RESUMEN
OBJECTIVES: Syntenin-1, a novel endogenous ligand, was discovered to be enriched in rheumatoid arthritis (RA) specimens compared with osteoarthritis synovial fluid and normal synovial tissue (ST). However, the cellular origin, immunoregulation and molecular mechanism of syntenin-1 are undescribed in RA. METHODS: RA patient myeloid and lymphoid cells, as well as preclinical models, were used to investigate the impact of syntenin-1/syndecan-1 on the inflammatory and metabolic landscape. RESULTS: Syntenin-1 and syndecan-1 (SDC-1) co-localise on RA ST macrophages (MΦs) and endothelial cells. Intriguingly, blood syntenin-1 and ST SDC-1 transcriptome are linked to cyclic citrullinated peptide, erythrocyte sedimentation rate, ST thickness and bone erosion. Metabolic CD14+CD86+GLUT1+MΦs reprogrammed by syntenin-1 exhibit a wide range of proinflammatory interferon transcription factors, monokines and glycolytic factors, along with reduced oxidative intermediates that are downregulated by blockade of SDC-1, glucose uptake and/or mTOR signalling. Inversely, IL-5R and PDZ1 inhibition are ineffective on RA MΦs-reprogrammed by syntenin-1. In syntenin-1-induced arthritis, F4/80+iNOS+RAPTOR+MΦs represent glycolytic RA MΦs, by amplifying the inflammatory and glycolytic networks. Those networks are abrogated in SDC-1-/- animals, while joint prorepair monokines are unaffected and the oxidative metabolites are moderately replenished. In RA cells and/or preclinical model, syntenin-1-induced arthritogenicity is dependent on mTOR-activated MΦ remodelling and its ability to cross-regulate Th1 cells via IL-12 and IL-18 induction. Moreover, RA and joint myeloid cells exposed to Syntenin-1 are primed to transform into osteoclasts via SDC-1 ligation and RANK, CTSK and NFATc1 transcriptional upregulation. CONCLUSION: The syntenin-1/SDC-1 pathway plays a critical role in the inflammatory and metabolic landscape of RA through glycolytic MΦ and Th1 cell cross-regulation (graphical abstract).
Asunto(s)
Artritis Reumatoide , Células TH1 , Animales , Humanos , Células Endoteliales/metabolismo , Macrófagos/metabolismo , Monocinas/metabolismo , Sindecano-1/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Sinteninas/metabolismo , Serina-Treonina Quinasas TORRESUMEN
Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (MÏ´) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human MÏ´s exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated MÏ´s nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of MÏ´s by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated MÏ´s, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated MÏ´s was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human MÏ´s as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated MÏ´ hyperactivation. IRAK4i therapy counteracts MÏ´ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human MÏ´s. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.
Asunto(s)
COVID-19 , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2 , Animales , Células HEK293 , Humanos , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/farmacología , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Ratones , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 7/metabolismoRESUMEN
The molecular mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein was characterized to identify novel therapies. The impact of tofacitinib, IL-6R Ab, or TNFi therapy was determined on Spike protein or LPS/IFN-γ-induced signaling, inflammation, and metabolic reprogramming in MΦs and/or rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS). ACE2 frequency was markedly expanded in MΦs compared to T cells and RA FLS. Tofacitinib suppresses Spike protein potentiated STAT1 signaling, whereas this function was unchanged by TNFi. Tofacitinib impairs IL-6/IFN/LPS-induced STAT1 and STAT3 phosphorylation in RA MΦs and FLS. Interestingly, tofacitinib had a broader inhibitory effect on the monokines, glycolytic regulators, or oxidative metabolites compared to IL-6R Ab and TNFi in Spike-protein-activated MΦs. In contrast, all three therapies disrupted IFN-α and IFN-ß secretion in response to Spike protein; nonetheless, the IFN-γ was only curtailed by tofacitinib or IL-6R Ab. While tofacitinib counteracted MΦ metabolic rewiring instigated by Spike protein, it was inconsequential on the glycolysis expansion mediated via HK2 and/or LDHA in the activated RA MΦ and FLS. Nevertheless, the potentiated inflammatory response and the diminished oxidative phosphorylation modulated by Spike protein and/or LPS/IFN-γ stimulation in MΦs or RA FLS were reversed by tofacitinib. In conclusion, tofacitinib suppresses MΦ inflammation and immunometabolism triggered by Spike protein and may provide a promising strategy for COVID-19 patients.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Macrófagos/efectos de los fármacos , Piperidinas/farmacología , Pirimidinas/farmacología , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Artritis Reumatoide/metabolismo , COVID-19/metabolismo , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Macrófagos/metabolismo , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Thirty percent of psoriasis patients develop psoriatic arthritis (PsA), nevertheless the mechanism remains unknown. Endogenous GU-rich miRNAs activate endosomal TLR7 that plays a critical role in autoimmune diseases. We found that endogenous TLR7 ligands, miR-29 and miR-Let7b, were markedly increased in PsA compared to osteoarthritis (OA) synovial fluid (SF)s. We showed that intradermal (i.d.) miR-Let7b injection promoted skin inflammation, which was characterized by amplified Th1 cells, CD68+ M1 macrophages, and transcriptional upregulation of glycolytic mediators, GLUT1, C-MYC, and HIF1α. Expansion of skin Th1 cells driven by miR-Let7b was also linked to elevated M1-associated IRFs. Interestingly, i.d. miR-Let7b administration exacerbated suboptimal joint inflammation along with metabolic reconfiguration of the PsA-like preclinical model. Moreover, TLR7 agonist, R837, potentiated metabolic reprogramming and expression of IL-1ß, IL-6, and IL-12 in murine macrophages, enabling myeloid-to-T-cell crosstalk. Consistently, treatment with glycolytic inhibitors, 2-DG and/or HIF1αi, reversed R837-induced metabolic remodeling and disrupted the TLR7-driven inflammatory phenotype in myeloid and lymphoid cells. Similar to miR-Let7b, R837 also differentiates progenitor cells into mature osteoclasts, primarily through RANKL induction. Taken together, this study indicates that TLR7-instigated metabolic rewiring of macrophages and their cross-regulation of T cells connects skin immunopathology to joint inflammation.
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Artritis Psoriásica/inmunología , Articulaciones/inmunología , Macrófagos/inmunología , Piel/inmunología , Receptor Toll-Like 7/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Citocinas/inmunología , Humanos , Inflamación/inmunología , Ligandos , Linfocitos/inmunología , Ratones , Ratones Endogámicos DBA , MicroARNs/inmunología , Células Mieloides/inmunología , Osteoclastos/inmunología , Transducción de Señal/inmunología , Líquido Sinovial/inmunología , Células TH1/inmunologíaRESUMEN
This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1ß and IL-6. CCR9 is also presented on IL-1ß and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.
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Artritis Reumatoide/inmunología , Diferenciación Celular/inmunología , Quimiocinas CC/inmunología , Macrófagos/inmunología , Osteoclastos/inmunología , Receptores CCR/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Quimiocinas CC/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Humanos , Interleucina-8/inmunología , Interleucina-8/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Fosforilación , Receptores CCR/metabolismo , Transducción de Señal/inmunología , Líquido Sinovial/citología , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
This study was designed to delineate the functional significance of CCL21 in metabolic reprogramming in experimental arthritis and differentiated rheumatoid arthritis (RA) macrophages (MΦs). To characterize the influence of CCL21 on immunometabolism, its mechanism of action was elucidated by dysregulating glucose uptake in preclinical arthritis and RA MΦs. In CCL21 arthritic joints, the glycolytic intermediates hypoxia-inducible factor 1α (HIF1α), cMYC and GLUT1 were overexpressed compared with oxidative regulators estrogen-related receptor γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1)-α. Interestingly, 2-deoxy-D-glucose (2-DG) therapy mitigated CCL21-induced arthritis by restraining the number of joint F4/80+ iNOS+ MΦs without impacting F4/80+ Arginase+ MΦs. Similar to the preclinical findings, blockade of glycolysis negated CCL21-polarized CD14+ CD86+ GLUT+ MΦ frequency; however, CD14+ CD206+ GLUT+ MΦs were not implicated in this process. In CCL21-induced arthritis and differentiated RA MΦs, the inflammatory imprint was uniquely intercepted by 2-DG via interleukin-6 (IL-6) downregulation. Despite the more expansive inflammatory response of CCL21 in the arthritic joints relative to the differentiated RA MΦs, 2-DG was ineffective in joint tumor necrosis factor-α, IL-1ß, CCL2 and CCL5 enrichment. By contrast, disruption of glycolysis markedly impaired CCL21-induced HIF1α and cMYC signaling in arthritic mice. Notably, in RA MΦs, glycolysis interception was directed toward dysregulating CCL21-enhanced HIF1α transcription. Nonetheless, in concurrence with the diminished IL-6 levels, CCL21 differentiation of CD14+ CD86+ GLUT1+ MΦs was reversed by glycolysis and HIIF1α inhibition. Moreover, in the CCL21 experimental arthritis or differentiated RA MΦs, the malfunctioning metabolic machinery was accompanied by impaired oxidative phosphorylation because of reduced PGC1α or peroxisome proliferator-activated receptor-γ expression. CCL21 reconfigures naïve myeloid cells into glycolytic RA CD14+ CD86+ GLUT+ IL-6high HIF1αhigh MΦs. Therefore, inhibiting the CCL21/CCR7 pathway may provide a promising therapeutic strategy.
Asunto(s)
Artritis Reumatoide , Macrófagos , Animales , Artritis Reumatoide/metabolismo , Glucólisis , Interleucina-6/metabolismo , Macrófagos/metabolismo , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Recent studies have shown the significance of metabolic reprogramming in immune and stromal cell function. Yet, the metabolic reconfiguration of RA macrophages (MΦs) is incompletely understood during active disease and in crosstalk with other cell types in experimental arthritis. This study elucidates a distinct regulation of glycolysis and oxidative phosphorylation in RA MΦs compared to fibroblast (FLS), although PPP (Pentose Phosphate pathway) is similarly reconfigured in both cell types. 2-DG treatment showed a more robust impact on impairing the RA M1 MΦ-mediated inflammatory phenotype than IACS-010759 (IACS, complexli), by reversing ERK, AKT and STAT1 signaling, IRF8/3 transcription and CCL2 or CCL5 secretion. This broader inhibitory effect of 2-DG therapy on RA M1 MΦs was linked to dysregulation of glycolysis (GLUT1, PFKFB3, LDHA, lactate) and oxidative PPP (NADP conversion to NADPH), while both compounds were ineffective on oxidative phosphorylation. Distinctly, in RA FLS, 2-DG and IACS therapies constrained LPS/IFNγ-induced AKT and JNK signaling, IRF5/7 and fibrokine expression. Disruption of RA FLS metabolic rewiring by 2-DG or IACS therapy was accompanied by a reduction of glycolysis (HIF1α, PFKFB3) and suppression of citrate or succinate buildup. We found that 2-DG therapy mitigated CIA pathology by intercepting joint F480+iNOS+MΦ, Vimentin+ fibroblast and CD3+T cell trafficking along with downregulation of IRFs and glycolytic intermediates. Surprisingly, IACS treatment was inconsequential on CIA swelling, cell infiltration, M1 and Th1/Th17 cytokines (IFN-γ/IL-17) and joint glycolytic mediators. Collectively, our results indicate that blockade of glycolysis is more effective than inhibition of complex 1 in CIA, in part due to its effectiveness on the MΦ inflammatory phenotype.
Asunto(s)
Artritis Reumatoide/fisiopatología , Desoxiglucosa/farmacología , Fibroblastos/inmunología , Glucólisis , Inflamación/prevención & control , Macrófagos/inmunología , Células Th17/inmunología , Animales , Antimetabolitos/farmacología , Artritis Experimental/fisiopatología , Movimiento Celular , Citocinas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos DBA , Vía de Pentosa Fosfato , FenotipoRESUMEN
In rheumatoid arthritis (RA), synovial tissue abundantly expresses CCL21, a chemokine strongly associated with RA susceptibility. In this study, we aimed to characterize the functional significance of CCL21/CCR7 signaling in different phases of RA pathogenesis. We determined that CCR7 is a hallmark of RA M1 synovial fluid (SF) macrophages, and its expression in RA monocytes and in vitro differentiated macrophages is closely associated with disease activity score (DAS28). In early stages of RA, monocytes infiltrate the synovial tissue. However, blockade of SF CCL21 or CCR7 prevents RA SF-mediated monocyte migration. CCR7 expression in the newly migrated macrophages can be accentuated by LPS and IFNγ and suppressed by IL-4 treatment. We also uncovered that CCL21 stimulation increases the number of M1-polarized macrophages (CD14+CD86+), resulting in elevated transcription of IL-6 and IL-23. These CCL21-induced M1 cytokines differentiate naïve T cells to Th17 cells, without affecting Th1 cell polarization. In the erosive stages of disease, CCL21 potentiates RA osteoclastogenesis through M1-driven Th17 polarization. Disruption of this intricate crosstalk, by blocking IL-6, IL-23, or IL-17 function, impairs the osteoclastogenic capacity of CCL21. Consistent with our in vitro findings, we establish that arthritis mediated by CCL21 expands the joint inflammation to bone erosion by connecting the differentiation of M1 macrophages with Th17 cells. Disease progression is further exacerbated by CCL21-induced neovascularization. We conclude that CCL21 is an attractive novel target for RA therapy, as blockade of its function may abrogate erosive arthritis modulated by M1 macrophages and Th17 cell crosstalk.
Asunto(s)
Artritis Reumatoide/inmunología , Quimiocina CCL21/metabolismo , Inflamación/patología , Articulaciones/patología , Macrófagos/metabolismo , Osteoclastos/patología , Receptores CCR7/metabolismo , Células Th17/inmunología , Animales , Artritis Reumatoide/sangre , Artritis Reumatoide/patología , Biomarcadores/metabolismo , Diferenciación Celular , Polaridad Celular , Quimiotaxis , Femenino , Humanos , Interleucinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Monocitos/patología , Células Mieloides/metabolismo , Osteogénesis , Receptores CCR7/sangre , Transducción de Señal , Líquido Sinovial/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: To prepare a novel nanoemulsion- Carbopol® 934 gel for Eugenol, in order to prevent the periodontitis. MATERIAL AND METHODS: Spontaneous emulsification method was used for the preparation of nanoemulsion in which it contain Eugenol (oil phase), Tween-80 (surfactant), and PEG (co-surfactant). To the development of best nanoemulsion, three-factor three-level central composite design was used in which %oil; %Smix and % water were optimized as independent variables. An optimized-nanoemulsion were converted to nanoemulsion-Carbopol® 934 gel. RESULTS: 5.5% oil, 35.5% Smix and 59.0% water were optimized as independent and dependent variables. Finally dependent variables optimized as a particle size (nm), PDI and %transmittance were observed 79.92⯱â¯6.33â¯nm, 0.229⯱â¯0.019, and 98.88⯱â¯1.31% respectively. The values of final results for dependent variables like particle size (nm), PDI and % transmittance were evaluated as 79.92⯱â¯6.33â¯nm, 0.229⯱â¯0.019, and 98.88⯱â¯1.31%, respectively. TEM and SEM showed a spherical shape of developed nanoemulsion with refractive index (1.63⯱â¯0.038), zeta potential (-19.16⯱â¯0.11), pH (7.4⯱â¯0.06), viscosity (34.28⯱â¯6 cp), and drug content of 98.8⯱â¯0.09%. After that a final optimized EUG-NE-Gel was assessed on the basis of their pH measurement, drug content, syringeability, and mucoadhesion on the goat buccal mucosa. Optimized EUG-NE-Gel (Tween-80 and Carbopol® 934 used) showed the results, to improve the periodontal drug delivery of EUG in future. CONCLUSION: EUG-NE-Gel showed a significant role in anti-inflammatory activity, analgesic, and anesthetic, antibacterial, and treatment of periodontal disease.
RESUMEN
BACKGROUND: Nomadic populations have a considerably higher risk of contracting a number of diseases but, despite the magnitude of the public health risks involved, they are mostly underserved with few health policies or plans to target them. Nomadic population movements are shown to be a niche for the transmission of diseases, including poliomyelitis. The nomadic routes traverse the northern states of Nigeria to other countries in the Lake Chad subregion. As part of the February 2016 polio supplemental immunization activity (SIA) plans in Bauchi state, a review of nomadic routes and populations identified a nomadic population who originated from outside the international borders of Nigeria. This study describes the engagement process for a transboundary nomadic population and the interventions provided to improve population immunity among them while traversing through Nigeria. METHODS: This was an intervention study which involved a cross-sectional mixed-method (quantitative and qualitative) survey. Information was collected on the nomadic pastoralists entry and exit points, resting points, and health-seeking behavior using key informant interviews and semistructured questionnaire. Transit vaccination teams targeted the groups with oral polio vaccines (OPVs) and other routine antigens along identified routes during the months of February to April 2016. Mobile health teams provided immunization and other child and maternal health survival interventions. RESULTS: A total of 2015 children aged under 5 years were vaccinated with OPV, of which 264 (13.1%) were zero-dose during the February 2016 SIAs while, in the March immunization plus days (IPDs), 1864 were immunized of which 211 (11.0%) were zero-dose. A total of 296 children aged under 1 year old were given the first dose of pentavalent vaccine (penta 1), while 119 received the third dose (penta 3), giving a dropout rate of 59.8%. CONCLUSIONS: Nomadic pastoralists move across international borders and there is a need for transboundary policies among the countries in the Lake Chad region to improve population immunity and disease surveillance through a holistic approach using the One-health concept.
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Programas de Inmunización , Poliomielitis/prevención & control , Poliomielitis/transmisión , Vacuna Antipolio Oral/administración & dosificación , Migrantes/estadística & datos numéricos , Agricultura , Chad/etnología , Preescolar , Estudios Transversales , Política de Salud , Humanos , Inmunización/estadística & datos numéricos , Lactante , Unidades Móviles de Salud , Nigeria/epidemiología , Poliomielitis/epidemiología , Evaluación de Programas y Proyectos de Salud , RiesgoRESUMEN
BACKGROUND: Irinotecan (IRN) (CPT-11) is a camptothecin derivative with low oral bioavailability due to active efflux by intestinal P-glycoprotein (p-gp) receptors. Hence, no oral formulation is marketed for IRN till date and its oral ingestion continues to remain a challenge. AIM OF STUDY: The study aims to develop a nanoformulation i.e. Chitosan (CS)-coated-IRN-loaded-poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) (CS-IRN-PLGA-NPs)in order to enhance oral bioavailability of IRN. RESULTS: Developed formulation revealed particle size, 166.9 ± 13.63 nm, zeta potential, 14.67 ± 1.08 mV and drug content (42.69 ± 1.97 µg/mg), with spherical shape and smooth surface. Cytotoxicity studies, performed against human breast adenocarcinoma cell lines (MCF-7), confirmed the superiority of IRN-CS-PLGA-NPs over free IRN solution (IRN-S). Cellular transport conducted on human colon adenocarcinoma cell line (Caco-2) exhibited a higher permeability of 1.33 folds for IRN through CS-IRN-PLGA-NPs as compared to IRN-S (p < 0.01) whereas the permeability for IRN was found to be higher at a rate of 4.32 folds, across rat ileum. Furthermore, pharmacokinetic studies demonstrated marked improvement of 3.53 fold and 8.03 fold in Wistar rat's plasma as well as brain higher oral bioavailability through IRN-CS-PLGA-NPs when compared with IRN-S. A simple, rapid UPLC-ESI-Q-TOF-MS/MS method for the determination of IRN (CPT-11) and SN-38 in both plasma and brain (over a range: 1.00-25000.00 ng/ml) was also developed and successfully applied for pharmacokinetic study. DISCUSSION: CS-IRN-PLGA-NPs approach may be effectively utilised, to replace pre-existing intravenous therapy thus providing 'patient care at home.
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Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacocinética , Camptotecina/análogos & derivados , Quitosano/análogos & derivados , Portadores de Fármacos/química , Ácido Láctico/química , Ácido Poliglicólico/química , Administración Oral , Animales , Antineoplásicos Fitogénicos/química , Disponibilidad Biológica , Encéfalo/metabolismo , Células CACO-2 , Camptotecina/administración & dosificación , Camptotecina/química , Camptotecina/farmacocinética , Humanos , Absorción Intestinal , Irinotecán , Células MCF-7 , Masculino , Nanopartículas/química , Nanopartículas/ultraestructura , Neoplasias/tratamiento farmacológico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas WistarRESUMEN
In search of potential therapeutics for inflammatory disease, we report herein the synthesis, characterization and anti-inflammatory activities of a new series of 1-{(5-substituted-1,3,4-oxadiazol-2-yl)methyl}-2-(morpholinomethyl)-1H-benzimidazoles (5a-r). The anti-inflammatory activity of the compounds was evaluated using carrageenan induced rat paw edema test. Some compounds showed excellent anti-inflammatory activity in carrageenan induced rat paw edema test. 1-{(5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl}-2-(morpholinomethyl)-1H-benzimidazole (5g) showed maximum anti-inflammatory (74.17±1.28% inhibition) with reduced ulcerogenic and lipid peroxidation profile and also showed significant COX-2 inhibition with IC50 values of 8.00µM. Compounds 5o and 5q were also found to exhibit good COX-2 inhibition with IC50 values of 11.4 and 13.7µM concentrations. Molecular docking study showed that morpholine and oxadiazole rings linked to the benzimidazole nucleus play an important role in binding with the COX-2.
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Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/uso terapéutico , Bencimidazoles/química , Bencimidazoles/uso terapéutico , Oxadiazoles/química , Oxadiazoles/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/farmacología , Bencimidazoles/efectos adversos , Bencimidazoles/farmacología , Inhibidores de la Ciclooxigenasa 2/efectos adversos , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/farmacología , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Edema/tratamiento farmacológico , Masculino , Modelos Moleculares , Simulación del Acoplamiento Molecular , Oxadiazoles/efectos adversos , Oxadiazoles/farmacología , Ratas Wistar , Úlcera/inducido químicamenteRESUMEN
Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5µM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.
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Antiinflamatorios/farmacología , Artritis Reumatoide/enzimología , Benzoquinonas/farmacología , Adhesión Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , MAP Quinasa Quinasa Quinasa 5/metabolismo , Membrana Sinovial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Artritis Reumatoide/inmunología , Artritis Reumatoide/cirugía , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/enzimología , Fibroblastos/inmunología , Humanos , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Interleucina-8/inmunología , Interleucina-8/metabolismo , MAP Quinasa Quinasa Quinasa 5/inmunología , Fosforilación , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/enzimología , Membrana Sinovial/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
AIM: Sepsis is a life-threatening clinical syndrome comprising multiorgan dysfunctions caused by a disproportionate body immune response. There are several animal sepsis models which are based on cecum ligation, cecal puncture, and cecum slurry injection. The major limitation of all current sepsis models is the high variability owing to the variable degree of ligation, puncture and inconsistent microbial composition used for sepsis initiation. The primary objective of this work is to demonstrate the feasibility of a standardized method for sepsis development. MATERIALS AND METHODS: The cecal slurry bacterial culture was developed and preserved in glycerol stocks. Antibiotics aztreonam and vancomycin were used for generating several defined, enriched cecal slurry bacterial cultures. Mice survival was assessed until 48 hrs post injection, and the tissue samples were collected after 10 hrs from sepsis initiation. KEY FINDINGS: The results indicate that increasing polymicrobial load resulted in lower survival rates and was associated with the higher number of infiltrating immune cells and necrosis. H&E (haematoxylin & eosin) staining & serum markers revealed that septic mice exhibited increased inflammation and significant damage to the liver and kidneys. The defined Gram-negative and Gram-positive specific cecal slurry bacterial cultures were developed and their efficiency in inducing sepsis was characterized. SIGNIFICANCE: Enriched cecal slurry bacterial cultures can be stored in glycerol stocks at -80 °C. This has an ethical advantage of avoiding unnecessary animal euthanasia for each experiment and provides a standardization capability of sepsis development.
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Glicerol , Sepsis , Ratones , Animales , Inyecciones Intraperitoneales , Sepsis/tratamiento farmacológico , Inflamación/complicaciones , Modelos Animales de Enfermedad , Ciego , Ligadura/efectos adversosRESUMEN
OBJECTIVES: Piperine, a main component of Piper species, is a plant alkaloid with a long history of medical use in a variety of inflammatory disorders like rheumatoid arthritis. Due to side effects in current treatment modalities of rheumatoid arthritis, the interest in alternative, well tolerated anti-inflammatory remedies has re-emerged. The aim of this work was to evaluate the anti-inflammatory and antiarthritic effects of piperine. METHODS: Arthritis was induced in male Wistar rats by collagen induced arthritis (CIA) method. Piperine was administered at a dose of 100mgkg(-1) and indomethacin at 1mgkg(-1) body weight once daily for 21days. The effects of treatment in the rats were assessed by biochemical (articular elastase, MPO, LPO, GSH, Catalase, SOD and NO), inflammatory mediators (IL-1ß, TNF-α, IL-10 and PGE2) and histological studies in joints. RESULTS: Piperine was effective in bringing significant changes on all the parameters (articular elastase, MPO, LPO, GSH, Catalase, SOD and NO) studied. Oral administration of piperine resulted in significantly reduced the levels of pro-inflammatory mediators (IL-1ß, TNF-α and PGE2) and increased level of IL-10. The protective effects of piperine against RA were also evident from the decrease in arthritis scoring and bone histology. CONCLUSIONS: In conclusion, the fact that piperine alter a number of factors known to be involved in RA pathogenesis indicates that piperine can be used similar to indomethacin as a safe and effective therapy for CIA and may be useful in the treatment of rheumatoid arthritis.
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Alcaloides/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Benzodioxoles/farmacología , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Animales , Artritis Experimental/enzimología , Artritis Experimental/inmunología , Artritis Reumatoide/enzimología , Artritis Reumatoide/inmunología , Catalasa/análisis , Dinoprostona/análisis , Glutatión/análisis , Histocitoquímica , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/inmunología , Interleucina-10/análisis , Interleucina-1beta/análisis , Masculino , Óxido Nítrico/análisis , Elastasa Pancreática/análisis , Peroxidasa/análisis , Distribución Aleatoria , Ratas , Ratas Wistar , Superóxido Dismutasa/análisis , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Rheumatoid arthritis is a chronic inflammatory disease characterized by the destruction of articular cartilage and bone in a chronic phase. Pathology of rheumatoid arthritis suggests autoimmunity linked to inflammation. In our study, rheumatoid arthritis was induced in Wistar rats by intradermal injections of 100 µl of emulsion containing bovine type II collagen in complete Freund's adjuvant at the base of the tail. Disease developed about 13 ± 1 days after immunization and treatment with hesperidin (HES) at a dose of 160 mg kg(-1) body weight was given after onset of disease daily until 20th day. The effect of treatment in the rats was monitored by clinical scoring, biochemical parameters and histological evaluations in joints. A steady increase in the articular elastase, nitric oxide and lipid peroxidation was observed in joints of arthritic rats as compared to control, whereas a significant decrease in reduced glutathione, superoxide dismutase activity and catalase was observed in collagen-induced arthritis rats as compared to control group. The results from the present work indicate that the treatment with hesperidin was effective in bringing about significant changes on all the parameters studied in collagen-induced arthritis rats. These data confirm that erosive destruction of the joint cartilage in collagen-induced arthritis is due free radicals released by activated neutrophils and produced by other biochemical pathways. In the present study, an attempt has been made to amelioration of the disease process by a natural product. These results suggest that oral administration of HES could be effective for treating human RA patients.
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Artritis Experimental/tratamiento farmacológico , Radicales Libres/metabolismo , Hesperidina/farmacología , Activación Neutrófila/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Catalasa/metabolismo , Hesperidina/uso terapéutico , Masculino , Óxido Nítrico/análisis , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
O-GlcNAcylation is a reversible post-translational modification that regulates numerous cellular processes, including embryonic development as well as immune responses. However, its role in inflammation remains ambiguous. This study was designed to examine the role of O-GlcNAcylation in rheumatoid arthritis (RA) and its regulation using human RA patient-derived synovial fibroblasts (RASFs). The efficacy of penta-O-galloyl-beta-D-glucose (PGG), a potent anti-inflammatory molecule, in regulating inflammatory processes in human RASFs was also evaluated. Human synovial tissues and RASFs exhibited higher expression of O-GlcNAcylation compared to their non-diseased counterparts. Pretreatment of RASFs with Thiamet G, an inhibitor of O-GlcNAcase, markedly increased the O-GlcNAc-modified proteins and concomitantly inhibited the IL-1ß-induced IL-6 and IL-8 production in human RASFs in vitro. Pretreatment of human RASFs with PGG (0.5-10 µM) abrogated IL-1ß-induced IL-6 and IL-8 production in a dose-dependent manner. Immunoprecipitation analysis showed that PGG inhibited O-GlcNAcylation of TAB1 to reduce its association with TGF ß-activated kinase 1 (TAK1) and its autophosphorylation, an essential signaling step in IL-1ß-induced signaling pathways. Molecular docking in silico studies shows that PGG occupies the C174 position, an ATP-binding site in the kinase domain to inhibit TAK1 kinase activity. Oral administration of PGG (25 mg/kg/day) for 10 days from disease onset significantly ameliorated rat adjuvant-induced (AIA) in rats. PGG treatment reduced the phosphorylation of TAK1 in the treated joints compared to AIA joints, which correlated with the reduced disease severity and suppressed levels of serum IL-1ß, GM-CSF, TNF-α, and RANKL. These findings suggest O-GlcNAcylation as a potential therapeutic target and provide the rationale for testing PGG or structurally similar molecule for their therapeutic efficacy.
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Artritis Experimental , Artritis Reumatoide , Glucosa , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Fibroblastos , Glucosa/farmacología , Humanos , Inflamación , Interleucina-6 , Interleucina-8 , Simulación del Acoplamiento Molecular , Ratas , Membrana SinovialRESUMEN
Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.
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Apoptosis , Encefalomielitis Autoinmune Experimental/patología , Neuronas/citología , Animales , Femenino , Inmunohistoquímica , Ratas , Ratas WistarRESUMEN
Scorpion and its organs have been used to cure epilepsy, rheumatism, and male impotency since medieval times. Scorpion venom which contains different compounds like enzyme and non-enzyme proteins, ions, free amino acids, and other organic inorganic substances have been reported to posses antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. We for the first time report the apoptotic and antiproliferative effects of scorpion venom (Odontobuthus doriae) in human neuroblastoma cells. After exposure of cells to medium containing varying concentrations of venom (10, 25, 50, 100, and 200 µg/ml), cell viability decreased to 90.75, 75.53, 55.52, 37.85, and 14.30%, respectively, after 24 h. Cells expressed morphological changes like swelling, inhibition of neurite outgrowth, irregular shape, aggregation, rupture of membrane, and release of cytosolic contents after treatment with venom. Lactate dehydrogenase (LDH) level increased in 50 and 100 µg/ml as compared to control, but there was no significant increase in LDH level at a dose of 10 and 20 µg/ml. Two concentrations viz. 50 and 100 µ/ml were selected because of the profound effect of these concentrations on the cellular health and population. Treatment with these two concentrations induced reactive nitrogen intermediates and depolarization in mitochondria. While caspase-3 activity increased in a concentration-dependent manner, only 50 µg/ml was able to fragment DNA. It was interesting to note that at higher dose, i.e., 100 µg/ml, the cells were killed, supposedly by acute necrosis. DNA synthesis evidenced by bromodeoxyuridine (BrdU) incorporation was inhibited in a concentration-dependent manner. The cells without treatment incorporated BrdU with high affinity confirming their cancerous nature whereas very less incorporation was noticed in treated cells. Our results show apoptotic and antiproliferative potential of scorpion venom (O. doriae) in human neuroblastoma cells. These properties make scorpion venom a valuable therapeutic agent in cancer research.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Neuroblastoma/patología , Venenos de Escorpión/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , L-Lactato Deshidrogenasa/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Factores de TiempoRESUMEN
The purpose of study was to examine the cytotoxic and anti-cancer properties along with addressing the plausible pathway followed by scorpion venom to reduce cell viability in SH-SY5Y and MCF-7 cells. Following exposure of cells with scorpion venom, cytotoxicity was estimated using MTT and lactate dehydrogenase assays. Apoptotic effects were measured by assessment of mitochondrial membrane potential, reactive nitrogen species, DNA fragmentation, and caspase-3 activity whereas antiproliferative effect was assayed using BrdU incorporation. Our results indicate that scorpion venom causes suppression of proliferation by arresting S-phase and induction of apoptosis through increased nitric oxide production, caspase-3 activity and depolarization of mitochondrial membrane. Induction of apoptosis and arrest of DNA synthesis are critical determinant factors for development of anti cancer drugs. These properties may lead to isolation of effective molecule(s) with potential anticancer activity from scorpion venom of Androctonus crassicauda.