Preconditioning by Low Dose LPS Prevents Subsequent LPS-Induced Severe Liver Injury via Nrf2 Activation in Mice

Abstract

Background: Sepsis is a syndrome triggered by endotoxin lipopolysaccharide (LPS) during bacterial infection. Sepsis sometimes recurs, with the second sepsis giving rise to a different phenotype because of disease modification by the preceding sepsis. Such a protective modification is called a preconditioning (PC) effect. PC is an endogenous protective mechanism by which sublethal damage confers tolerance to a subsequent lethal load. Oxidative stress is one of the important pathogenetic mechanisms that occur in sepsis. The nuclear factor erythroid 2 (NF-E2)-related factor-2 (Nrf2) system is a key regulatory transcription factor that protects organs and cells against oxidative stress and may be associated with the PC effect in repeated sepsis.

Methods: The effect of PC induced by low-dose LPS on survival rate and liver injury against subsequent high-dose LPS stimulation was examined using a mouse model of sepsis. In order to understand the detailed mechanism(s) involved in the PC effect within the liver, gene expression array was performed. As a candidate mechanism of PC, the activation of the Nrf2 system was analyzed using Nrf2 reporter mice. Furthermore, the induction of heme oxygenase-1 (HO-1), one of the main targets of Nrf2, in the liver was examined by immunoblotting and immunohistochemistry. The PC effect on liver injury induced by LPS was further examined using Nrf2-deficient mice.

Results: PC by LPS (1.7 or 5.0 mg/kg body weight, intraperitoneally) increased the survival rate of mice and decreased liver injury in response to a subsequent injection of a lethal level of LPS (20 mg/kg body weight). DNA array revealed that the gene ontology term "antioxidant activity" as one of the candidate mechanisms of the PC effect by LPS. In Nrf2 reporter mice, PC immediately and intensely enhanced luminescence that indicated Nrf2 activation after subsequent LPS injection. The induction of HO-1 by LPS was also enhanced by preceding PC, and its induction was observed mainly in Kupffer cells of the liver. In Nrf2-deficient mice, the induction of HO-1 in Kupffer cells and the hepatoprotective effect of PC were decreased as compared with wild-type mice.

Conclusion: Our results suggest that activation of the Nrf2 system is, at least in part, one of the mechanisms of a PC effect in the mouse liver in the case of repeated LPS stimulation.

Keywords: Kupffer cell; NF-E2-related factor 2; heme oxygenase-1; lipopolysaccharide; preconditioning.

Fig. 1.

Experimental protocol performed in the present study. (A) PC (0, 1.7, or 5.0 mg/kg BW LPS, i.p.) was carried out at day –5 in C57BL/6 mice. At day 0, a high dose (20 mg/kg BW) of LPS was injected i.p. as a MS. Survival rates were followed for 7 days (days 0–7). Tissue sampling for DNA array was carried out 12 h after MS. Western blotting (WB), histological analyses (HA), and plasma aminotransferase assay (ALT and AST) were carried out at days 1, 1, and 2, respectively. Abbreviations of each condition and how LPS was injected are described in the figure. (B) Time course of in vivo Nrf2 reporter assay. Luminescence (Lumi) was detected 24 h after PC (on day –4) and 16 h after MS by LPS. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BW, body weight; HA, histological analyses; i.p., intraperitoneal injection; LPS, lipopolysaccharide; Lumi, luminescence; MS, main stimulation; Nrf2, NF-E2-related factor-2; PC, preconditioning; WB, western blotting.

Fig. 2.

PC by low-dose LPS induces tolerance against later high-dose LPS stimulation. (A) Life spans of LPS-injected mice (n = 8, each group). The life spans of PC1.7-LPS20 and PC5.0-LPS20 mice groups are significantly longer than that of the PC0-LPS20 group. *: P < 0.01 vs. PC0-LPS20, **: P < 0.05 vs. PC0-LPS20. (B, C) Plasma ALT and AST levels in PC0-LPS0 (n = 3), PC0-LPS20 (n = 4), and PC1.7-LPS20 groups (n = 7). *: P < 0.05 vs. PC0-LPS0, **: P < 0.05 vs. PC0-LPS20. (ANOVA) (D–H) HE staining of liver tissues. (D–H) HE staining of the liver. (D) The liver from a representative PC0-LPS0 mouse. (E) The liver from a representative PC0-LPS20 mouse. Sites of focal necrosis are scattered within the liver (arrowheads). (F) The liver from a representative PC0-LPS20 mouse at higher magnification. Neutrophil infiltration is observed in the sinusoids (arrows). (G) The liver from a representative PC1.7-LPS20 mouse. A small area of focal necrosis (arrowhead) is observed in the liver. (H) The liver from a representative PC1.7-LPS20 mouse at higher magnification. A small number of infiltrating neutrophils is observed in a sinusoid (arrow). Scale bars (D)(E)(G) represent 200 μm and (F)(H) 100 μm. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CV, central vein; HE, hematoxylin-eosin; LPS, lipopolysaccharide; PC, preconditioning; PV, portal vein.

Fig. 3.

PC by low-dose LPS enhances Nrf2 activation during subsequent stimulation by high-dose LPS. (A) An in vivo Nrf2 reporter assay using OKD48 mice. The upper photograph shows luminescence 24 h after PC (day –4). The lower photograph shows luminescence 16 h after MS with LPS (20 mg/kg BW). An immediate and intense luminescence is observed in PC1.7-LPS20 and PC5.0-LPS20 mice. (B) The induction of HO-1 protein is shown. Immunoblots are representative of three independent experiments. HO-1 protein bands in liver samples from PC0-LPS0, PC1.7-LPS0, and PC0-LPS20 mice appear faint. The induced expression of HO-1 protein in liver from PC1.7-LPS20 mice is intense. (C, D) Immunohistochemistry using anti-HO-1 and anti-CD163 antibodies in the liver from PC0-LPS20 (C) and PC1.7-LPS20 mice (D). The expression of HO-1 (brown) is observed mainly in CD163-positive Kupffer cells. Small window in (D) represents a double staining for HO-1 (red) and CD163 (brown). Scale bars represent 100 μm. BW, body weight; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; MS, main stimulation; Nrf2, NF-E2-related factor-2; PC, preconditioning.

Fig. 4.

Nrf2 deficiency abolished the protective effect of PC against liver damage by high-dose LPS in mice. (A) Immunoblot using anti-HO-1 antibody. The immunoblot shown is representative of three independent experiments. In Nrf2-deficient mice, the expression of HO-1 protein bands in liver samples from PC0-LPS0, PC1.7-LPS0, and PC0-LPS20 mice are faint, unlike the case for wild-type mice (far left lane). Liver samples from PC1.7-LPS20 treated Nrf2-deficient mice show a lower induction of HO-1 protein (far right lane) than PC1.7-LPS20 treated wild-type mice (far left lane). (B, C) Immunohistochemistry using anti-HO-1 antibody at the same time of western blotting. (B) The liver from the Nrf2-deficient mice with PC0-LPS20, (C) The liver from the Nrf2-deficient mice with PC1.7-LPS20. In Nrf2-deficient mice, the induction of HO-1 in Kupffer cells is reduced even if samples are from PC1.7-LPS20 treatment group. (D–F) HE staining of the liver from Nrf2-deficient mice. (D) The liver from Nrf2-deficient mice with PC0-LPS20 treatment shows severe necrosis in a large area (arrowheads), severe neutrophil infiltration (arrows), and microhemorrhage (white arrowhead). (E, F) In a liver from an Nrf2-deficient mouse after PC1.7-LPS20 treatment, the protective effect of PC is abolished, and focal necrosis (arrowheads), neurotrophil infiltration in sinusoids (arrows), and microhemorrhage (white arrowhead) are observed. Scale bars (D)(E) represent 200 μm and (B, C, F) 100 μm. CV, central vein; HE, hematoxylin-eosin; LPS, lipopolysaccharide; MS, main stimulation; Nrf2, NF-E2-related factor-2; PC, preconditioning.