Flurbiprofen, a cyclooxygenase inhibitor, protects mice from hepatic ischemia/reperfusion injury by inhibiting GSK-3β signaling and mitochondrial permeability transition

Abstract

Flurbiprofen acts as a nonselective inhibitor for cyclooxygenases (COX-1 and COX-2), but its impact on hepatic ischemia/reperfusion (I/R) injury remains unclear. Mice were randomized into sham, I/R and flurbiprofen (Flurb) groups. The hepatic artery and portal vein to the left and median liver lobes were occluded for 90 min and unclamped for reperfusion to establish a model of segmental (70%) warm hepatic ischemia. Pretreatment of animals with flurbiprofen prior to I/R insult significantly decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), and prevented hepatocytes from I/R-induced apoptosis/necrosis. Moreover, flurbiprofen dramatically inhibited mitochondrial permeability transition (MPT) pore opening, and thus prevented mitochondrial-related cell death and apoptosis. Mechanistic studies revealed that flurbiprofen markedly inhibited glycogen synthase kinase (GSK)-3β activity and increased phosphorylation of GSK-3β at Ser9, which, consequently, could modulate the adenine nucleotide translocase (ANT)-cyclophilin D (CyP-D) complex and the susceptibility to MPT induction. Therefore, administration of flurbiprofen prior to hepatic I/R ameliorates mitochondrial and hepatocellular damage through inhibition of MPT and inactivation of GSK-3β, and provides experimental evidence for clinical use of flurbiprofen to protect liver function in surgical settings in addition to its conventional use for pain relief.

Figure 1

The effects of flurbiprofen pretreatment prior to ischemia on liver damage. (A) H&E staining results of liver sections 6 h after reperfusion (100× magnification). (B) TUNEL assay results of liver sections 6 h after reperfusion (100× magnification). (C) Bar graphs showing the percentage of necrotic cells in the tissue sections. (D) Bar graphs showing the percentages of apoptotic cells in the tissue sections. (E) Serum levels for ALT and AST. (F) Serum levels for LDH. Six mice were included in each study group. **p < 0.05 versus I/R.

Figure 2

Flurbiprofen protects mitochondria from morphological changes after ischemic insult. The mitochondria isolated from I/R animals revealed greater contortion and increased vacuoles than that from mice with flurbiprofen pretreatment.

Figure 3

The effects of flurbiprofen treatment on mitochondrial calcium tolerance. Mitochondria were isolated from animals euthanized after 90 min of hepatic ischemia plus 2 h or 6 h of reperfusion in each group. Calcium pulse were fluorometrically monitored using the probe Ca²⁺-Green 5N. (A) Determination of extramitochondrial Ca²⁺ after subsequent addition of 10 μmol/L CaCl₂ pulses in mitochondria isolated from 6 h of reperfusion. (B) and (C) Calcium retention capacity after 2 h and 6 h of reperfusion in each groups (n = 6 per group), the CRC was measured without (−CsA) or with (+CsA) addition of 0.8 mmol/L of CsA in the cuvette to fully inhibit the interaction of CyP-D with the MPT pore. **p < 0.05 versus CRC in the I/R group (same strain).

Figure 4

The effect of flurbiprofen on cytochrome c release and caspase-3/9 activation. (A) A representative results for Western blot analysis of mitochondrial and cytoplasmic cytochrome c. (B) Relative amount of mitochondrial cytochrome c. (C) Relative amount of cytoplasmic cytochrome c. (D) Representative Western blot results for the cleaved caspase 3 and caspase 9. (E) Relative amount for the cleaved caspase 3. (F) Relative amount for the cleaved caspase 9. The studies were carried out with three replications. **p < 0.05 versus I/R.

Figure 5

The effects of flurbiprofen administered before ischemia on GSK-3β activity on liver homogenates. Tau phosphorylation by GSK-3β was detected using a Tau (pS199) phosphoELISA kit. Animals were euthanized after 90 min of hepatic ischemia plus 2 h or 6 h of reperfusion, with (Flurb) or without (I/R) flurbiprofen (10 mg/kg) in the caudal vein 20 min before ischemia. I/R livers exhibited increased GSK-3β activity, when compared with both Sham and Flurb animals. **p < 0.05 versus I/R, n = 6.

Figure 6

The effects of flurbiprofen administered before ischemia on GSK-3β and phosphorylated GSK-3β at Ser9 (pSer9-GSK-3β) content in liver homogenates. (A) Western blot results for the total GSK-3β and pSer9-GSK-3β. (B) Relative amount for the pSer9-GSK-3β. **p < 0.05 versus I/R, n = 6. I/R animals exhibited decreased content of phosphorylated GSK-3β at Ser9, when compared with both Sham and Flurb animals.

Figure 7

The effects of flurbiprofen on interaction of ANT with CyP-D in mitochondria. The effects of flurbiprofen administered before ischemia on interaction of ANT with CyP-D in mitochondria isolated from animals euthanized after 90 min of hepatic ischemia plus 2 h or 6 h of reperfusion in each group. (A) A representative immunoblot results for the ANT and CyP-D. (B) Relative amount for the CyP-D compared with ANT. **p < 0.05 versus I/R, n = 6. I/R animals exhibited increased content of CyP-D–ANT complex, when compared with both Sham and Flurb animals. IP: immunoprecipitation; IB: immunoblotting.

Figure 8

The impact of flurbiprofen on COX expressions. (A) Representative Western blot results for COX-1 and COX-2, (B) Relative expression levels for COX1. (C) Relative expression levels for COX2. The relative expression levels for each target were assessed by densitometry and normalized by β-actin. Three replicates were included for the analyses. **p < 0.05 versus I/R.