Protection of rat liver against hepatic ischemia-reperfusion injury by a novel selenocysteine-containing 7-mer peptide

Abstract

Hepatic ischemia-reperfusion (I-R) injury causes acute organ damage or dysfunction, and remains a problem for liver transplantation. In the I-R phase, the generation of reactive oxygen species aggravates the injury. In the current study, a novel selenocysteine-containing 7‑mer peptide (H-Arg-Sec-Gly-Arg-Asn-Ala-Gln-OH) was constructed to imitate the active site of an antioxidant enzyme, glutathione peroxidase (GPX). The 7‑mer peptide which has a lower molecular weight, and improved water‑solubility, higher stability and improved cell membrane permeability compared with other GPX mimics. Its GPX activity reached 13 U/µmol, which was 13 times that of ebselen (a representative GPX mimic). The effect of this GPX mimic on I‑R injury of the liver was assessed in rats. The 7‑mer peptide significantly inhibited the increase in serum hepatic amino‑transferases, tissue malondialdehyde, nitric oxide contents, myeloperoxidase activity and decrease of GPX activity compared with I‑R tissue. Following treatment with the 7‑mer peptide, the expression of B‑cell CLL/lymphoma‑2 (Bcl‑2) was significantly upregulated at the mRNA and protein level compared with the I‑R group, as determined by reverse transcription‑polymerase chain reaction and immunohistochemistry, respectively. By contrast, Bcl‑2 associated X protein (Bax) was downregulated by the 7‑mer peptide compared the I‑R group. Histological and ultrastructural changes of the rat liver tissue were also compared among the experimental groups. The results of the current study suggest that the 7‑mer peptide protected the liver against hepatic I‑R injury via suppression of oxygen‑derived free radicals and regulation of Bcl‑2 and Bax expression, which are involved in the apoptosis of liver cells. The findings of the present study will further the investigation of the 7-mer peptide as an effective therapeutic agent in hepatic I-R injury.

Figure 1

Effect of the 7p on ALT activity in serum following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. sham group; ^##P<0.01 vs. I-R injury group. ALT, alanine aminotransferase; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 2

Effect of the 7p on AST activity in serum following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. sham group; ^##P<0.01 vs. I-R injury group. AST, aspartate aminotransferase; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 3

Effect of the 7p on LDH activity in serum following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. sham group; ^##P<0.01 vs. I-R injury group. LDH, lactate dehydrogenase; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 4

Effect of the 7p on liver MPO activity following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. control group; ^##P<0.01 vs. I-R injury group. MPO, myeloperoxidase; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 5

Effect of the 7p on liver MDA content following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. sham group; ^##P<0.01 vs. I-R injury group. MDA, malondialdehyde; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 6

Effect of the 7p on liver NO content following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. sham group; ^#P<0.05 vs. I-R injury group. NO, nitric oxide; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 7

Effect of the 7p on liver GPX activity following hepatic reperfusion. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^**P<0.01 vs. sham group; ^##P<0.01 vs. I-R injury group. GPX, glutathione peroxidase; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 8

Immunohistochemical assay of B-cell CLL/lymphoma-2 in liver tissue. (A) Sham control group; (B) 7p control group; (C) I-R group; (D) 7p treated I-R group. Original magnification, ×400. 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 9

Immunohistochemical assay of Bcl-2 associated X protein in liver tissue. (A) Sham control group; (B) 7p control group; (C) I-R group; (D) 7p treated I-R group. Original magnification: ×400. 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 10

The IHC levels of Bcl-2 and Bax expression in liver tissue. Student's t test was used, values are mean ± standard deviation of three independent experiments. ^*P<0.05, ^**P<0.01 vs. sham group; ^##P<0.01 vs. I-R injury group. IHC, immunohistochemistry; Bcl-2, B-cell CLL/lymphoma-2; Bax, Bcl-2 associated X protein; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 11

Semi-quantitative reverse transcription-polymerase chain reaction analysis of (A) Bcl-2 and (B) Bax mRNA expression in the liver tissue. β-actin was used as control. M: marker; Lane 1: sham control group; Lane 2: 7p control group; Lane 3: I-R group; Lane 4: 7p-treated I-R group. (C) Relative expression of Bcl-2 and Bax mRNA (% β-actin) in the four groups were compared. Student's t test was used, ^*P<0.05, ^**P<0.01 vs. control group; ^##P<0.01 vs. I-R injury group. Bcl-2, B-cell CLL/lymphoma-2; Bax, Bcl-2 associated X protein; 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 12

Microscopic histological changes of liver tissue following hematoxylin and eosin staining. (A) Sham control group; (B) 7p control group; (C) I-R group; (D) 7p treated I-R group. Original magnification, ×400. 7p, 7-mer peptide; I-R, ischemia-reperfusion.

Figure 13

Hepatic ultrastructure changes following reperfusion under electron microscopy. (A) Sham control group; (B) 7p control group; (C) I-R group; (D) 7p treated I-R group. Original magnification, ×12 K. 7p, 7-mer peptide; I-R, ischemia-reperfusion.