RESUMO
UNLABELLED: Leptin signaling is involved in T-cell polarization and is required for profibrotic function of hepatic stellate cells (HSCs). Leptin-deficient ob/ob mice do not develop liver fibrosis despite the presence of severe long-standing steatohepatitis. Here, we blocked leptin signaling with our recently generated mouse leptin antagonist (MLA), and examined the effects on chronic liver fibrosis in vivo using the chronic thioacetamide (TAA) fibrosis model, and in vitro using freshly-isolated primary HSCs. In the chronic TAA fibrosis model, leptin administration was associated with significantly enhanced liver disease and a 100% 5-week to 8-week mortality rate, while administration or coadministration of MLA markedly improved survival, attenuated liver fibrosis, and reduced interferon gamma (IFN-gamma) levels. No significant changes in weight, serum cholesterol, or triglycerides were noted. In vitro administration of rat leptin antagonist (RLA), either alone or with leptin, to rat primary HSCs reduced leptin-stimulated effects such as increased expression of alpha-smooth muscle actin (alpha-SMA), and activation of alpha1 procollagen promoter. CONCLUSION: Inhibition of leptin-enhanced hepatic fibrosis may hold promise as a future antifibrotic therapeutic modality.
Assuntos
Células Estreladas do Fígado/fisiologia , Antagonistas de Hormônios/farmacologia , Leptina/antagonistas & inibidores , Leptina/fisiologia , Cirrose Hepática/tratamento farmacológico , Animais , Feminino , Células Estreladas do Fígado/efeitos dos fármacos , Antagonistas de Hormônios/uso terapêutico , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Camundongos , Modelos Animais , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Transdução de Sinais/fisiologia , TioacetamidaRESUMO
DNA inserts encoding human interleukin 10 (hIL-10), optimized for codon usage and secondary RNA structure, were purchased from several commercial sources and subcloned into a pMon vector. Despite the optimization, protein expression was nil. We therefore subjected the 5' segment of the cDNA encoding N-terminal amino acids 2-11 to degenerate PCR in order to create a small library of 130K theoretical cDNA combinations that would not change the respective amino acid sequence and tested their expression. After screening over 320 colonies 10 hIL-10 clones encoding the original amino acid sequence were identified. Three nucleotide substitutions were sufficient to ensure reasonable protein expression. Subsequently, hIL-10 was expressed in Escherichia coli, refolded and purified to homogeneity, yielding over 95% electrophoretically pure noncovalent homodimeric protein, which was biologically active in MC/9 cells. The yield of recombinant hIL-10 from 10L of fermentation culture was 60mg and a protocol for its long-term storage as a carrier-free lyophilized powder at -20 degrees was developed.