Preservation of Hepatocyte Nuclear Factor-4α Is Associated with Zinc Protection Against TNF-α Hepatotoxicity in Mice

Abstract

Hepatocyte nuclear factor-4α (HNF-4α), a zinc finger protein, is the most abundant transcription factor in the liver. HNF-4α regulates a large number of genes involved in most aspects of hepatocyte functions. The present study was undertaken to determine the role of HNF-4α in zinc protection against tumor necrosis factor-α (TNF-α) hepatotoxicity. Mice were treated with murine TNF-α via intravenous injection at 20 μg/kg body wt 30 mins after d-galactosamine (d-Gal) sensitization (800 mg/kg body wt). Two doses of zinc sulfate (5 mg elemental zinc/kg body wt) were administered at 36 and 12 hrs before TNF-α treatment via subcutaneous injection. TNF-α treatment after sensitization induced liver injury as detected by plasma alanine aminotransferase activity and apoptotic cell death in the liver. Zinc pretreatment attenuated TNF-α–induced liver injury. Furthermore, TNF-α–induced activations of caspase 3 and caspase 8 in the liver were significantly inhibited by zinc pretreatment. The mRNA and protein levels of HNF-4α in the liver were remarkably decreased by TNF-α treatment, which was suppressed by zinc. To determine if HNF-4α depletion is involved in d-Gal sensitization to TNF-α toxicity, mice were administered either d-Gal or TNF-α. Immunohistochemistry demonstrated that HNF-4α depletion in the liver is associated with d-Gal sensitization but not TNF-α treatment. To define the link between HNF-4α depletion and TNF-α–induced cell death, the effect of silencing the HNF-4α gene by siRNA transfection on TNF-α cytotoxicity was determined in HepG2 cells. A lactate dehydrogenase cytotoxicity assay showed that neither TNF-α nor HNF-4α siRNA transfection had a toxic effect, but TNF-α treatment after HNF-4α siRNA transfection caused HepG2 cell death. These results suggest that zinc protects against TNF-α hepatotoxicity, at least partially, through preservation of the zinc finger protein HNF-4α.

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