Sevoflurane Preconditioning Reduces Intestinal Ischemia-Reperfusion Injury: Role of Protein Kinase C and Mitochondrial ATP-Sensitive Potassium Channel

<div><p>Ischemic preconditioning (IPC) has been considered to be a potential therapy to reduce ischemia-reperfusion injury (IRI) since the 1980s. Our previous study indicated that sevoflurane preconditioning (SPC) also reduced intestinal IRI in rats. However, whether the protective effect of SPC is similar to IPC and the mechanisms of SPC are unclear. Thus, we compared the efficacy of SPC and IPC against intestinal IRI and the role of protein kinase C (PKC) and mitochondrial ATP-sensitive potassium channel (mK_ATP) in SPC. A rat model of intestinal IRI was used in this study. The superior mesenteric artery (SMA) was clamped for 60 min followed by 120 min of reperfusion. Rats with IPC underwent three cycles of SMA occlusion for 5 min and reperfusion for 5 min before intestinal ischemia. Rats with SPC inhaled sevoflurane at 0.5 minimum alveolar concentration (MAC) for 30 min before the intestinal ischemic insult. Additionally, the PKC inhibitor Chelerythrine (CHE) or mK_ATP inhibitor 5-Hydroxydecanoic (5-HD) was injected intraperitoneally before sevoflurane inhalation. Both SPC and IPC ameliorated intestinal IRI-induced histopathological changes, decreased Chiu’s scores, reduced terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive cells in the epithelium, and inhibited the expression of malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α). These protective effects of SPC were similar to those of IPC. Pretreatment with PKC or mK_ATP inhibitor abolished SPC—induced protective effects by increasing Chiu’s scores, down-regulated the expression of Bcl-2 and activated caspase-3. Our results suggest that pretreatment with 0.5 MAC sevoflurane is as effective as IPC against intestinal IRI. The activation of PKC and mK_ATP may be involved in the protective mechanisms of SPC.</p></div

Both SPC and IPC inhibited the increase in IRI-induced TUNEL positive cells in the intestine of rats.

<p>(A) Representative images of TUNEL fluorescent staining in the intestinal mucosa (×200) (scan bar = 100 μm). Green staining indicates TUNEL-positive cells, blue staining indicates nuclear. (B) Quantification of TUNEL positive cells in the intestinal mucosal. Data are expressed as the mean ± SD, n = 9. Results were compared using one-way ANOVA with Bonferroni’s posttest. *** <i>P < 0</i>.<i>001</i> vs. Sham, ^&&&<i>P</i> < 0.001 vs. SPC-Sham. ^▲▲▲<i>P</i> < 0.001 vs. IPC-Sham, ^###<i>P < 0</i>.<i>001</i> vs. IRI. IPC = ischemic preconditioning; IRI = ischemia-reperfusion injury; SPC = sevoflurane preconditioning.</p

Both SPC and IPC attenuated the increased plasma MDA and TNF-α induced by IRI, but did not significantly change the level of SOD.

<p>(A) Quantification of the plasma MDA level; (B) Quantification of the plasma SOD level; (C) Quantification of the plasma TNF-α level. Data are expressed as the mean ± SD, n = 9. Results were compared using one-way ANOVA with Bonferroni’s posttest. ** <i>P < 0</i>.<i>01</i>, *** <i>P < 0</i>.<i>001</i> vs. Sham; ^&<i>P</i> < <i>0</i>.<i>05</i>, ^&&<i>P</i> < <i>0</i>.<i>01</i>, ^&&&<i>P</i> < 0.001 vs. SPC-Sham; ^▲▲<i>P</i> < <i>0</i>.<i>01</i>, ^▲▲▲<i>P</i> < 0.001, vs. IPC-Sham; ^##<i>P < 0</i>.<i>01</i>, ^###<i>P < 0</i>.<i>001</i> vs. IRI. IPC = ischemic preconditioning; IRI = ischemia-reperfusion injury; SPC = sevoflurane preconditioning.</p

The protocol of the experiment one and two.

<p>Sham: involving isolation of SMA without occlusion; IRI: performed SMA occlusion for 60 min followed by reperfusion for 120 min without any interventions; SPC-sham: Rats were pretreated with 0.5 MAC sevoflurane for 30 min before sham operation; IPC-sham: Rats underwent intestinal IPC without 60 min ischemia; SPC: Rats were pretreated with 0.5 MAC sevoflurane for 30 min before 60 min ischemia; IPC: Rats underwent intestinal IPC prior to 60 min ischemia; SPC + CHE: Rats received intraperitoneal injection of the PKC inhibitor Chelerythrine (5 mg/kg) 15 min before SPC; SPC + 5-HD: Rats received intraperitoneal injection of the mK_ATP inhibitor 5-Hydroxydecanoic (10 mg/kg) 15 min before SPC. 5-HD = 5-Hydroxydecanoic; CHE = Chelerythrine; IPC = ischemic preconditioning; IRI = ischemia-reperfusion injury; SPC = sevoflurane preconditioning; SMA = superior mesenteric artery. * = SMA occlusion for 5 min; # = reperfusion for 5 min.</p

CHE and 5-HD reversed the inhibition of SPC on intestinal apoptosis induced by intestinal IRI in rats.

<p>(A) Representative immunohistochemical images of cleaved caspase-3 in the intestinal mucosa (×400), caspase-3 positive cells are stained dark brown under light microscopy shown by the arrows; (B) Representative western blots of cleaved caspase-3 and Bcl-2; (C) The quantitative analysis of Bcl-2 and caspase-3. Data are expressed as the mean ± SD, n = 9. Results were compared using one-way ANOVA with Bonferroni’s posttest. * <i>P < 0</i>.<i>05</i>, ** <i>P < 0</i>.<i>01</i>, *** <i>P < 0</i>.<i>001</i> vs. the Sham; ^###<i>P < 0</i>.<i>001</i> vs. IRI; ^$<i>P < 0</i>.<i>05</i>, ^$$<i>P < 0</i>.<i>01</i>, ^$$$<i>P < 0</i>.<i>001</i> vs. SPC. 5-HD = 5-Hydroxydecanoic; CHE = Chelerythrine; IRI = ischemia-reperfusion injury; SPC = sevoflurane preconditioning.</p

CHE and 5-HD inhibited the protective effect of SPC against intestinal IRI.

<p>(A) Morphologic changes of intestinal mucosa under light microscopy (×200); (B) The evaluation of intestinal injury with Chiu’s scores. The Chiu’s score data are expressed as the mean ± SD, n = 9. Results were compared using one-way ANOVA with Bonferroni’s posttest. *** <i>P < 0</i>.<i>001</i> vs. Sham, ^###<i>P < 0</i>.<i>001</i> vs. IRI, ^$<i>P < 0</i>.<i>05</i>, ^$$$<i>P < 0</i>.<i>001</i> vs. SPC. 5-HD = 5-Hydroxydecanoic; CHE = Chelerythrine; IRI = ischemia-reperfusion injury; SPC = sevoflurane preconditioning.</p

Both SPC and IPC inhibited intestinal IRI.

<p>(A-F) Histopathological changes of intestinal mucosa under light microscopy (×200); (G) The evaluation of intestinal injury with Chiu’s scores. In the sham (A) and SPC- sham (B) groups, there were no injuries to the villi and glands, whereas mildly injured villi and glands were observed in the IPC-sham group (C). However, severe intestinal glands injury, mucosa villi disintegration or edema, increased gap of epithelial cells and severe hemorrhage were observed in the IRI group (D). In the SPC (E) and IPC (F) groups, the damageto intestinal villi and glands was much slighter than that in the IRI group. The Chiu’s score data are expressed as the mean ± SD, n = 9. Results were compared using one-way ANOVA with Bonferroni’s posttest. *** <i>P < 0</i>.<i>001</i> vs. the Sham group, * <i>P < 0</i>.<i>05</i> vs. the Sham group, ^&&&<i>P</i> < 0.001 vs. SPC-Sham. ^▲▲▲<i>P</i> < 0.001 vs. IPC-Sham, ^###<i>P</i> < 0.001 vs. the IRI group. IPC = ischemic preconditioning; IRI = ischemia-reperfusion injury; SPC = sevoflurane preconditioning.</p