Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy

Abstract

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

Figure 1

Effect of oral administration of zileuton in liver proliferation in PH-rats. (a) Liver weight to body weight (LW/BW) ratio 24 and 48 h post-PH. (b) Representative images of Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry from rat liver tissue 24 h post-PH obtained by optical microscopy (200X). (c) Proliferation Index (PI) determined by PCNA staining 24 h post-PH. PI was calculated as the number of cells in G₁, S, G₂ and M phases of the cell cycle per 100 hepatocytes. (d) Number of PCNA-positive cells in each phase of the cell cycle per 100 hepatocytes 24 h post-PH. (e) Quantification of mitotic figures by analysis of H&E images 24 h post-PH. Analysis of cyclin D1 24 h post-PH by (f) immunoblotting studies in liver nuclear extracts and (g) immunohistochemistry (100X). Selected lanes were cropped from different parts of the same gel and they are shown after cropping, aligning and separating them by white space. Full-length blots are available in Supplementary Information. (h) Proliferation Index determination by PCNA immunohistochemistry in 48 h post-PH rat liver tissue. (i) PCNA-positive cells in each phase of the cell cycle per 100 hepatocytes 48 h post-PH. Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi10: PH-animals treated with zileuton 10 mg/Kg body weight, PHZi40: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n ≥ 4 per experimental group). *p < 0.05 vs. Sh, ^#p < 0.05 vs. PH.

Figure 2

Hepatic LTB4 and LTC4 levels detected by ELISA. (a) LTB4 and LTC4 levels in the removed lobes (time zero for surgery and 2 h after zileuton administration). (b) LTB4 and (c) LTC4 hepatic levels at 1, 5 and 24 h post-partial hepatectomy (PH) in animals treated with vehicle or zileuton 40 mg/Kg body weight. Pre-PH: Removed tissue from animals treated with vehicle, Pre-PHZi40: Removed tissue from animals treated with zileuton 40 mg/Kg body weight, Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n = 4 per experimental group). *p < 0.05 vs. Sh or Pre-PH, ^#p < 0.05 vs. PH for each time.

Figure 3

mRNA and protein expression of main enzymes for LT synthesis in rat liver 1, 5 and 24 h post-PH in animals treated with vehicle or zileuton. RT-qPCR of (a) 5-LOX, (c) LTA4-H and (e) LTC4-S. Immunoblotting of (b) 5-LOX, (d) LTA4-H and (f) LTC4-S. Selected lanes were cropped from different gels and they are shown after cropping, aligning and separating them by white space. Full-length blots are available in Supplementary Information. The amount of every protein band was corrected by β-actin but not shown in order to avoid a more complex image. Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n = 4 per experimental group). *p < 0.05 vs. Sh, ^#p < 0.05 vs. PH for each time.

Figure 4

Analysis of zileuton effect on NF-κB activation 1 h post-PH. (a) NF-κB binding activity measured with a commercial kit. (b) Immunoblotting of the NF-κB inhibitor, IκB-α, in total lysates. (c) Analysis of mRNA expression by RT-qPCR of IL-6. Selected lanes were cropped from different parts of the same gel and they are shown after cropping, aligning and separating them by white space. Full-length blots are available in Supplementary Information. Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n = 4 per experimental group). *p < 0.05 vs. Sh, ^#p < 0.05 vs. PH.

Figure 5

Effect of zileuton treatment on neutrophils recruitment after partial hepatectomy. (a) Mieloperoxydase (MPO)-positive cells quantification in liver tissue from Sh, PH and PHZi rats at 1 and 5 h post-PH. (b) Representative images of MPO immunostaining 5 h post-PH obtained by optical microscopy (400X). Analysis of mRNA expression of (c) MPO and (d) elastase in isolated liver leukocytes 5 h after PH. (d) Quantification of recruited liver neutrophils 5 h post-PH assessed by immunofluorescence and confocal microscopy. (e) Representative images from immunofluorescence detection of neutrophils (100X magnification). Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n = 4 per experimental group). *p < 0.05 vs. Sh, ^#p < 0.05 vs. PH.

Figure 6

Analysis of macrophages/Kuppfer cells markers in liver NPCs population. Immunoblotting of (a) CLEC4F (b) CD68 and (c) CD11b in NPCs lysates 24 h post-PH. Selected lanes were cropped from different parts of the same gel and they are shown after cropping, aligning and separating them by white space. Full-length blots are available in Supplementary Information. Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n = 4 per experimental group). *p < 0.05 vs. Sh, ^#p < 0.05 vs. PH.

Figure 7

Analysis of NPCs apoptosis 24 h post-PH. Apoptosis was assessed by (a) AnnexinV/propidium iodide staining, (b) caspase-3 activity, and (c) immunoblotting study of active caspase-3. Selected lanes were cropped from different parts of the same gel and they are shown after cropping, aligning and separating them by white space. Full-length blots are available in Supplementary Information. Sh: sham animals, PH: partial-hepatectomized animals treated with vehicle, PHZi: PH-animals treated with zileuton 40 mg/Kg body weight. Bars represent mean ± SEM (n = 4 per experimental group). *p < 0.05 vs. Sh, ^#p < 0.05 vs. PH.