RESUMO
Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.
Assuntos
Resistência à Insulina , Tecido Adiposo/metabolismo , Animais , Homeostase , Gordura Intra-Abdominal/metabolismo , Camundongos , Obesidade/genética , Obesidade/metabolismo , ProteômicaRESUMO
Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3'- or 5'-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3'- and 5'-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.
Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Oligonucleotídeos/síntese química , Compostos Organofosforados/química , Interferência de RNA , RNA Interferente Pequeno/síntese química , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Camundongos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Projetos de Pesquisa , Sonicação , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Fluxo de TrabalhoRESUMO
Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors.
Assuntos
Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Carcinoma Epitelial do Ovário , Feminino , Recombinação Homóloga , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Estresse Oxidativo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
BACKGROUND: Butyrate-producing gut bacteria are reduced in patients with active inflammatory bowel disease (IBD), supporting the hypothesis that butyrate supplementation may be beneficial in this setting. Nonetheless, earlier studies suggest that the oxidation of butyrate in IBD patients is altered. We propose that inflammation may decrease epithelial butyrate consumption. METHODS: Non-IBD controls and IBD patients were recruited for the study. Stool samples were used for short-chain fatty acid and bacterial butyryl CoA:acetate CoA-transferase quantification. Colonic biopsies and ex vivo differentiated epithelial organoids (d-EpOCs) treated with butyrate and/or tumor necrosis factor alpha (TNFα) were used for analyzing the expression of transporters MCT1 and ABCG2, metabolic enzyme ACADS, and butyrate receptor GPR43, and for butyrate metabolism and consumption assays. RESULTS: We observed that lower stool content of butyrate-producing bacteria in active IBD patients did not correlate with decreased butyrate concentrations. Indeed, the intestinal epithelial expression of MCT1, ABCG2, ACADS, and GPR43 was altered in active IBD patients. Nonetheless, d-EpOCs derived from IBD patients showed SLC16A1 (gene encoding for MCT1 protein), ABCG2, ACADS, and GPR43 expression levels comparable to controls. Moreover, IBD- and non-IBD-derived d-EpOCs responded similarly to butyrate, as assessed by transcriptional regulation. TNFα significantly altered SLC16A1, ABCG2, and GPR43 transcription in d-EpOCs, mimicking the expression profile observed in biopsies from active IBD patients and resulting in reduced butyrate consumption. CONCLUSIONS: We provide evidence that the response to butyrate is not intrinsically altered in IBD patients. However, TNFα renders the epithelium less responsive to this metabolite, defeating the purpose of butyrate supplementation during active inflammation.
Assuntos
Butiratos/metabolismo , Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos de Casos e Controles , Técnicas de Cultura de Células , Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologia , Fezes/química , Fezes/microbiologia , Feminino , Humanos , Inflamação , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/patologia , Intestinos/patologia , Masculino , Pessoa de Meia-Idade , Organoides/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Transforming Growth Factor beta (TGF-ß) induces tumor cell migration and invasion. However, its role in inducing metabolic reprogramming is poorly understood. Here we analyzed the metabolic profile of hepatocellular carcinoma (HCC) cells that show differences in TGF-ß expression. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), metabolomics and transcriptomics were performed. Results indicated that the switch from an epithelial to a mesenchymal/migratory phenotype in HCC cells is characterized by reduced mitochondrial respiration, without significant differences in glycolytic activity. Concomitantly, enhanced glutamine anaplerosis and biosynthetic use of TCA metabolites were proved through analysis of metabolite levels, as well as metabolic fluxes from U-13C6-Glucose and U-13C5-Glutamine. This correlated with increase in glutaminase 1 (GLS1) expression, whose inhibition reduced cell migration. Experiments where TGF-ß function was activated with extracellular TGF-ß1 or inhibited through TGF-ß receptor I silencing showed that TGF-ß induces a switch from oxidative metabolism, coincident with a decrease in OCR and the upregulation of glutamine transporter Solute Carrier Family 7 Member 5 (SLC7A5) and GLS1. TGF-ß also regulated the expression of key genes involved in the flux of glycolytic intermediates and fatty acid metabolism. Together, these results indicate that autocrine activation of the TGF-ß pathway regulates oxidative metabolism in HCC cells.
Assuntos
Glicólise/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Transcriptoma , Fator de Crescimento Transformador beta1/farmacologia , Comunicação Autócrina , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Ácidos Graxos/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/metabolismo , Glutamina/farmacologia , Glicólise/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Metaboloma , Consumo de Oxigênio/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
This study investigated the metabolic requirements for neuronal progenitor maintenance in vitro and in vivo by examining the metabolic adaptations that support neuronal progenitors and neural stem cells (NSCs) in their undifferentiated state. We demonstrate that neuronal progenitors are strictly dependent on lactate metabolism, while glucose induces their neuronal differentiation. Lactate signaling is not by itself capable of maintaining the progenitor phenotype. The consequences of lactate metabolism include increased mitochondrial and oxidative metabolism, with a strict reliance on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway to support anabolic functions, such as the production of extracellular matrix. In vivo, lactate maintains/induces populations of postnatal neuronal progenitors/NSCs in a PEPCK-M-dependent manner. Taken together, our data demonstrate that, lactate alone or together with other physical/biochemical cues maintain NSCs/progenitors with a metabolic signature that is classically found in tissues with high anabolic capacity.
Assuntos
Ácido Láctico/metabolismo , Células-Tronco Neurais/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Bromodesoxiuridina , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Glucose/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidoresRESUMO
The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter ß-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Intestinos/virologia , Proteínas Virais/genética , Tropismo Viral , Animais , Linhagem Celular , Suco Gástrico/química , Suco Gástrico/metabolismo , Marcação de Genes , Humanos , Concentração de Íons de Hidrogênio , Mucosa Intestinal/metabolismo , Camundongos , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribuição Tecidual , Transdução Genética , Proteínas Virais/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared with parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein-protein interaction network and differential expression. The protein-protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub that retained four down-regulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E, and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias da Mama/metabolismo , Metástase Neoplásica , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos SCID , Mapeamento de Interação de Proteínas , Proteoma , Transcriptoma , Vimentina/metabolismoRESUMO
Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked.
Assuntos
Aminoácidos/metabolismo , Glicólise/fisiologia , Miócitos Cardíacos/enzimologia , Fosfofrutoquinase-2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Aminoácidos/genética , Animais , Ativação Enzimática/fisiologia , Frutosedifosfatos/genética , Frutosedifosfatos/metabolismo , Glucose/genética , Glucose/metabolismo , Células HEK293 , Células HeLa , Humanos , Ácido Láctico/metabolismo , Masculino , Miócitos Cardíacos/citologia , Fosfofrutoquinase-2/genética , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Serina/genética , Serina/metabolismoRESUMO
North bicyclo methanocarba thymidine (T(N)) nucleosides were substituted into siRNAs to investigate the effect of bicyclo[3.1.0]hexane 2'-deoxy-pseudosugars on RNA interference activity. Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery. We studied the effect of the T(N) modification in a screen involving residue-specific changes in an siRNA targeting Renilla luciferase and we applied the most effective pattern of modification to the knockdown of murine tumor necrosis factor (TNF-α). We also showed that incorporation of T(N) units into siRNA duplexes increased their thermal stabilities, substantially enhanced serum stabilities, and decreased innate immunostimulation. Comparative RNAi studies involving the T(N) substitution and locked nucleic acids (LNAs) showed that the gene-silencing activities of T(N) -modified siRNAs were comparable to those obtained with the LNA modification. An advantage of the North 2'-deoxy-methanocarba modification is that it may be explored further in the future by changing the 2'-position. The results from these studies suggest that this modification might be valuable for the development of siRNAs for therapeutic applications.
Assuntos
Compostos Bicíclicos com Pontes/química , Interferência de RNA , RNA Interferente Pequeno/química , Animais , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Camundongos , Estrutura Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/síntese química , Temperatura , Timidina/análogos & derivados , Timidina/químicaRESUMO
Oligoribonucleotide conjugates and the corresponding siRNA duplexes against tumor necrosis factor carrying one, two, or four glucose and galactose residues at the 5'-end have been prepared using phosphoramidite chemistry. Carbohydrate-modified siRNA duplexes have similar inhibitory properties than unmodified RNA duplexes in HeLa cells transfected with oligofectamine. When HeLa cells were treated with siRNA carrying one, two, or four glucose residues without oligofectamine, no inhibition was observed. The inhibitory properties of siRNA carrying galactose residues without transfecting agent were tested on HuH-7 cells that have abundant asialoglycoprotein receptors. In these cells siRNA carrying galactose residues have slight anti-TNF inhibitory properties (25% in the best case) that are eliminated if the receptors are blocked with a competitor. These results demonstrate receptor-mediated uptake of siRNA carrying galactose residues, although the efficacy of the process is not enough for efficient RNA interference experiments.
Assuntos
Desenho de Fármacos , RNA Interferente Pequeno , Fatores de Necrose Tumoral/genética , Linhagem Celular Tumoral , Galactose/química , Glucose/química , Células HeLa , Humanos , Lipídeos/farmacologia , Compostos Organofosforados/química , Interferência de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Necrose Tumoral/químicaRESUMO
Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3'-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3'-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.
Assuntos
Sinais de Localização Nuclear/síntese química , Oligorribonucleotídeos/síntese química , Peptídeos/síntese química , Interferência de RNA , RNA Interferente Pequeno/síntese química , Análise de Variância , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Células HeLa , Humanos , Nucleoplasminas/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The up-regulation of glycolysis to enhance the production of energy under reduced pO(2) is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5'-flanking region. Extensive analysis of the 5' pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1alpha null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.
Assuntos
Hipóxia Celular/fisiologia , Regiões Promotoras Genéticas , Proteínas/genética , Proteínas/fisiologia , Fatores de Transcrição/química , Animais , Sítios de Ligação , Biotinilação , Western Blotting , Linhagem Celular , Proliferação de Células , Cobalto/química , Desferroxamina/química , Elementos Facilitadores Genéticos , Frutosedifosfatos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes Reporter , Glicina/química , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Quelantes de Ferro/farmacologia , Luciferases/metabolismo , Camundongos , Modelos Genéticos , Oligonucleotídeos/química , Oxigênio/metabolismo , Fosfofrutoquinase-2 , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Estrogênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Software , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
The present study evaluates the effectiveness of ischemic preconditioning and Bcl-2 overexpression against the liver and lung damage that follow hepatic ischemia-reperfusion and investigates the underlying protective mechanisms. Preconditioning and Bcl-2, respectively, reduced the increased tumor necrosis factor (TNF) and macrophage inflammatory protein-2 (MIP)-2 levels observed after hepatic reperfusion. Bcl-2 overexpression or anti-MIP-2 pretreatment seems to be more effective than preconditioning or anti-TNF pretreatment against inflammatory response, microcirculatory disorders, and subsequent hepatic ischemia-reperfusion injury. Furthermore, each one of these strategies individually was unable to completely inhibit hepatic injury. The combination of preconditioning and Bcl-2 overexpression as well as the combined anti-TNF and anti-MIP-2 pretreatment totally prevented hepatic injury, whereas the benefits of preconditioning and Bcl-2 were abolished by TNF and MIP-2. In contrast to preconditioning, Bcl-2 did not modify lung damage induced by hepatic reperfusion. This could be explained by the differential effect of both treatments on TNF release. Anti-TNF therapy or preconditioning, by reducing TNF release, reduced pulmonary inflammatory response, whereas the benefits of preconditioning on lung damage were abolished by TNF. Thus, the induction of both Bcl-2 overexpression in liver and preconditioning, as well as pharmacological strategies that simulated their benefits, such as anti-TNF and anti-MIP-2 therapies, could be new strategies aimed to reduce lung damage and inhibit the hepatic injury associated with hepatic ischemia-reperfusion.