Z. Chang1, W. He1, B. Liu1, I. Halaweish1, T. Bambakidis1, Y. Li1, H. B. Alam1 1University Of Michigan,Surgery,Ann Arbor, MI, USA
Introduction: We have previously shown that treatment with histone deacetylase (HDAC) 6 inhibitor Tubasatin A (Tub A) can improve outcome in a rat model of septic shock. Also, we have demonstrated that circulating levels of claudin 3, a tight junction (TJ) protein, increase after shock and return to normal after treatment with VPA (pan-HDAC inhibitor). This study investigated whether specific inhibition of HDAC 6 could protect intestinal cellular TJ and improve survival after HS.
Methods: Experiment I: Sprague Dawley rats underwent hemorrhagic shock (40% blood loss over 10 min) followed by treatment with Tub A (70 mg/kg), without any fluid resuscitation. The experimental groups were: (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, no treatment), and (3) treatment (hemorrhage with Tub A treatment). Animals were sacrificed 6 hrs later, and intestinal tissues were used to create whole cell lysate, which were analyzed for acetyl-tubulin, total tubulin, claudin 3 and zonula occludens 1 (ZO-1) proteins by Western blot. Experiment II: human intestinal epithelial cells (Caco-2) were divided into 3 groups: (1) sham (no hypoxia), (2) control (hypoxia, no treatment), (3) treatment (hypoxia, treatment with tub A). After 12 hours in a hypoxia chamber, cells were analyzed for viability, Lactate dehydrogenase (LDH) levels measuring cellular injury, and TJ protein (claudin 3 and ZO-1) levels.
Results: Hemorrhage decreased, whereas treatment with Tub A increased, expression of claudin 3 and ZO-1. Administration of Tub A also acetylated the tubulin protein. In vitro study showed that hypoxia decreased the viability of caco- 2 cells, which was prevented by Tub A treatment. Similar results were seen when cellular cytotoxicity was determined by the LDH activity. The expression of TJ protein claudin 3 and ZO-1 was significantly decreased by hypoxia, which was significantly prevented by Tub A treatment. Immunofluorescent study further confirmed that Tub A attenuated the hypoxia-induced claudin 3 and ZO-1 signal loss in the caco-2 cells (Figure).
Conclusion: Selective inhibition of HDAC6 with Tub A preserves the expression of intestinal TJ proteins in models of hemorrhagic shock in vivo and cellular hypoxia in vitro.
