RESUMO
AIM: Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Effective therapies for liver fibrosis are lacking. Interleukin-10 (IL-10) is a cytokine that downregulates pro-inflammatory responses and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to investigate whether electroporative IL-10 gene therapy has a hepatic fibrolytic effect on mice. METHODS: Hepatic fibrosis was induced by administering carbon tetrachloride (CCl4) for 10 weeks in mice. The human IL-10 expression plasmid was delivered via electroporation after hepatic fibrosis was established. Histopathology, reverse transcription polymerase chain reaction (RT-PCR), immunoblotting, and gelatin zymography were used to investigate the possible mechanisms of action of IL-10. RESULTS: Human IL-10 gene therapy reversed CCl4-induced liver fibrosis in mice. RT-PCR revealed that IL-10 gene therapy attenuated liver TGF-beta1, collagen alpha1, fibronectin, and cell adhesion molecule mRNA upregulation. Following gene transfer, both the activation of alpha-smooth muscle actin and cyclooxygenase-2 were significantly attenuated. Furthermore, IL-10 significantly inhibited matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase (TIMP) activation after CCl4 intoxication. CONCLUSIONS: We demonstrated that IL-10 gene therapy attenuated CCl4-induced liver fibrosis in mice. IL-10 prevented upregulated fibrogenic and pro-inflammatory gene responses. Its collagenolytic effect may be attributed to MMP and TIMP modulation. IL-10 gene therapy may be an effective therapeutic modality against liver fibrosis with potential clinical use.
Assuntos
Terapia Genética , Interleucina-10/genética , Cirrose Hepática Experimental , Actinas/metabolismo , Animais , Colágeno Tipo I/metabolismo , Ciclo-Oxigenase 2/metabolismo , Eletroporação , Fibronectinas/metabolismo , Técnicas de Transferência de Genes , Molécula 1 de Adesão Intercelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Experimental/terapia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Inibidores Teciduais de Metaloproteinases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Interleukin-10 (IL-10) is a cytokine that downregulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to investigate whether IL-10 gene therapy possesses anti-hepatic fibrogenesis in mice. Liver fibrosis was induced by long-term thioacetamide administration in mice. Human IL-10 expression plasmid was delivered via electroporation after liver fibrosis established. IL-10 gene therapy reversed hepatic fibrosis and prevented cell apoptosis in a thioacetamide-treated liver. RT-PCR revealed IL-10 gene therapy to reduce liver transforming growth factor-beta1, tumor necrosis factor-alpha, collagen alpha1, cell adhesion molecule, and tissue inhibitors of metalloproteinase mRNA upregulation. Following gene transfer, the activation of alpha-smooth muscle actin and cyclooxygenase-2 was significantly attenuated. In brief, IL-10 gene therapy might be an effective therapeutic reagent for liver fibrosis with potential future clinical applications.
Assuntos
Terapia Genética , Interleucina-10/genética , Cirrose Hepática/terapia , Tioacetamida/toxicidade , Animais , Sequência de Bases , Western Blotting , Ciclo-Oxigenase 2 , Primers do DNA , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Cirrose Hepática/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos ICR , Prostaglandina-Endoperóxido Sintases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: To investigate effects of the cyclin-dependent kinase5 (Cdk5) inhibitor roscovitine on formalin-induced nociceptive responses in rats. METHODS: The flinch response as a methood of pain threshold measurement and intrathecal injection techniques were used. Cdk5 and phosphorylation of its downstream target, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa), were investigated by Western blot analysis. RESULTS: Rats demonstrated a typical flinch response after formalin injection. Intrathecal roscovitine injections significantly suppressed the flinch response in a dose-dependent manner. Western blot analysis showed that phosphorylated DARPP-32 at Thr75 increased in concentration after formalin hyperalgesia, with this effect reduced by roscovitine administration. This antinociception was partially attenuated by administration of naloxone before the formalin test. CONCLUSION: DARPP-32 phosphorylation is involved in acute inflammatory pain response. Intrathecal roscovitine administration attenuates formalin-induced nociceptive responses and there is potential for further application.