative therapeutics against VL. On this quest, from a series of eugenol derivatives, we had demonstrated eugenol oleate as a lead immunomodulatory anti-VL molecule earlier. In this report, the oral efficacy and mechanism of eugenol oleate in inducing immunomodulatory anti-VL activity has been studied in BALB/c mice model. The plasma pharmacokinetic and acute toxicity studies suggested that the eugenol oleate is safe with an appreciable pharmacokinetic profile. Eugenol oleate (30 mg/kg B.W.) showed 86.5% of hepatic and 84.1% of splenic parasite clearance. The increased Th1 cytokine profile and decreased Th2 cytokine profile observed from ELISA and qRTPCR suggested that the eugenol oleate induced the parasite clearance through the activation of the host immune system. Subsequently, the mechanistic insights behind the anti-leishmanial activity of eugenol oleate were studied in peritoneal macrophages in vitro by inhibitor response study and immunoblotting. The results inferred that eugenol oleate activated the PKC-ßII-p38 MAPK and produced IL-12 and IFN-γ which intern activated the iNOS2 to produce NO free radicals that cleared the intracellular parasite.
Assuntos
Eugenol/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Ácido Oleico/farmacologia , Administração Oral , Animais , Citocinas , Modelos Animais de Doenças , Feminino , Sistema Imunitário/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/parasitologiaRESUMO
In the present work, 16 different plant drugs used by traditional healers from West Bengal were screened through in vitro cell line model. Herbal drugs used by traditional tribal healers in Purulia, Birbhum and Bankura districts of West Bengal were collected and screening against acute myeloid leukemia (AML) cell line (HL-60). Among 16 plant extracts, bark of Flacourtia indica (66.67%), leaf of Madhuca longifolia (69.17%), and leaf of Prosopis cineraria (68.08%) showed better cytotoxicity results than other herbals. Further, time-dependent study showed maximum cytotoxicity of the selected herbal extracts between 36 and 48 hours of treatment in both acute and chronic myeloid leukemia (CML) cell lines (HL-60 and K562). The LC-MS/MS analysis of the selected drugs revealed the presence of picrotoxinin and 10-deacetylbaccatin from F. indica, isoorientin and hirsutrin from M. longifolia, vitexin and rhoifolin in P. cineraria.
Assuntos
Antineoplásicos Fitogênicos/química , Leucemia/tratamento farmacológico , Compostos Fitoquímicos/análise , Plantas Medicinais/química , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Cromatografia Líquida , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Índia/etnologia , Luteolina/análise , Medicina Tradicional , Compostos Fitoquímicos/farmacologia , Picrotoxina/análogos & derivados , Picrotoxina/análise , Extratos Vegetais/análise , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Sesterterpenos , Espectrometria de Massas em TandemRESUMO
Obesity is considered as an independent risk factor for breast cancer (BCa) and plays a major role in the breast tumor microenvironment. The etiology and mechanisms by which obesity contributes to BCa development is not yet understood. Herein, we show that in vitro coculture of BCa cells with mature adipocytes (MA-BCa) increased proliferation, migration, and invasive phenotype of BCa cells. MA-BCa coculture led to increased production of proinflammatory cytokines and chemokines. To identify microRNAs (miRNAs) in BCa cells that are modulated by the presence of adipocytes, we used small RNA sequencing analysis. Sequencing data revealed that 98 miRNAs were differentially expressed in MA-BCa. Among them, miR-3184-5p and miR-181c-3p were found to be the most upregulated and downregulated miRNAs, and direct targets are FOXP4 and PPARα, respectively. In vitro functional assays using a combination of miR-3184-5p inhibitor and miR-181c-3p mimic synergistically decreased adipocytes-induced cell proliferation and invasive capacity of BCa cells. Gene Set Enrichment analysis indicated that transcription factors were highly enriched followed by protein kinases, oncogene, and protein regulators in MA-BCa. GeneGo Metacore pathway analysis uncovered "NOTCH-induced EMT pathway" was found to be the most abundant in MA-BCa. Consistently, epithelial-mesenchymal transition-associated markers were also increased in MA-BCa. The disease enrichment analysis of the predict target genes revealed that diabetes mellitus was significantly affected disease in MA-BCa. Taken together, our data suggest that miRNA-based regulatory mechanism associated with deregulation of pathways and biological functions orchestrated by adipocytes-secreted factors might drive the BCa progression and metastasis in obese patients.
Assuntos
Adenocarcinoma/metabolismo , Adipócitos/metabolismo , Neoplasias da Mama/metabolismo , MicroRNAs/metabolismo , Obesidade/metabolismo , Microambiente Tumoral , Células 3T3 , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adipócitos/patologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Camundongos , MicroRNAs/genética , Invasividade Neoplásica , Obesidade/genética , Obesidade/patologia , PPAR alfa/genética , PPAR alfa/metabolismo , Transdução de SinaisRESUMO
Visceral leishmaniasis (VL) is a life threatening infectious disease caused by Leishmania donovani. It leads to the severe immune suppression in the host defense system. Higher cytotoxicity, rigorous side effects and lower therapeutic indexes (TI) of current antileishmanial drugs have created a necessity to develop new molecules with better antileishmanial activity and high TI value. In this study, we have synthesized 36 derivatives of eugenol and screened them for their activity against promastigote and amastigote forms of L. donovani. Among the synthesized derivatives, comp.35 showed better antileishmanial activity against extra cellular promastigotes (IC50- 20.13 ± 0.91 µM) and intracellular amastigotes (EC50-4.25 ± 0.26 µM). The TI value (82.24 ± 3.77) was found to improve by 10-13 fold compared to Amphotericin B and Miltefosine respectively. Treatment with comp.35 (5 µg/ml) enhanced the nitric oxide (NO) generation, iNOS2 mRNA expression (â¼8 folds increase) and decreased the arginase-1 activity (â¼4 folds) in L. donovani infected peritoneal macrophages. Comp.35 had also increased the IL-12 (â¼6 folds) and decreased the IL-10 (â¼3 folds) mRNA expression and release in vitro. Results of in vivo studies revealed that comp.35 treatment at 25 mg/kg body weight efficiently cleared the hepatic and splenic parasite burden with enhanced Th1 response in L. donovani infected BALB/c mice. Hence, this study clearly represents comp.35, as an immunomodulatory molecule, can induce host protective immune response against visceral leishmaniasis through enhanced NO generation and Th1 response, which are essentials against this deadly disease.
Assuntos
Antiprotozoários/farmacologia , Eugenol/farmacologia , Imunomodulação , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eugenol/síntese química , Eugenol/química , Leishmania donovani/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Testes de Sensibilidade Parasitária , Células RAW 264.7 , Relação Estrutura-AtividadeRESUMO
Chloroquine (CQ) is highly effective against P. vivax, due to the rapid spread of CQ resistance in P. falciparum parasites; it is no longer the drug of choice against P. falciparum. This study elucidates the scenario of chloroquine efficacy at times that coincided with a new drug policy and especially assessed the chloroquine resistant molecular markers after withdrawal of chloroquine in Kolkata and Purulia, two malaria endemic zones of West Bengal, India. In vitro CQ susceptibility was tested in 781 patients with P. falciparum mono infections between 2008 and 2013, of which 338 patients had received CQ in 2008-2009. Genotyping of the pfcrt and the pfmdr1 gene was carried out in all isolates. Early treatment failure was detected in 114 patients {43 (31·39%) from Kolkata and 71 (35·32%) from Purulia} while recrudescence was identified in 13 (9.49%) and 17 (8.46%) patients from Kolkata and Purulia respectively. In vivo chloroquine resistance was strongly associated with CVMNT-YYSNY (p < 0.01) and SVMNT-YYSNY (p < 0.05) allele in Kolkata. In Purulia chloroquine resistance was associated with CVMNK-YYSNY (P < 0.005), SVMNT-YYSNY (P < 0.01) allele. The proportion of in vitro chloroquine resistance increased in subsequent years to 87.23% and 93·10% in 2013, in Kolkata and Purulia, respectively. Isolates with SVMNT-YFSND, SVMNT-YFSNY, CVIET-YFSND and CVIET-YYSNY haplotypes increased gradually (p < 0.05) from 2010 to 2013, leading to a rise in IC50 (p < 0.05) of chloroquine. An increase in in vitro chloroquine resistance and candidate gene mutations even after five years of chloroquine withdrawal against P. falciparum calls for synchronized research surveillance and proper containment strategies.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/genética , Mutação Puntual , Adolescente , Adulto , Idoso , Antimaláricos/uso terapêutico , Criança , Pré-Escolar , Cloroquina/uso terapêutico , Coinfecção/tratamento farmacológico , Coinfecção/epidemiologia , Coinfecção/parasitologia , Feminino , Haplótipos , Humanos , Índia/epidemiologia , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Malária Vivax/tratamento farmacológico , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Adulto JovemRESUMO
The present study portrays the isolation of four phenylpropanoids - ferulic acid (FA), sinapic acid (SA), caffeic acid (CA), and chlorogenic acid (CHA) from the water extract of Suaeda maritima (L.) Dumort, a phytochemically less explored Indian medicinal plant. Further, synthesis and characterization of silver and gold nanoparticles using the isolated phenylpropanoids were done. The silver nanoparticles synthesized from S. maritima water extract along with silver nano-conjugated forms of the isolated compounds exhibited appreciable anti-leukemic activity against K562 cells (human myeloid leukemia). Especially, the ferulic and CA-conjugated silver nanoparticles showed significant (P < .01) activity against leukemia.
Assuntos
1-Propanol/química , 1-Propanol/farmacologia , Amaranthaceae/química , Leucemia Mieloide/patologia , Nanopartículas Metálicas/química , Plantas Comestíveis/química , Prata/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Células K562 , Folhas de Planta/químicaRESUMO
Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 µg/ml against H37Rv and mc26030 strains. The best molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta position of the second aromatic ring showed the MIC value of 5.2 µg/ml and 1 µg/ml against H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 µg/ml. The effective compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in 14C-radiolabelled assay. At the same time these molecules also executed their potent immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10.
Assuntos
Macrófagos/microbiologia , Mycobacterium/efeitos dos fármacos , Ureia/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-12/biossíntese , Interleucina-12/genética , Macrófagos/metabolismo , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/antagonistas & inibidores , Ácidos Micólicos/metabolismo , Relação Estrutura-Atividade , Ureia/análogos & derivadosRESUMO
M2 polarization of macrophages is predominant in case of tumors and some other infectious diseases for disease progression. Repolarization of the M2 phenotype to the M1 state may be required to cure diseases. Hence, it is of great interest to find out a material that would repolarize the M2 phenotype to the M1 state. Herein, the arabinogalactan protein from Andrographis paniculata (ApAGP) was used to prepare a silver nanoparticle-ApAGP (SNP-ApAGP) bioconjugate, which was characterized via UV-vis spectroscopy, zeta potential analysis, FT-IR spectroscopy, and HR-TEM. Studies suggest that SNP-ApAGP (2.5 µg mL-1) up-regulates ROS generation, NO generation, and pro-inflammatory cytokine release (IL-12, IFN-γ, TNF-α, and IL-6). SNP-ApAGP also down-regulates the arginase-1 activity and anti-inflammatory cytokine release (IL-4 & IL-10) in M0, M1, and M2-polarized peritoneal macrophages in vitro. Therefore, SNP-ApAGP induces M1 polarization in M0 macrophages, enhances the pro-inflammatory activity of the M1 phenotype, and can also repolarize M2 macrophages into the M1 phenotype. Therefore, SNP-ApAGP could be used for treating various infectious diseases and cancers where repolarization of M2 macrophages may be required to cure the disease.
RESUMO
Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL), characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2) ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+), Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL.
Assuntos
Linfócitos T CD8-Positivos/parasitologia , Leishmaniose Visceral/imunologia , Proteínas com Domínio T/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Antígenos CD28/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Granzimas/genética , Histonas/metabolismo , Interferon gama/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leishmania donovani , Leishmaniose Visceral/sangue , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Regiões Promotoras Genéticas , Receptores de Interferon/metabolismo , Receptores de Interleucina-12/metabolismo , Transdução de Sinais , Regulação para Cima , Receptor de Interferon gamaRESUMO
The purpose of this study was to determine the intracellular signaling transduction pathways involved in oxidative stress induced by nanoparticles in cancer cells. Activation of reactive oxygen species (ROS) has some therapeutic benefits in arresting the growth of cancer cells. Cobalt oxide nanoparticles (CoO NPs) are an interesting compound for oxidative cancer therapy. Our results showed that CoO NPs elicited a significant (P <0.05) amount of ROS in cancer cells. Co-treatment with N-aceyltine cystine (an inhibitor of ROS) had a protective role in cancer cell death induced by CoO NPs. In cultured cells, the elevated level of tumor necrosis factor-alpha (TNF-α) was noted after CoO NPs treatment. This TNF-α persuaded activation of caspase-8 followed by phosphorylation of p38 mitogen-activated protein kinase and induced cell death. This study showed that CoO NPs induced oxidative stress and activated the signaling pathway of TNF-α-Caspase-8-p38-Caspase-3 to cancer cells.
Assuntos
Caspase 8/fisiologia , Cobalto/efeitos adversos , Leucemia Experimental/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas Metálicas/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Óxidos/efeitos adversos , Fator de Necrose Tumoral alfa/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Microscopia Eletrônica de Transmissão , Transplante de Neoplasias , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The objective of this study was to develop chitosan-based delivery of cobalt oxide nanoparticles to human leukemic cells and investigate their specific induction of apoptosis. The physicochemical properties of the chitosan-coated cobalt oxide nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, X-ray diffraction, and Fourier transform infrared spectroscopy. The solubility of chitosan-coated cobalt oxide nanoparticles was higher at acidic pH, which helps to release more cobalt ions into the medium. Chitosan-coated cobalt oxide nanoparticles showed good compatibility with normal cells. However, our results showed that exposure of leukemic cells (Jurkat cells) to chitosan-coated cobalt oxide nanoparticles caused an increase in reactive oxygen species generation that was abolished by pretreatment of cells with the reactive oxygen species scavenger N-acetyl-L-cysteine. The apoptosis of Jurkat cells was confirmed by flow-cytometric analysis. Induction of TNF-α secretion was observed from stimulation of Jurkat cells with chitosan-coated cobalt oxide nanoparticles. We also tested the role of TNF-α in the induction of Jurkat cell death in the presence of TNF-α and caspase inhibitors. Treatment of leukemic cells with a blocker had a greater effect on cancer cell viability. From our findings, oxidative stress and caspase activation are involved in cancer cell death induced by chitosan-coated cobalt oxide nanoparticles.
Assuntos
Apoptose/fisiologia , Quitosana/química , Cobalto/química , Leucemia de Células T/metabolismo , Nanopartículas Metálicas/química , Óxidos/química , Fator de Necrose Tumoral alfa/fisiologia , Apoptose/efeitos dos fármacos , Quitosana/farmacologia , Quitosana/uso terapêutico , Cobalto/farmacologia , Cobalto/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Células Jurkat , Leucemia de Células T/tratamento farmacológico , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Óxidos/farmacologia , Óxidos/uso terapêutico , Fator de Necrose Tumoral alfa/farmacologia , Difração de Raios XRESUMO
Visceral leishmaniasis (VL) caused by the protozoan parasite, Leishmania donovani, is associated with irregular fever, weight loss, hepatosplenomegaly and anemia. The therapeutic arsenal against VL is limited and the recent advent of a novel immunomodulatory drug, Miltefosine has shown promising results for effective treatment of VL but its dependence on Toll like receptors (TLR) has not been explored. In this study, we have shown that the non-cytotoxic dose (5 µM) of Miltefosine could render significant protection corresponding to 88% and 95% reduction in intracellular parasite load at 24 h and 48 h in L. donovani infected THP1 cells. This was accompanied by a strong proinflammatory cytokine response in the form of IFN-γ, IL-12 and TNF-α as evident by enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR). This Miltefosine induced proinflammatory cytokine response in infected THP1 cells was also accompanied by simultaneous 10- and 12-fold increase in TLR4 mRNA and TLR9 mRNA. These changes in cytokine response and TLR expression were also studied in peripheral blood mononuclear cells (PBMC) of VL patients treated with Miltefosine by RT-PCR which showed similar results as in THP1 cells. Thereby, suggesting a probable dependence of Miltefosine on TLR4 and TLR9 in triggering a proinflammatory response.
Assuntos
Antiprotozoários/farmacologia , Leishmaniose Visceral/imunologia , Fosforilcolina/análogos & derivados , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Adulto , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Feminino , Humanos , Leishmania donovani , Leishmaniose Visceral/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Fosforilcolina/farmacologia , RNA Mensageiro/imunologia , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/genética , Adulto JovemRESUMO
Staphylococcus aureus is the most frequently isolated pathogen causing bloodstream infections, skin and soft tissue infections and pneumonia. Lymphocyte is an important immune cell. The aim of the present paper was to test the ameliorative role of nanoconjugated vancomycin against Vancomycin-sensitive Staphylococcus aureus (VSSA) and vancomycin-resistant Staphylococcus aureus (VRSA) infection-induced oxidative stress in lymphocytes. VSSA and VRSA infections were developed in Swiss mice by intraperitoneal injection of 5 × 10(6) CFU/mL bacterial solutions. Nanoconjugated vancomycin was adminstrated to VSSA- and VRSA-infected mice at its effective dose for 10 days. Vancomycin was adminstrated to VSSA- and VRSA-infected mice at a similar dose, respectively, for 10 days. Vancomycin and nanoconjugated vancomycin were adminstrated to normal mice at their effective doses for 10 days. The result of this study reveals that in vivo VSSA and VRSA infection significantly increases the level of lipid peroxidation, protein oxidation, oxidized glutathione level, nitrite generation, nitrite release, and DNA damage and decreases the level of reduced glutathione, antioxidant enzyme status, and glutathione-dependent enzymes as compared to control group, which were increased or decreased significantly near to normal in nanoconjugated vancomycin-treated group. These findings suggest the potential use and beneficial role of nanoconjugated vancomycin against VSSA and VRSA infection-induced oxidative stress in lymphocytes.
Assuntos
Antibacterianos/farmacologia , Fragmentação do DNA , Portadores de Fármacos/química , Linfócitos/microbiologia , Estresse Oxidativo , Staphylococcus aureus/isolamento & purificação , Vancomicina/farmacologia , Animais , Enzimas/metabolismo , Glutationa/metabolismo , Linfócitos/efeitos dos fármacos , Masculino , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Camundongos , Nanopartículas/química , Nanotecnologia , Óxido Nítrico/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
The aim of this study was to evaluate the immune functions by nicotine-induced murine peritoneal macrophages, and Th1/Th2 cytokine balance in it, and concurrently to establish the immunomodulatory role of eugenol, and N-acetylcysteine in nicotine-induced macrophages. Eugenol was isolated from Ocimum gratissimum, and characterized by HPLC, FTIR, and (1)H NMR. The cytotoxic effect of isolated eugenol was studied in murine peritoneal macrophages at various concentrations (0.1-50 µg/ml) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. To evaluate the immunomodulatory role of eugenol and N-acetylcysteine, ROS and nitrite generations, phenotype functions by macrophages were studied. The effect of eugenol and N-acetylcysteine on the release of Th1 cytokines (TNF-α, IL-12) and Th2 cytokines (IL-10, TGF-ß) was measured by ELISA, and the expression of these cytokines at mRNA level were analyzed by real-time PCR. Eugenol, at a dose of 15 µg/ml, showed less cytotoxicity to the macrophages and it significantly reduced the nicotine-induced ROS, NO generation, and iNOSII expression. Similar kinds of response were observed in the presence of N-acetylcysteine (1 µg/ml). We have found the decreased adherence, chemotaxis, phagocytosis and intracellular killing of bacteria in nicotine treated macrophages, whereas eugenol and N-acetylcysteine with nicotine treatment enhanced these cellular functions by macrophages significantly (p < 0.05). Eugenol and N-acetylcysteine were found to down regulate the Th1 cytokines in nicotine treated macrophages with concurrent activation of Th2 responses. These findings strongly enhanced our understanding of the molecular mechanism leading to nicotine-induced suppression of immune functions, and provide additional rationale for the application of anti-inflammatory therapeutic approaches by eugenol, and N-acetylcysteine for different inflammatory diseases prevention and treatment during nicotine toxicity.