Quantification of the Expression of HIF-1alpha by Real-Time PCR in Rat Hepatocytes Cultures Invaded by <i>Shigella flexneri</i> under Normoxic and Hypoxic Conditions

Abstract

Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epithelium. Hypoxia is present in many human diseases and can induce the expression of the transcription factor HIF-1alpha that may have a cell protective role. The influence of hypoxia and HIF-1alpha on bacterial infection, studied in this work, is unclear. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Methods: We compared the ability of S. flexneri to invade rat hepatocytes in primary culture both in normoxic and hypoxic conditions. We evaluated TNF-alpha released by hepatocytes, apoptosis rate and HIF-1alpha expression by confocal microscopy as well as real time PCR technique. Results: We showed that S. flexneri invaded less hepatocytes previously submitted to 24 h hypoxia (6.5% O2) than those cultivated in normoxia (21% O2). S. flexneri also induced HIF-1α expression in hepatocytes, TNF-α secretion and apoptosis. Conclusion: a) Hypoxia alone was not a stimulus to TNF-α secretion, but induced cell apoptosis and HIF-1α expression; b) S. flexneri was able to invade rat hepatocytes and hypoxia apparently influenced significantly bacterial cell invasiveness; c) HIF-1α was expressed in hypoxic conditions, and it was also stimulated by S. flexneri.