692 NOS-3 REGULATION BY OXIDATIVE STRESS IN A CELLULAR MODEL OF CHOLESTATIC DAMAGE

Abstract

Listen

Translate article

Background: During cholestatic liver disease, excessive accumulation of hydrophobic bile acids exerts a cytotoxic effect leading to cell death and tissue damage. Oxidative stress plays a key role in this process by promoting the development of fibrosis, cirrhosis, portal hypertension and chronic liver failure. Furthermore, in cellular models of cholestatic damage it has been established a cytoprotective role for nitric oxide (NO). The aim of the study was to evaluate the regulation of endothelial nitric oxide synthase (NOS-3) in a cellular model of cytotoxicity by glycochenodeoxycholic acid (GCDCA) and its relationship with the oxidative stress and cell death. Materials and Methods: A kinetic study was performed (0–24 hours) for induction of cell death by GCDCA (0.5 mM) in the human hepatocarcinoma cell line HepG2. The compound Mn (III) tetrakis (4-benzoic acid) porphyrinchloride (MnTBAP, 1mg/mL) was tested as an antioxidant molecule. The detection of reactive oxygen species and assessment of cell death was performed spectrophotometrically by using the probes 2,7-dichlorofluorescein diacetate and dihydroetidium, and by measuring caspase-3 activation and lactate dehydrogenase cellular release, respectively. NOS activity was determined by analyzing nitrite and nitrate accumulation in the extracellular medium. NOS-3 expression was measured by RT-qPCR and western-blot. The promoter activity of Nos-3 gene (1601 bp) was assessed using the luciferase activity assay. The identification of transcription factors (TFs) that could be involved in the NOS-3 regulation was performed using prediction programs. Chromatin immunoprecipitation assay and western-blot were used for further analysis and for the identification of the TFs binding sites in the Nos-3 promoter. Results: GCDCA administration was associated to oxidative stress increase and Nos-3 promoter activity decrease, with a reduction in NOS-3 expression and cellular NO production. The expression and the binding of TFs cJun, cFos and SP1 to the Nos-3 promoter (identified positions), as well as the phosphorylation of protein kinases JNK and ERK1/2, were related to GCDCA-induced hepatocellular damage. MnTBAP treatment prevented the cellular effects of GCDCA. Conclusions: GCDCA-induced cell death was associated to NOS-3 expression/activity decrease by oxidative-stress. This fact was related to JNK and ERK1/2 phosphorilation, and Nos-3 promoter binding increase of TFs cJun, cFos and SP1.