Salutary effect of pre-treatment with an Nrf2 inducer on ischemia reperfusion injury in the rat liver

Abstract

Background: Ischemia-reperfusion injury (IRI) is a common phenomenon occurring during liver surgery, transplantation, and trauma. IRI causes oxidative stress which plays a critical role in causing organ damage. The Nrf2 is the master regulator of numerous genes, encoding antioxidant, detoxifying, and cytoprotective molecules. Nrf2 dysfunction has been implicated in the pathogenesis of several inflammatory disorders, cancer, and aging. This study was undertaken to investigate the effect of Nrf2 pathway activator (dh404) on warm liver IRI in a rodent model.

Methods: Ten Sprague-Dawley rats were treated with dh404 or vehicle. Dh404 was dissolved in sesame oil and was given orally (1.5mg/kg) the night before and 5 hours before procedures. Rat livers were subjected to 60 minutes of 70% ischemia followed by 3 hours of reperfusion. Serum ALT and Malondialdehyde (MDA) were determined and liver tissue was processed for histological examination, and determination of apoptosis, myeloperoxidase (MPO) activity, ADP/ATP ratio, and expressions of Nrf2, eNOS, anti-oxidant enzymes, and inflammatory mediators.

Results: Serum ALT and MDA levels and tissue MPO activity were significantly lower, expression of the anti-oxidant enzyme, glutamate cysteine ligase were significantly higher, whereas expression of NFkB and COX-2 was unchanged in the dh404-treated group. Although the total Suzuki histology score did not differ significantly, the extent of sinusoidal congestion, vacuolization, and apoptosis was significantly reduced in the dh404 treated compared to the untreated group (P<0.01).

Conclusions: Pre-treatment with dh404 resulted in partial attenuation of hepatic ischemia reperfusion injury in rats.

Keywords: Reactive Oxygen Species; bardoxolone methyl analogue; inflammation; ischemia reperfusion injury; liver; oxidative stress.

Figure 1. Liver damage evaluation by histological assay

(A). Liver tissue specimens were stained with hematoxylin and eosin. Representative specimens are shown (×40 - ×200). (B) Suzuki score The level of sinusoidal congestion and vacuolization in the dh404 treated group was significantly improved compared to the untreated group but not necrosis level. There was no significant difference in the total score. Data represent mean±S.D.

Figure 2. TUNEL staining in liver post ischemia/reperfusion injury

(A). a-d. TUNEL staining in the control group (a; DAPI ×63, b; TUNEL×63, c; Merged×63, d; Merged ×20). e-h. Dh404 group. (Arrows indicate apoptotic cells) Apoptotic index indicates that the dh404 treated group had significantly less TUNEL-positive cells when compared to the control group (P<0.05). (B). Apoptotic index. Data represent mean±S.D.

Figure 3. Biochemical makers for liver damage

The levels of serum ALT (A), MDA (B) and MPO(C) in the liver tissue were determined. All of them in the dh404 treated group showed significantly lower compared to the control group. However, there was no significant difference in ADP/ATP ratio (D).

Figure 4. Nrf2 expression in both cytoplasm and nucleus in the liver

The data were expressed as relative values standardized by GAPDH for cytoplasmic protein and histone for nucleus protein. There was no significant difference in Nrf2 expression in both cytoplasm and nucleus.

Figure 5. Western blotting analysis

The data were expressed as relative values standardized by GAPDH. The expression of GCLM and GCLC in the dh404 treated group was significantly increased. However, there was no significant difference in other Nrf2 target antioxidants. There were no significant reduction of both NF-κB, COX-2 and eNOS expressions in the dh404-treated animals’ liver tissues