Up-regulation of FOXO1 and reduced inflammation by β-hydroxybutyric acid are essential diet restriction benefits against liver injury

Abstract

Liver ischemia and reperfusion injury (IRI) is a major challenge in liver surgery. Diet restriction reduces liver damage by increasing stress resistance; however, the underlying molecular mechanisms remain unclear. We investigated the preventive effect of 12-h fasting on mouse liver IRI. Partial warm hepatic IRI model in wild-type male C57BL/6 mice was used. The control ischemia and reperfusion (IR) group of mice was given food and water ad libitum, while the fasting IR group was given water but not food for 12 h before ischemic insult. In 12-h fasting mice, serum liver-derived enzyme level and tissue damages due to IR were strongly suppressed. Serum β-hydroxybutyric acid (BHB) was significantly raised before ischemia and during reperfusion. Up-regulated BHB induced an increment in the expression of FOXO1 transcription factor by raising the level of acetylated histone. Antioxidative enzyme heme oxigenase 1 (HO-1), a target gene of FOXO1, then increased. Autophagy activity was also enhanced. Serum high-mobility group box 1 was remarkably lowered by the 12-h fasting, and activation of NF-κB and NLRP3 inflammasome was suppressed. Consequently, inflammatory cytokine production and liver injury were reduced. Exogenous BHB administration or histone deacetylase inhibitor administration into the control fed mice ameliorated liver IRI, while FOXO1 inhibitor administration to the 12-h fasting group exacerbated liver IRI. The 12-h fasting exerted beneficial effects on the prevention of liver IRI by increasing BHB, thus up-regulating FOXO1 and HO-1, and by reducing the inflammatory responses and apoptotic cell death via the down-regulation of NF-κB and NLRP3 inflammasome.

Keywords: FOXO1; fasting; ischemia and reperfusion injury; β-hydroxybutyric acid.

Fig. 1.

Hepatocellular damage induced by IR in the controls and the 12-h fasting mice. (A) The sALT levels were measured. Means and SD are shown (n = 8 mice per group; two-way ANOVA; P < 0.0001, Bonferroni’s posttest: ***P < 0.001 vs. control mice). (B-1) Liver histology (H & E staining) after IR and sham-operated (magnification 200×; in box, 400×). (B-2) Suzuki’s histological score after IR. Means and SD are shown (n = 8 mice per group; ***P < 0.001). (C-1) TUNEL-assisted detection of hepatic apoptosis after IR (magnification 200×; in box, 400×). (C-2) Quantification of TUNEL positive cells. Means and SD are shown (n = 8 mice per group; ***P < 0.001). (D) Western blot-assisted expression of cleaved caspase-3, Bcl-2 at 6 h of reperfusion. (E) Quantification of Western blot bands shown in D. Means and SD are shown (*P < 0.05).

Fig. 2.

Preoperative fasting suppressed the release of HMGB1 and proinflammatory cytokines. (A) Serum HMGB1 levels were measured. Means and SD are shown (n = 7 mice per group; two-way ANOVA: P < 0.0001; Bonferroni’s posttest: **P < 0.01, ***P < 0.001 vs. control mice). (B) Serum levels of proinflammatory cytokines (IL-6, TNFα, IL-1β, IL-18, and IFNγ) were measured. Means and SD are shown (n = 8 mice per group; two-way ANOVA; IL-6, P < 0.0001; TNFα, P < 0.0001; IFNγ, P < 0.001; IL-1β, P < 0.0001; IL-18, P < 0.001; Bonferroni’s posttest: **P < 0.01, ***P < 0.001 vs. control mice). (C) Quantitative PCR detection of proinflammatory cytokines (IL-6, TNFα, IL-1β, and IFNγ) was performed at 3 h of reperfusion after 60 min of ischemia. Data were normalized to HPRT gene expression. Means and SD are shown (sham, n = 4; IR 3 h, n = 8 mice per group; *P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 3.

Preoperative fasting protected liver from injury through the up-regulation of FOXO1 and suppressed NF-κB and NLRP3 signaling induced by IR. (A) Western blot-assisted analyses of the total and phosphorylated protein levels of FOXO1, IκBα and acetylated histone-3, histone-3, FOXO3a, HO-1, LC3B, and NLRP3 before ischemia and during reperfusion. β-actin was used as the internal control. (B) Representative of immunohistochemistry of FOXO1 (dark brown) at 6 h of reperfusion (magnification 400×). (C) Representative of immunofluorescence staining of FOXO1 (green) and DAPI (blue) at 6 h of reperfusion (magnification 400×). (D) Representative immunohistochemistry of 4-HNE (dark brown) at 6 h of reperfusion (magnification 200×; in box, 400×). (E) The sALT levels were measured for mice pretreated with FOXO1 inhibitor and the 12-h fasting mice pretreated with DMSO only. Means and SD are shown (n = 8 mice per group; **P < 0.01). (F) Serum levels of proinflammatory cytokines (IL-6, TNFα, IL-1β, and IFNγ) were measured at 6 h of reperfusion. Means and SD are shown (sham, n = 4; IR 6 h, n = 8 mice per group; *P < 0.05; **P < 0.01; ***P < 0.001). (G) Western blot-assisted expression of HO-1 and LC3B at 6 h of reperfusion.

Fig. 4.

BHB controls the benefits of 12-h fasting. (A) Serum BHB levels were measured in control fed mice and 12-h fasting mice. Means and SD are shown (n = 8 mice per group; two-way ANOVA: P < 0.0001; Bonferroni’s posttest: ***P < 0.001 vs. control fed mice). (B) After the administration of BHB or PBS into the fed mice, serum BHB levels were measured. Means and SD are shown (n = 8 mice per group; two-way ANOVA: P < 0.0001; Bonferroni’s posttest: **P < 0.01, ***P < 0.001 vs. PBS-administered mice). (C) The sALT levels were measured. Means and SD are shown (n = 8 mice per group; ***P < 0.001). (D) After the administration of BHB or PBS, serum levels of proinflammatory cytokines (IL-6, TNFα, IL-1β, and IFNγ) were measured. Means and SD are shown (sham, n = 4; IR 6 h, n = 8 mice per group; *P < 0.05; **P < 0.01). (E-1) Liver histology (H & E staining) after IR (magnification 200×). (E-2) Suzuki’s histological score after IR. Means and SD are shown (n = 8 mice per group; **P < 0.01). (F) Western blot-assisted analyses of the FOXO1, HO-1 acetylated histone-3, and NLRP3.

Fig. 5.

HDAC inhibition reduces liver IRI through the up-regulation of FOXO1. (A) After the administration of DMSO or HDAC inhibitor in DMSO, sALT levels were measured. Means and SD are shown (n = 8 mice per group; ***P < 0.001). (B) Serum levels of proinflammatory cytokines (IL-6, TNFα, IL-1β, and IFNγ) were measured. Means and SD are shown (sham, n = 4; IR 6 h, n = 8 mice per group; *P < 0.05; **P < 0.01). (C) Western blot-assisted analyses of the FOXO1, acetylated histone-3, HO-1, and LC3B.

Fig. 6.

Addition of glucose to drinking water reversed the preventive effects of the 12-h fast on liver IRI. (A) Serum BHB levels were measured in 12-h fasting mice fed with water only or 10% glucose water. Means and SD are shown (n = 8 mice per group; two-way ANOVA; P < 0.0001, Bonferroni’s posttest: ***P < 0.001 vs. 12-h fasting without glucose). (B) The sALT levels were measured in the control fed mice, 12-h fasting mice, and 12-h fasting mice fed with 10% glucose water. Means and SD are shown (n = 8 mice per group; *P < 0.05; **P < 0.01; ***P < 0.001). (C) Serum levels of proinflammatory cytokines (IL-6, TNFα, IL-1β, and IFNγ) were measured. Means and SD are shown (sham, n = 4; IR 6 h, n = 8 mice per group; *P < 0.05; **P < 0.01). (D) Western blot-assisted analyses of FOXO1 and HO-1.

Fig. 7.

Schematic summary of the present study.