Interaction of L-arginine-methyl ester and Sonic hedgehog in liver ischemia-reperfusion injury in the rats

Abstract

Aim: To investigate the role of Sonic hedgehog (Shh) on the course of liver ischemia and reperfusion (I/R) in rats, and the interaction between treatment with nitric oxide donor L-Arginine-methyl ester (L-Arg) and up-regulation of Shh expression.

Methods: A total of 30 male Sprague-Dawley rats weighing 220-240 g were used in this study. Sham-control group (G1, n = 10): a sham operation was performed (except for liver I/R). I/R-untreated group (G2, n = 10): rats underwent liver ischemia for 1 h followed by reperfusion for 45 min. I/R-L-Arg group (G3, n = 10): after performing the same surgical procedure as in group 2, animals were treated with L-Arg. Liver tissues were taken for determination of malondialdehyde (MDA) levels, and biochemical and histological evaluations were made.

Results: Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and gamma-glutamyltranspeptidase (GGT) activities were higher in group 2 than in group 3. MDA values and the hepatic injury score decreased in the L-Arg treated group compared to the I/R-untreated group. In group 2, the hepatocytes were swollen with marked vacuolization. Group 3 rats showed well-preserved liver parenchyma, with hepatocytes extending from the central vein. The morphology of the hepatocytes and the sinusoidal structures was normal, without any signs of congestion. Mild Shh positive immunostaining was detected in group 2 animals. The expression of immunoreactive cells was increased markedly in liver tissue from I/R-L-Arg rats.

Conclusion: Our findings suggest that Shh molecules are critical factors in the pathophysiology of inflammatory liver injury induced by I/R. In addition, NO plays an important role in the immunohistochemical expression of these molecules.

Figure 1

Effects of L-Arg on liver function after liver I/R. AST (A), LDH (B), ALT(C) and GGT (D) values in group 2 were significantly higher compared to groups 1 and 3 (^aP < 0.05 vs group 1; ^cP < 0.05 vs group 3). Values are expressed as mean ± SE. GGT: γ-glutamyltranspeptidase; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Group 1 (n = 10), Sham-control group; Group 2 (n = 10), I/R-untreated group; Group 3 (n = 10), I/R-L-Arg group.

Figure 2

Effects of ischemia/reperfusion and L-Arg on MDA levels (A) and IRS (B) of liver tissue. ^aP < 0.05 group 2 vs group 1; ^cP < 0.05 group 2 vs group 3. Values are expressed as mean ± SE, MDA; Malondialdehyde. IRS; Immunoreactivity scores, Group 1 (n = 10), Sham-control group; Group 2 (n = 10), I/R-untreated group; Group 3 (n = 10), I/R-L-Arg group.

Figure 3

A: Group 1 shows normal liver parenchyma with regular morphology of both the hepatocytes and the sinusoids around the central vein (HE; x 200); B: Group 2 shows increase in necrosis (↑) and cell infiltration (↑↑); C: Swollen hepatocytes with marked vacuolization; D: necrosis (↑) (HE, x 200); E: Group 3 rats showing normal morphology of hepatocytes and sinusoids, reflecting well-preserved liver parenchyma (HE, x 200).

Figure 4

A: No expression of Shh in the control group (Immunoperoxidase. X 200); B: Mild Shh-positive immunostaining in group 2 (arrows) (Immunoperoxidase, X 200), C, D, E, F: Intense Shh expression in liver tissue from I/R-L-Arg treated rats (arrows) (Immunoperoxidase, X 200).