Intestinal Ischemia Reperfusion Injury Is Alleviated by Remote Ischemic Conditioning via the Inhibition of Autophagy Through Endoplasmic Reticulum Stress Signaling

Background: Remote ischemic conditioning (RIC) involves the application of brief cycles of ischemia and reperfusion to a remote tissue/organ and has been shown to alleviate ischemia reperfusion (IR) injury in many intestinal diseases such as volvulus, intussusception, and other intestinal obstructions. Autophagy is a recycling process that plays an important role in cell survival. However, excessive autophagy induces apoptosis. Endoplasmic reticulum (ER) stress is activated in response to cellular stress triggered by a variety of conditions that disturb ER homeostasis. This study aims to investigate the protective mechanisms of RIC in an injury mouse model of IR.

Methods: Following ethical approval, 3-week-old C57BL/6 mice were divided into 3 groups, (i) Sham (laparotomy only, n=7), (ii) IR (mice receiving IR, n=5), (iii) IR+perRIC (mice receiving RIC during RIC, n=5). Mice subjected to IR injury was induced by 45 minutes occlusion of superior mesenteric artery followed by 2 hours of reperfusion. RIC was induced by transient occlusion of femoral vessels of one hind-limb during the ischemia phase of IR. The RIC stimulus was given in four cycles, each consisting of 5 min occlusion followed by 5 min reperfusion. Sham group did not receive IR or RIC. Terminal ileum was harvested and processed for autophagy and ER stress makers using Western Blot. Groups were compared using one-way ANOVA.

Results: Animals receiving IR induced intestinal injury as shown by the increased in pro-apoptosis protein expression of CHOP (Fig. 1A). Up-regulation of LC3B II/I expression (Fig. 1B) during IR indicates that autophagy was activated and CHOP induction was induced by the autophagy pathway rather than ER stress pathway as shown in ER stress marker BiP (Fig. 1C). However, mice subjected to RIC during IR up-regulated ER stress marker BiP and decreased protein expression of LC3B II/I, and CHOP, indicating that RIC prevents IR-induced autophagy and decreased apoptosis through ER stress pathway (Fig. 1).

Conclusion: Intestinal ischemic reperfusion induced autophagy and apoptosis. However, remote ischemic conditioning up-regulated ER stress, thereby decreased apoptosis and subsequently down-regulated autophagy. This study provides insights of the protective mechanisms of RIC in intestinal ischemia reperfusion injury and the cross talk between autophagy and apoptosis during ER stress.