Naofen promotes TNF-α-mediated apoptosis of hepatocytes by activating caspase-3 in lipopolysaccharide-treated rats In vivo in vitro

AIM: To investigate whether naofen is involved in tumor necrosis factor (TNF)-alpha-mediated apoptosis of hepatocytes induced by lipopolysaccharide (LPS). METHODS: In vivo, rats were treated with LPS or anti-TNF-alpha antibody, whereas in vitro, primary hepatocytes and Kupffer cells (KCs) were separately isolated from rat livers using collagenase perfusion, and primary hepatocytes were cultured in medium containing LPS or TNF-alpha, or in conditioned medium from LPS-treated KCs (KC-CM)/KC-CM + anti-TNF-alpha antibody. Naofen and TNF-alpha mRNA expression was examined by real-time reverse transcription-polymerase chain reaction. Immunoblotting was used to measure protein expression. Hepatocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: LPS significantly induced both naofen expression and caspase-3 activity in the rat liver, which coincided with an increase in the number of TUNEL-positive hepatocytes. The increase of TNF-alpha expression induced by LPS was preceded by increases in naofen and caspase-3 activity. Elevation of naofen expression and caspase-3 activity was abrogated by pretreatment with anti-TNF-alpha antibody. In KCs, LPS caused an increase in TNF-alpha that was almost consistent with that in the liver of LPS-treated rats. In hepatocytes, neither LPS nor TNF-alpha alone affected either naofen expression or caspase-3 activation. The incubation of hepatocytes with KC-CM significantly enhanced both naofen expression and caspase-3 activity. Moreover, the effects of the KC-CM-induced increase in naofen expression and caspase-3 activity were blocked by anti-TNF-alpha antibody. CONCLUSION: TNF-alpha released from KCs treated with LPS may induce hepatic naofen expression, which then stimulates hepatocellular apoptosis through activation of caspase-3. (C) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.