Doxycycline protects human intestinal cells from hypoxia/reoxygenation injury: Implications from an in-vitro hypoxia model

- Hypoxia/reoxygenation leads to damage of in-vitro cultured intestinal CaCo-2 cells.
- Preincubation with doxycycline protects CaCo-2 from hypoxia/reoxygenation injury.
- Doxycycline attenuates hypoxia/reoxygenation induced caspase-3 protein expression.

Intestinal ischemia/reperfusion (I/R) injury is a grave clinical emergency and associated with high morbidity and mortality rates. Based on the complex underlying mechanisms, a multimodal pharmacological approach seems necessary to prevent intestinal I/R injury. The antibiotic drug doxycycline, which exhibits a wide range of pleiotropic therapeutic properties, might be a promising candidate for also reducing I/R injury in the intestine.To investigate possible protective effects of doxycycline on intestinal I/R injury, human intestinal CaCo-2 cells were exposed to doxycycline at clinically relevant concentrations. In order to mimic I/R injury, CaCo-2 were thereafter subjected to hypoxia/reoxygenation by using our recently described two-enzyme in-vitro hypoxia model. Investigations of cell morphology, cell damage, apoptosis and hydrogen peroxide formation were performed 24 h after the hypoxic insult.Hypoxia/reoxygenation injury resulted in morphological signs of cell damage, elevated LDH concentrations in the respective culture media (P<0.001) and increased protein expression of proapoptotic caspase-3 (P<0.05) in the intestinal cultures. These events were associated with increased levels hydrogen peroxide (P<0.001). Preincubation of CaCo-2 cells with different concentrations of doxycycline (5 µM, 10 µM, 50 µM) reduced the hypoxia induced signs of cell damage and LDH release (P<0.001 for all concentrations). The reduction of cellular damage was associated with a reduced expression of caspase-3 (5 µM, P<0.01; 10 µM, P<0.01; 50 µM, P<0.05), while hydrogen peroxide levels remained unchanged.In summary, doxycycline protects human intestinal cells from hypoxia/reoxygenation injury in-vitro. Further animal and clinical studies are required to prove the protective potential of doxycycline on intestinal I/R injury under in-vivo conditions.