RESUMO
We hypothesized whether propofol or active propofol component (2,6-diisopropylphenol [DIPPH] and lipid excipient [LIP-EXC]) separately may alter inflammatory mediators expressed by macrophages and neutrophils in lean and obese rats. Male Wistar rats (n = 10) were randomly assigned to receive a standard (lean) or obesity-inducing diet (obese) for 12 weeks. Animals were euthanized, and alveolar macrophages and neutrophils from lean and obese animals were exposed to propofol (50 µM), active propofol component (50 µM, 2,6-DIPPH), and lipid excipient (soybean oil, purified egg phospholipid, and glycerol) for 1 h. The primary outcome was IL-6 expression after propofol and its components exposure by alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were the production of mediators released by macrophages from adipose tissue, and neutrophils from lung and adipose tissues, and neutrophil migration. IL-6 increased after the exposure to both propofol (median [interquartile range] 4.14[1.95-5.20]; p = .04) and its active component (2,6-DIPPH) (4.09[1.67-5.91]; p = .04) in alveolar macrophages from obese animals. However, only 2,6-DIPPH increased IL-10 expression (7.59[6.28-12.95]; p = .001) in adipose tissue-derived macrophages. Additionally, 2,6-DIPPH increased C-X-C chemokine receptor 2 and 4 (CXCR2 and CXCR4, respectively) in lung (10.08[8.23-29.01]; p = .02; 1.55[1.49-3.43]; p = .02) and adipose tissues (8.78[4.15-11.57]; p = .03; 2.86[2.17-3.71]; p = .01), as well as improved lung-derived neutrophil migration (28.00[-3.42 to 45.07]; p = .001). In obesity, the active component of propofol affected both the M1 and M2 markers as well as neutrophils in both alveolar and adipose tissue cells, suggesting that lipid excipient may hinder the effects of active propofol.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Anestésicos Intravenosos/farmacologia , Excipientes/farmacologia , Interleucina-6/metabolismo , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Obesidade/metabolismo , Propofol/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Glicerol/farmacologia , Interleucina-10/metabolismo , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Neutrófilos/metabolismo , Fosfolipídeos/farmacologia , Ratos , Receptores CXCR4/efeitos dos fármacos , Receptores CXCR4/metabolismo , Receptores de Interleucina-8B/efeitos dos fármacos , Receptores de Interleucina-8B/metabolismo , Óleo de Soja/farmacologiaRESUMO
BACKGROUND: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. METHODS: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. RESULTS: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. CONCLUSION: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates.
Assuntos
Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Vacinas Protozoárias/farmacologia , Baço/efeitos dos fármacos , Animais , Anticorpos Anti-Helmínticos/imunologia , Calgranulina A/efeitos dos fármacos , Calgranulina A/genética , Epitopos/imunologia , Feminino , Perfilação da Expressão Gênica , Interleucina-12/genética , Interleucina-8/efeitos dos fármacos , Interleucina-8/genética , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Camundongos , Peptídeos/imunologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Interleucina-8B/efeitos dos fármacos , Receptores de Interleucina-8B/genética , Baço/metabolismo , Regulação para Cima , VacinaçãoRESUMO
A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.