Pharmacological postconditioning with diazoxide attenuates ischemia/reperfusion-induced injury in rat liver

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Abstract

It has been demonstrated that ischemic postconditioning (IPO) is capable of attenuating ischemia/reperfusion (I/R) injury in the heart. However, the novel role of pharmacological postconditioning in the liver remains unclear. In this study, the hypothesis that diazoxide postconditioning reduces I/R-induced injury in rat liver was tested. Rats were assigned randomly to the sham-operated control, I/R (occlusion of the porta hepatis for 60 min, followed by a persistent reperfusion for 120 min), diazoxide ischemic postconditioning (DIPO; occlusion of the porta hepatis for 60 min, then treat- ment with diazoxide for 10 min reperfusion, followed by a persistent reperfusion for 110 min) or 5-hydroxydecanoate (5-HD)+DIPO (occlusion of the porta hepatis for 60 min, then treatment with diazoxide and 5-HD for 10 min reperfusion, followed by a persistent reperfusion for 110 min) groups. The alanine aminotransferase (ALT) and aspartate transaminase (AST) levels were assayed. The expression levels of protein kinase c-ε (pkc-ε), cytochrome c (cyt-c), caspase-3 and bcl-2 protein were determined by western blotting. The serum levels of ALT and AST and expression levels of cyt-c and caspase-3 were significantly lower in the DIPO group (P<0.05). However, the protein expression levels of pkc-ε and bcl-2 were markedly increased in the DIPO group (P<0.05). 5-HD abrogated the protective effects of DIPO. The data of the present study provide the first evidence that DIPO protects the liver from I/R injury by opening the mitochondrial KATP channels, activating and upregulating pkc-ε and inhibiting the activation of the apoptotic pathway by decreasing the release of cyt-c and the expression of caspase-3 and increasing bcl-2 expression.

Figures

  • Figure 1. Experimental groups and protocols. I/R, ischemia/reperfusion; DIPO, diazoxide ischemic preconditioning; 5-HD, 5-hydroxydecanoate.
  • Figure 2. ALT and AST levels. After 60 min of ischemia and 2 h of reperfusion, serum levels of (A) ALT and (B) AST were determined. Compared with
  • Figure 3. Expression levels of pkc-ε by western blotting. After 60 min of ischemia and 2 h of reperfusion, expression levels of pkc-ε in DIPO-treated hepatic tissues were determined by western blot analysis. Treatment with diazoxide (30 µmol/l) activated the pkc-ε protein. However, 5-HD (300 µmol/l) significantly abrogated the expression levels of pkc‑ε. β-actin was used as an equal loading control. (A) The corresponding mean optical densities in each group are shown. (B) Bar graph shows the ratio of pkc-ε to β-actin. All values are means ± SD; n=7. *P<0.05 vs. sham; **P<0.05 vs. I/R; ***P<0.05 vs. DIPO. DIPO, diazoxide ischemic postconditioning; 5‑HD, 5-hydroxydecanoate; I/R, ischemia-reperfusion; pkc-ε, protein kinase c‑ε.
  • Figure 5. Western blot analysis of caspase-3 expression. After 60 min of ischemia and 2 h of reperfusion, expression levels of caspase-3 in DIPO-treated hepatic tissues were determined by western blot analysis. I/R treatment activated the rat liver apoptotic pathway which led to an increase in caspase-3 protein expression levels. Diazoxide (30 µmol/l) postconditioning inhibited the increases of caspase-3. However, 5-HD (300 µmol/l) significantly abrogated the effect of diazoxide postconditioning. β-actin was used as an equal loading control. (A) The corresponding mean optical densities in each group are shown. (B) Bar graph shows the ratio of caspase-3 to β-actin. All values are means ± SD; n=7. *P<0.05 vs. sham; **P<0.05 vs. I/R; ***P<0.05 vs. DIPO. DIPO, diazoxide ischemic postconditioning; 5-HD, 5-hydroxydecanoate; I/R, ischemia-reperfusion.
  • Figure 4. Western blot analysis of cyt-c. After 60 min of ischemia and 2 h of reperfusion, expression levels of cyt-c in DIPO-treated hepatic tissues were determined by western blot analysis. I/R treatment activated the rat liver apoptotic pathway. Diazoxide (30 µmol/l) postconditioning inhibited the release of cyt-c from the mitochondrion to the cytoplasm. However, 5-HD (300 µmol/l) significantly abrogated the effect of diazoxide postconditioning. β-actin was used as an equal loading control. (A) The corresponding mean optical densities in each group are shown. (B) Bar graph shows the ratio of cyt-c to β-actin. All values are means ± SD; n=7. *P<0.05 vs. sham; **P<0.05 vs. I/R; ***P<0.05 vs. DIPO. DIPO, diazoxide ischemic postconditioning; 5-HD, 5-hydroxydecanoate; I/R, ischemia-reperfusion; cyt-c, cytochrome c.
  • Figure 6. Western blot analysis of bcl-2 expression. After 60 min of ischemia and 2 h of reperfusion, expression levels of bcl-2 in DIPO-treated hepatic tissues were determined by western blot analysis. Diazoxide (30 µmol/l) postconditioning activated the antiapoptotic pathway which led to significant increases in bcl-2 expression levels. Treatment with 5-HD (300 µmol/l) significantly abrogated the effect of diazoxide postconditioning. β-actin was used as an equal loading control. (A) The corresponding mean optical densities in each group are shown. (B) Bar graph shows the ratio of bcl-2 to β-actin. All values are means ± SD; n=7. *P<0.05 vs. sham; **P<0.05 vs. I/R; ***P<0.05 vs. DIPO. DIPO, diazoxide ischemic postconditioning; 5‑HD, 5-hydroxydecanoate; I/R, ischemia-reperfusion.

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APA

Tian, Y. S., Rong, T. Z., Hong, Y. L., Min, L., & Jian, P. G. (2013). Pharmacological postconditioning with diazoxide attenuates ischemia/reperfusion-induced injury in rat liver. Experimental and Therapeutic Medicine, 5(4), 1169–1173. https://doi.org/10.3892/etm.2013.941

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