Su1649 Dynamics of Mitochondrial Network in Helicobacter pylori Infection

INTRODUCTION: Mitochondria are dynamic organelles that move around and can fuse with one another or can break off from these unions in a continuous process called fusion/ fission. The fusion/fission balance determines the mitochondrial connectivity degree and the mitochondrial network morphology inside the cells. It is known that a fusion/fission imbalance is involved in several pathological processes that occur as a result of an increased cellular death by apoptosis, as it is observed in Helicobacter pylori (HP)-infection. AIMS: To evaluate in gastric epithelial cells the effect of HP on mitochondrial fission/fusion modulation and apoptosis. Relationship with oxidative stress. MATERIALS AND METHODS: Human gastric epithelial cells (AGS, ATCC CRL-1739) were cultured with a HP strain (ATCC51932) for 24h at 108CFU/mL, with and without VitE (10-4M). It was determined: -Cellular (ROS) and mitochondrial (O2.-) oxidative stress using H2DCFDA and MitoSOX Red probes, respectively. -Mitochondrial network phenotype, visualized with NAO staining. -mRNA levels of mitofusin-2 (fusion-involved protein) and drp-1 (dynamin family protein, related to mitochondrial fission). -Number of apoptotic cells visualized with Hoechst dye. Technologies used: Flow Cytometry (FACScan, Becton Dickson), Confocal Microscopy (Olympus FV 1000) and Real Time-PCR (ABI Prism 7000, Applied Biosystems). RESULTS: HP caused an increase of H2DCFDA and MitoSOX fluorescences (2.5 and 1.8-fold, respectively) in HPinfected AGS cells. The reticulotubular phenotype, characteristic of viable cells and composed by long chains of fusedmitochondria, was transformed into a punctiform phenotype (rounded and isolated mitochondria) typical of an increased fission. A 13.6-fold increase in drp-1/ mitofusin-2 mRNA ratio it was observed. Hoechst showed a homogeneous staining in controls, whereas a 30-40% of infected cells displayed a phenotype related to apoptotic features (condensation, peripheral location and shape resembling a half-moon of nuclear chromatin). VitE pre-treatment avoided all these alterations, with statistically significant differences. CONCLUSIONS: The increased oxidative stress in HP-infected gastric epithelial cells is associated with fission-mediated fragmentation of mitochondrial network. The recovery of fusion/fission balance by the antioxidant Vit.E, was accompanied by the inhibition of apoptosis. So, these data could indicate that the mitochondrial dynamic is other stage in the apoptotic cascade triggered by oxidative stress and therefore, it is involved in the development of gastric pathologies related to HP-infection.