Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

Abstract

Aim: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antioxidant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R).

Methods: Rats were divided randomly into four experimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfusion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the operation. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis.

Results: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a significant increase in serum AST and ALT levels (AST: 260.13 +/- 40.17 U/L vs 186.00 +/- 24.21 U/L, P < 0.01; ALT: 139.63 +/- 11.35 U/L vs 48.38 +/- 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 +/- 40.17 U/L vs 216.63 +/- 22.65 U/L, P < 0.05; ALT: 139.63 +/- 11.35 U/L vs 97.63 +/- 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In addition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevated liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression.

Conclusion: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Figure 1

Changes in histology of intestine (A1-D1) and liver (A2-D2) tissues (HE stain, × 200) in the control (A), sulforaphane (SFN) control (B), intestinal ischemia/reperfusion (I/R) (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. A, B: Normal structure of intestine and liver; C: Edema, hemorrhage and neutrophil infiltration were observed in intestinal mucosa and liver tissue; D: Relatively normal histology of intestine and liver with less inflammatory cell infiltration.

Figure 2

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (mean ± SD, U/L) levels in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs intestinal I/R group.

Figure 3

Liver tissue superoxide dismutase (SOD) (mean ± SD, U/mgprot) and myeloperoxidase (MPO) (mean ± SD, U/g) level in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. ^bP < 0.01 vs control group; ^dP < 0.01 vs intestinal I/R group.

Figure 4

Liver tissue glutathione (GSH) and glutathione peroxidase (GSH-Px) (mean ± SD, U/gprot) level in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. ^aP < 0.05, ^bP < 0.01 vs control group; ^cP < 0.05 vs intestinal I/R group.

Figure 5

Immunohistochemical staining of liver NF-E2-related factor-2 (Nrf2) expression (original magnification × 400) in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups.

Figure 6

Immunohistochemical results (semi-quantitative analysis) of liver Nrf2 expression in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. Data are presented as mean ± SD of eight animals. ^bP < 0.01 vs control group; ^dP < 0.01 vs intestinal I/R group.

Figure 7

Immunohistochemical staining of liver heme oxygenase-1 (HO-1) expression (original magnification × 400) in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups.

Figure 8

Immunohistochemical results (semi-quantitative analysis) of liver HO-1 expression in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. Data are presented as mean ± SD of eight animals. ^bP < 0.01 vs control group; ^dP < 0.01 vs intestinal I/R group.

Figure 9

Western blotting analysis of expression of liver Nrf2 and HO-1 in the control (A), SFN control (B), intestinal I/R (C) (1 h ischemia and 2 h reperfusion) and SFN pretreatment (D) groups. PCNA and GAPDH were as the internal control respectively. ^aP < 0.05, ^bP < 0.01 vs control group; ^cP < 0.05, ^dP < 0.01 vs intestinal I/R group.