Estudo da invasão de hepatócitos de rato por Shigella flexneri: análise da influência da hipóxia sobre a injúria celular

Abstract

Lima, Camila Barbara Cantalupo. Study of rat hepatocytes invasion by Shigella flexneri: analysis of hypoxia influence on cellular injury. [Dissertation]. Sao Paulo: Faculdade de Medicina, Universidade de Sao Paulo; 2011. This study evaluated the invasiveness of rat hepatocytes by Shigella flexneri (S. flexneri) in normoxia and hypoxia conditions. The study of hypoxia microenvironment is of great importance, since hypoxia is present in many liver diseases and increases bacterial translocation when present in intestinal lumen. Invasive bacteria such as S. flexneri can disrupt the intestinal barrier and reach the liver through portal circulation. The effect of bacterial invasion in liver cells is poorly understood. In this study we investigated the morphological and functional changes of rat hepatocytes after infection with S. flexneri in the presence and absence of hypoxia. For this purpose we used primary cultures of rat hepatocytes. Several parameters were analyzed, such as: bacterial invasion cell rate, quantification of LDH production and release, TNF-α production, cell death rate by apoptosis and expression of the transcription factor HIF-1α. The results showed that the methodology used to obtain the hypoxic microenvironment was satisfactory, with 70% reduction of initial pO2 (to 43.2 mmHg in vitro or 6.5% O2). The invasion of rat hepatocytes by S. flexneri was lower in cells previously exposed to hypoxia compared with the invasion of cells grown in normoxia. The viability of hepatocytes showed no significant differences between experimental groups, ranging between 74% and 86%. The release of TNF-α in situations of normoxia and hypoxia was similar, although the infected cells in normoxia have increased the release levels of this cytokine. In hypoxia + infection condition the release of TNF-α was lower than normoxia + infection condition, but both groups produced a significant increase in cytokine release when compared to normoxic and hypoxic controls. This result suggests that the presence of bacteria inside the cells significantly increases the release of TNF-α by hepatocytes. DHL production was also significantly greater in the hypoxic group compared to the normoxic group, but had no change in the groups infected with S. flexneri after an hour. The apoptosis rates increased in hypoxia and infected groups in a similar way, varying between 24% and 31% when compared with control group in normoxia. The expression of HIF1α transcription factor occurred in hypoxia, normoxia + infection and hypoxia + infection groups, indicating that infection with S. flexneri induces the expression of this factor. Overall, our results sought to contribute to a greater understanding of the interaction between S. flexneri and hepatocytes under hypoxia and normoxia conditions. Such knowledge may be useful in building future strategies to assist in combating these major invasive bacteria. Descriptors: Shigella flexneri; Hypoxia; Hypoxia-inducible factor 1, alpha subunit; Tumor necrosis factor alpha, Apoptosis; Rats, Wistar