Protective effects of 2-methoxycinnamaldehyde an active ingredients of Cinnamomum cassia on warm hepatic ischemia reperfusion injury in rat model

Abstract

Objectives: Hepatic ischemia/reperfusion injury (IRI) is one of the major causes of hepatic failure during liver transplantation, trauma, and infections. The present study investigated the protective effect of intra-portal administration of 2-methoxycinnamaldehyde (2-MCA) on hepatic IRI in rats.

Materials and methods: Twenty-four rats were equally divided into four groups; 1) sham group, (no IRI or transfusion), 2) Hepatic IRI (60 min ischemia + 120 min reperfusion, 3) Hepatic IRI+ NS (IRI + normal saline), 4) Hepatic IRI+2-MCA, (IRI + 2-MCA). In groups 3 and 4, 1 ml/kg normal saline and 2-MCA were administered slowly into the vein of the left lateral and median lobes of the liver 10 min before induction of hepatic reperfusion (upper the site of clumping), respectively. The harvest time points were at 2 hours post-reperfusion in all groups.

Results: Histologically, cell death, degenerative changes, sinusoidal dilatation, congestion, hemorrhage, and infiltration of inflammatory cells were observed in IRI group, while these pathological changes were attenuated in the 2-MCA administrated group. The level of alanine transaminase, aspartate transaminase, tumor necrosis factor- α and interleukin-6 in serum and hepatic malondialdehyde were significantly increased by IRI, and 2-MCA administration reduced all these markers. In addition, caspase-3 and nuclear factor κB (NF-κB) expression were investigated immunohistochemically. Administration of 2-MCA considerably decreased caspase-3 positive cells and NF-κB activity in comparison with IRI group.

Conclusion: As a conclusion, in situ administration of 2-MCA protects against hepatic IRI via anti-inflammatory, and anti-apoptotic properties.

Keywords: Ischemia reperfusion injury; Liver; Oxidative stress; Rat 2-Methoxycinnamaldehyde.

Figure 1

Effect of intra-portal administration of 2-MCA on the level of AST and ALT in IRI model. **P<0.001.a versus group 2, b versus group 3. All the values were expressed as mean±SEM. 2-MCA: 2-methoxycinnamaldehyde, ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; IRI: Ischemic-reperfusion injury

Figure 2

Effect of intra-portal administration of 2-MCA on the level of hepatic MDA in IRI model. *P<0.05, **P<0.001.a versus group 2, b versus group 3. All the values were expressed as mean±SEM. MDA: Malondialdehyde, 2-MCA: 2-methoxycinnamaldehyde, IRI: Ischemic-reperfusion injury

Figure 3

Effect of intra-portal administration of 2-MCA on the level of TNF-α and IL-6 in IRI model. *P<0.05, **P<0.001.a versus group 2, b versus group 3. All the values were expressed as mean±SEM. TNF-α: Tumor necrosis factor-alpha, IL-6: Interleukin-6, IRI: Ischemic-reperfusion injury, 2-MCA: 2-methoxycinnamaldehyde

Figure 4

Male Wistar rats were subject to partial warm hepatic IRI with intra-portal administration of NS and 2-MCA at dose of 1 ml/kg 10 min before finishing of ischemic period. Liver damage was assessed 2 hr after reperfusion (A & B):Sham-operated rat, cords of hepatocytes are arranged radially around the central vein (circle), (C): Rat undergoing 1 hr of ischemia followed by 2 hr of reperfusion, note to serve injury characterized by necrotic hepatocytes (rectangle) and degenerative changes (circle), (D): Higher magnification of previous Figure, note to hydropic degeneration (circle), pyknotic nuclei (black arrow), and congestion (blue arrow), (E & F): (IRI+ NS), Note to karyolysis in the nuclei of hepatocytes (arrow), (G & H): (IRI+ 2-MCA), Noticeable restoration of hepatocyte integrity and slight congestion (arrow) were detected (H& E, scale bar: A, C, E, and G: 200 μm , B, D, F, and H: 100 μm). IRI: Ischemic-reperfusion injury; 2-MCA: 2-methoxycinnamaldehyde; NS: Normal saline

Figure 5

Immunohistochemical staining of cleaved caspase-3 in hepatic tissue, (A):Sham-operated rats showing no expression of cleaved caspase-3, (B) IRI demonstrating significant caspase-3 activity, (C): (IRI+ NS), Note to prominent immunopositivity in cytoplasm of hepatocytes, (D): (IRI+ 2-MCA), scatter expression of caspase-3 (IHC, scale bar: 100 μm). IRI: Ischemic-reperfusion injury; 2-MCA: 2-methoxycinnamaldehyde; NS: Normal saline

Figure 6

Immunohistochemical staining of NF-kB in rat liver, (A): Sham-operated rats showing no expression of NF-KB. (B): IRI showing a significant increase in NF-kB immunoreactivity in the cytoplasm of hepatocytes. (C): (IRI+ NS), Brown color indicates NF-kB positivity, (D): (IRI+ 2-MCA), demonstrating a significant reduction in NF-kB immunostaining (IHC, scale bar: 100 μm). IRI: Ischemic-reperfusion injury, 2-MCA: 2-methoxycinnamaldehyde, NS: Normal saline, NF-kB: Nuclear factor-kB