Background: The small intestine is highly sensitive to ischemia reperfusion (I/R)-induced injury, which is associated with high morbidity and mortality. Apoptosis, or programmed cell death, is a major mode of cell death occurring during I/R-induced injury. However, the mechanisms by which I/R cause apoptosis in small intestine are poorly understood. PUMA is a p53 downstream target and a member of BH3-only group of Bcl-2 family proteins. It has been shown that PUMA plays an essential role in apoptosis induced by a variety of stimuli in different tissues through a mitochondrial pathway.
Aims: In this study, we investigated the role of PUMA in I/R-induced injury and apoptosis in small intestine. We also studied the mechanisms by which PUMA is regulated in I/R-induced intestinal apoptosis.
Methods: Ischemia was induced by superior mesenteric artery occlusion in mouse small intestine. Induction of PUMA in response to ischemia alone, or ischemia followed by reperfusion (I/R) was examined. I/R-induced intestinal apoptosis and injury were compared between PUMA knockout and wild-type mice. The mechanisms of I/R-induced and PUMA-mediated apoptosis were investigated through analysis of caspase activation, cytosolic release of mitochondrial cytochrome c, and alterations of the proapoptotic Bcl-2 family proteins Bax and Bak. To determine whether PUMA is induced by reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) generated by I/R, superoxide dismutase (SOD) and N-nitro-L-arginine methyl ester (L-NAME) were used to treat animals before I/R. To determine whether p53 is involved in regulating PUMA during I/R-induced apoptosis, PUMA induction and apoptosis in response to I/R were examined in p53 knockout mice.
Results: PUMA was markedly induced following ischemia reperfusion in the mucosa of the mouse small intestine. I/R-induced intestinal apoptosis was significantly attenuated in the PUMA knockout mice compared with that in the wild-type mice. I/R-induced caspase 3 activation, cytochrome c release, Bax mitochondrial translocation, and Bak multimerzation were also inhibited in the PUMA knockout mice. SOD or L-NAME significantly blunted I/R-induced PUMA expression and apoptosis. Furthermore, I/R-induced PUMA expression and apoptosis in small intestine were not affected in the p53 knockout mice.
Conclusions: Our data demonstrated that PUMA is activated by oxidative stress in response to I/R to promote p53-independent apoptosis in small intestine through the mitochondrial pathway. Inhibition of PUMA is potentially useful for protecting against I/R-induced intestinal injury and apoptosis.
- ischemia reperfusion
- oxidative stress
- small intestine