RESUMO
BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.
Assuntos
Reação de Fase Aguda/genética , Dano ao DNA/genética , Estresse do Retículo Endoplasmático/genética , Regeneração Hepática/genética , Fígado/metabolismo , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Células Hep G2 , Hepatectomia , Humanos , Interleucina-6/genética , Camundongos , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT3/metabolismoRESUMO
Discovery of potent and safe therapeutics that improve upon currently available antifibrinolytics, e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix metalloproteinases (MMPs) participate in thrombus dissolution. Then we designed a novel series of optimized MMP inhibitors that went through phenotypic screening consisting of thromboelastometry and mouse tail bleeding. Our optimized lead compound, CM-352 (2), inhibited fibrinolysis in human whole blood functional assays and was more effective than the current standard of care, 1, in the tail-bleeding model using a 30â¯000 times lower dose. Moreover, 2 reduced blood loss during liver hepatectomy, while 1 and aprotinin had no effect. Molecule 2 displayed optimal pharmacokinetic and safety profiles with no evidence of thrombosis or coagulation impairment. This novel mechanism of action, targeting MMP, defines a new class of antihemorrhagic agents without interfering with normal hemostatic function. Furthermore, 2 represents a preclinical candidate for the acute treatment of bleeding.
Assuntos
Benzamidas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Hemorragia/prevenção & controle , Hemostáticos/química , Hemostáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Animais , Antifibrinolíticos/química , Antifibrinolíticos/farmacologia , Benzamidas/química , Células CACO-2/efeitos dos fármacos , Descoberta de Drogas/métodos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Hemorragia/tratamento farmacológico , Hemorragia/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos Endogâmicos C57BL , Estrutura Molecular , Terapia de Alvo Molecular/métodosRESUMO
OBJECTIVE: Only a limited proportion of patients needing pharmacological control of portal hypertension are hemodynamic responders to propranolol. Here we analyzed the effects of zolmitriptan on portal pressure and its potential interaction with propranolol. METHODS: ZOLMITRIPTAN, PROPRANOLOL OR BOTH WERE TESTED IN TWO RAT MODELS OF PORTAL HYPERTENSION: common bile duct ligation (CBDL) and CCl4-induced cirrhosis. In these animals we measured different hemodynamic parameters including portal venous pressure, arterial renal flow, portal blood flow and cardiac output. We also studied the changes in superior mesenteric artery perfusion pressure and in arterial wall cAMP levels induced by zolmitriptan, propranolol or both. Moreover, we determined the effect of splanchnic sympathectomy on the response of PVP to zolmitriptan. RESULTS: In both models of portal hypertension zolmitriptan induced a dose-dependent transient descent of portal pressure accompanied by reduction of portal flow with only slight decrease in renal flow. In cirrhotic rats, splanchnic sympathectomy intensified and prolonged zolmitriptan-induced portal pressure descent. Also, propranolol caused more intense and durable portal pressure fall when combined with zolmitriptan. Mesenteric artery perfusion pressure peaked for about 1 min upon zolmitriptan administration but showed no change with propranolol. However propranolol enhanced and prolonged the elevation in mesenteric artery perfusion pressure induced by zolmitriptan. In vitro studies showed that propranolol prevented the inhibitory effects of ß2-agonists on zolmitriptan-induced vasoconstriction and the combination of propranolol and zolmitriptan significantly reduced the elevation of cAMP caused by ß2-agonists. CONCLUSION: Zolmitriptan reduces portal hypertension and non-selective beta-blockers can improve this effect. Combination therapy deserves consideration for patients with portal hypertension failing to respond to non-selective beta-blockers.
Assuntos
Anti-Hipertensivos/farmacologia , Hipertensão Portal/tratamento farmacológico , Hipertensão Portal/fisiopatologia , Oxazolidinonas/farmacologia , Pressão na Veia Porta/efeitos dos fármacos , Propranolol/farmacologia , Triptaminas/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Anti-Hipertensivos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Tetracloreto de Carbono , Catecolaminas/farmacologia , Ducto Colédoco/efeitos dos fármacos , Ducto Colédoco/patologia , Ducto Colédoco/fisiopatologia , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Infusões Intravenosas , Ligadura , Cirrose Hepática Experimental/tratamento farmacológico , Cirrose Hepática Experimental/fisiopatologia , Lipressina/análogos & derivados , Lipressina/farmacologia , Lipressina/uso terapêutico , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/patologia , Artéria Mesentérica Superior/fisiopatologia , Oxazolidinonas/administração & dosagem , Oxazolidinonas/uso terapêutico , Perfusão , Propranolol/uso terapêutico , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Artéria Renal/efeitos dos fármacos , Artéria Renal/fisiopatologia , Circulação Esplâncnica/efeitos dos fármacos , Simpatectomia , Terlipressina , Triptaminas/administração & dosagem , Triptaminas/uso terapêutico , Vasodilatação/efeitos dos fármacosRESUMO
OBJECTIVE: Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. Fibroblast growth factor 15 (Fgf15, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf15 in the preservation of BA homeostasis after PH and its potential role in the regenerative process. DESIGN: Liver regeneration after PH was studied in Fgf15 (-/-) and Fgf15 (+/+) mice. The effects of the BA sequestrant cholestyramine and adenovirally delivered Fgf15 were examined in this model. The role of Fgf15 in BA-induced liver growth was tested in Fgf15 (-/-) mice upon cholic acid (CA) feeding. The direct mitogenic effect of Fgf15 was evaluated in cultured mouse hepatocytes and cholangiocytes. RESULTS: Fgf15 (-/-) mice showed marked liver injury and mortality after PH accompanied by persistently elevated intrahepatic BA levels. Cholestyramine feeding and adenovirally delivered Fgf15 reduced BA levels and significantly prevented this lethal outcome. Fgf15 also reduced mortality after extensive hepatectomy in Fgf15(+/+) animals. Liver growth elicited by CA feeding was significantly diminished in Fgf15 (-/-) mice. Proliferation of hepatocytes and cholangiocytes was also noticeably reduced in CA-fed Fgf15 (-/-) mice. Fgf15 induced intracellular signalling and proliferation of cultured hepatocytes and cholangiocytes. CONCLUSIONS: Fgf15 is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf15 is an essential mediator of the liver growth-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.