Modulation of Glutathione Hemostasis by Inhibition of 12/15-Lipoxygenase Prevents ROS-Mediated Cell Death after Hepatic Ischemia and Reperfusion

Abstract

Background: Reactive oxygen species- (ROS-) mediated ischemia-reperfusion injury (IRI) detrimentally impacts liver transplantation and resection. 12/15-Lipoxygenase (12/15-LOX), an antagonistic protein of the glutathione peroxidase 4 (GPX4) signaling cascade, was proven to mediate cell death in postischemic cerebral and myocardial tissue. The aim of this study was to investigate the impact of 12/15-LOX inhibition on hepatic IRI.

Methods: Livers of C57BL/6 mice were exposed to 60 minutes of partial warm ischemia and 90 minutes of reperfusion after previous Baicalein administration, an inhibitor of 12/15-LOX. Tissue samples were analyzed by TUNEL assay, Western blot, and spectral photometry.

Results: TUNEL labeling showed a significant reduction of hepatic cell death following baicalein pretreatment. Western Blot analysis revealed a significant downregulation of Jun-amino-terminal-kinase (JNK), caspase-3, and poly-ADP-ribose-polymerase (PARP), besides considerably lowered p44/42-MAP-kinase (ERK1/2) expression after Baicalein administration. A significant elevation of glutathione oxidation was measured in Baicalein pretreated livers.

Conclusion: Our data show that inhibition of 12/15-lipoxygenase causes significant cell death reduction after hepatic ischemia and reperfusion by enhancing glutathione metabolism. We conclude that GPX4-dependent cell death signaling cascade might play a major role in development of hepatic IRI, in which the investigated proteins JNK, caspase-3, ERK1/2, and PARP might contribute to tissue damage.

Figure 1

Measurements of macrohemodynamics while inducing ischemia and reperfusion in the liver throughout the experiment. Intra-arterial measurements of blood pressure (a) and heart rate (b) revealed a brief decrease in mean arterial blood pressure by 35.4% at the beginning of the experiment after Baicalein administration and after sole DMSO pretreatment (−34.4%) compared to the control. A heart rate elevation at the same time point by 104.2% (Baicalein) and 93.7% (DMSO) compared to control could also be measured. In further progress, no significant differences in macrohemodynamics between the groups could be monitored. A self-limiting decrease of blood pressure five minutes after reperfusion could repeatedly be observed in all groups. Data are presented as the mean ± SEM.

Figure 2

TUNEL assay of liver tissue (left posterior segment) exposed to ischemia-reperfusion injury. Representative images of TUNEL stainings demonstrating the differences in cell death after no/saline pretreatment (a), pretreatment with DMSO (b), and pretreatment with Baicalein (c). Quantitive planimetric analyses of TUNEL positive cells confirm a significant reduction of cell death after Baicalein pretreatment (Baicalein versus control by −64.8%, ^∗∗∗p = 0.0007; Baicalein versus vehicle DMSO by −54.1%, ^∗p = 0.0471) (d). Data are presented as the mean ± SEM.

Figure 3

Western blot assays of liver tissue (right anterior segment) exposed to ischemia-reperfusion injury for detection of activity levels of proapoptotic proteins JNK (stress-activated protein kinase/Jun-amino-terminal kinase), ERK (mitogen-activated protein kinase p44/42), caspase-3, and PARP (poly-ADP-ribose-polymerase). (a) Representative images of Western blots demonstrating the differences in enzyme activity levels for each experimental group. Inactive proteins are shown on top for ERK (total ERK, 42 + 44 kDa), PARP (uncleaved, 116 kDa), caspase-3 (loading control GAPDH, 37 kDa), and JNK (total JNK, 46 + 54 kDa). Activated protein forms are shown in the middle for ERK (phosphorylated ERK, 42 + 44 kDa), PARP (cleaved, 89 kDa), caspase-3 (cleaved, 20 kDa), and JNK (phosphorylated JNK, 46 + 54 kDa). Loading controls are shown below in each group for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 37 kDa). (b) Quantitative photometric analyses of Western blots prove the mostly significant downregulation of proapoptotic enzyme activity levels after Baicalein pretreatment. Activated enzyme ratio is calculated as a quotient of active/inactive protein form. Changes in protein level activation after sole vehicle administration (DMSO) could also be observed. Data are presented as the mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 4

Quantitative serum analyses of caval blood gained after inducing hepatic IRI or, respectively, after sham operation. (a) Quantification of the increase of liver enzymes in sham-operated mice after DMSO and Baicalein administration by calculating the toxic factor. The toxic factor is calculated as a ratio of enzyme elevation after drug administration compared to the control group over the control group itself. The data show that Baicalein and DMSO administration relevantly increase AST, ALT, and GLDH levels. (b) Assessment of attenuation of the liver enzyme elevation post DMSO and Baicalein pretreatment after inducing hepatic IRI by calculating a relative increase in enzyme activation. This is generated as ratio of the calculated toxic factor after IRI over the previously calculated toxic factor of the respective sham-operated mice. Therefore, the relative enzyme increase for the sham group matches 100% for each enzyme and pretreatment to be investigated. Hence, it can be stated that Baicalein pretreatment after IRI leads to an acknowledgeable decrease of liver enzyme elevation compared to the respective sham-operated mice, significant for AST activation (−50.3%; ^∗p = 0.0438) and GLDH activation (−65.8%; ^∗p = 0.0481).

Figure 5

Quantification of spectral photometric analyses of intracellular activity of glutathione oxidation using Tietze's kinetic test. Activity was calculated as a ratio of measured amount of oxidized glutathione disulfide (GSSG), the product of GPX4, over its substrate glutathione (GSH). Pretreatment with Baicalein showed a significant increase in glutathione oxidation by +54.5% (^∗p = 0.0438), DMSO solely in a more discrete fashion (+16.7%).