Oxidative stress-stimulated nuclear factor-kappa B (NF-kappa B) activation has been associated with rapid transcription of TNF-alpha and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-kappa B and release of TNF-alpha secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 +/- 0.08 vs. 0.8 +/- 0.06* microM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-kappa B to nuclei (167% +/- 21% vs. 142% +/- 18%*), decreased the level of plasma TNF-alpha (1,994 +/- 447 vs. 667 +/- 130* pg/mL), and inhibited caspase-3 activity (0.57% +/- 0.1% vs. 0.21% +/- 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% +/- 1% vs. 11% +/- 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-kappa B expression (42% +/- 8%*) and reduction of release of TNF-alpha (231 +/- 72* pg/mL). Caspase-3 activity (0.33% +/- 0.1%*) and apoptotic cells (12% +/- 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-kappa B-TNF-alpha signaling pathway. *P < 0.05 Postcon and NAC vs. Control.
Summary
Significant organ injury occurs after transplantation and reflow (i.e., reperfusion injury). Postconditioning (PoC), consisting of alternating periods of reperfusion and re‐occlusion at onset of reperfusion, attenuates reperfusion injury in organs including heart and brain. We tested whether PoC attenuates renal ischemia–reperfusion (I/R) injury in the kidney by activating adenosine receptors (AR) and protein kinase C (PKC). The single kidney rat I/R model was used. Groups: (1) sham: time‐matched surgical protocol only. In all others, the left renal artery (RA) was occluded for 45 min and reperfused for 24 h. (2) Control: I/R with no intervention at R. All antagonists were administered 5 min before reperfusion. (3) PoC: I/R + four cycles of 45 s of R and 45 s of re‐occlusion before full R. (4) PoC + ARi: PoC plus the AR antagonist 8‐ρ‐(sulfophenyl) theophylline (8‐SPT). (5) PoC + PKCi: PoC plus the PKC antagonist chelerythrine (Che). In shams, plasma blood urea nitrogen (BUN mg/dl) at 24 h averaged 23.2 ± 5.3 and creatinine (Cr mg/dl) averaged 1.28 ± 0.2. PoC reduced BUN (87.2 ± 10 in Control vs. 38.8 ± 9, P = 0.001) and Cr (4.2 ± 0.6 in Control vs. 1.5 ± 0.2, P < 0.001). 8‐SPT and Che reversed renal protection indices after PoC. I/R increased apoptosis, which was reduced by PoC, which was reversed by 8‐SPT and Che. Postconditioning attenuates renal I/R injury by adenosine receptor activation and PKC signaling.
Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given 10 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERK1/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 +/- 2% vs. 53 +/- 1%, P< 0.05), and SP had no effect on infarct size (53 +/- 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 +/- 2% vs. PD + PAR-2 AP, 50 +/- 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 +/- 2%) but not by LY (PAR-2 AP, 36 +/- 2% vs. LY + PAR-2 AP, 38 +/- 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC.
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