Original Article
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World J Gastroenterol. May 7, 2014; 20(17): 4963-4971
Published online May 7, 2014. doi: 10.3748/wjg.v20.i17.4963
Naofen promotes TNF-α-mediated apoptosis of hepatocytes by activating caspase-3 in lipopolysaccharide-treated rats
Jun-Hua Fan, Guo-Gang Feng, Lei Huang, Guo-Duo Tang, Hai-Xing Jiang, Jing Xu
Jun-Hua Fan, Guo-Duo Tang, Hai-Xing Jiang, Department of Gastroenterology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
Guo-Gang Feng, Lei Huang, Department of Pharmacology, Aichi Medical University School of Medicine, Nagakute, Aichi-gun 480-1195, Japan
Jing Xu, Department of Hepatobiliary Surgery, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
Author contributions: Fan JH, Feng GG and Huang L performed the majority of experiments; Tang GG, Jiang HX and Xu J analyzed the data; Fan JH and Feng GG designed the study and wrote the paper.
Correspondence to: Jun-Hua Fan, MD, PhD, Department of Gastroenterology, the First Affiliated Hospital of Guangxi Medical University, 6 Shuangyong Road, Nanning 530021, Guangxi Zhuang Autonomous Region, China. fanjunhuaxiaoshe@hotmail.com
Telephone: +86-771-5356501 Fax: +86-771-5356585
Received: October 25, 2013
Revised: December 25, 2013
Accepted: March 5, 2014
Published online: May 7, 2014
Processing time: 193 Days and 20 Hours
Abstract

AIM: To investigate whether naofen is involved in tumor necrosis factor (TNF)-α-mediated apoptosis of hepatocytes induced by lipopolysaccharide (LPS).

METHODS: In vivo, rats were treated with LPS or anti-TNF-α 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-α, or in conditioned medium from LPS-treated KCs (KC-CM)/KC-CM + anti-TNF-α antibody. Naofen and TNF-α 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-α 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-α antibody. In KCs, LPS caused an increase in TNF-α that was almost consistent with that in the liver of LPS-treated rats. In hepatocytes, neither LPS nor TNF-α 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-α antibody.

CONCLUSION: TNF-α released from KCs treated with LPS may induce hepatic naofen expression, which then stimulates hepatocellular apoptosis through activation of caspase-3.

Keywords: Naofen; Tumor necrosis factor-α; Apoptosis; Lipopolysaccharide; Kupffer cells; Caspase-3

Core tip: Naofen, a WD40-repeat protein, is increased in the liver but not in the kidneys, thymus or spleen of rats injected with lipopolysaccharide (LPS). Increased naofen expression is blocked by pretreatment with anti-tumor necrosis factor (TNF)-α antibody. TNF-α has no effect on naofen expression or caspase-3 activation in primary hepatocytes, but conditioned medium from LPS-treated Kupffer cells (KC-CM) significantly enhances both. KC-CM-induced increase in naofen expression and caspase-3 activity is blocked by anti-TNF-α antibody. LPS in the liver may enhance release of TNF-α from KCs, and induce hepatocyte apoptosis, for which naofen promotes caspase-3 activity through the mitochondrial pathway.