Oligonol Ameliorates CCl₄-Induced Liver Injury in Rats via the NF-Kappa B and MAPK Signaling Pathways

Abstract

Oxidative stress is thought to be a key risk factor in the development of hepatic diseases. Blocking or retarding the reactions of oxidation and the inflammatory process by antioxidants could be a promising therapeutic intervention for prevention or treatment of liver injuries. Oligonol is a low molecular weight polyphenol containing catechin-type monomers and oligomers derived from lychee fruit. In this study, we investigated the anti-inflammatory effect of oligonol on carbon tetrachloride- (CCl4-) induced acute hepatic injury in rats. Oral administration of oligonol (10 or 50 mg/kg) reduced CCl4-induced abnormalities in liver histology and serum AST and serum ALT levels. Oligonol treatment attenuated the CCl4-induced production of inflammatory mediators, including TNF-α, IL-1β, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA levels. Western blot analysis showed that oligonol suppressed proinflammatory nuclear factor-kappa B (NF-κB) p65 activation, phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) as well as Akt. Oligonol exhibited strong antioxidative activity in vitro and in vivo, and hepatoprotective activity against t-butyl hydroperoxide-induced HepG2 cells. Taken together, oligonol showed antioxidative and anti-inflammatory effects in CCl4-intoxicated rats by inhibiting oxidative stress and NF-κB activation via blockade of the activation of upstream kinases including MAPKs and Akt.

Figure 1

Effect of oligonol on HepG2 cell damage induced by t-BHP. Cell viability was assessed using MTT assays. Data shown represent means ± standard deviation of triplicate experiments.

Figure 2

Effect of oligonol on t-BHP-induced COX-2 expression on HepG2 cells. HepG2 cells were treated with different concentrations of oligonol (0, 0.5, and 2 μg/mL) for 4 h before being exposed to t-BHP (200 μM) for 24 h. Western blotting was performed to detect COX-2 in whole protein (30 μg) from HepG2 cell. One representative blot of each protein is shown from three experiments that yielded similar result, respectively. Values are normalized as percentage of β-actin. Values are mean ± SE of n = 3. ^∗ p < 0.05 compared with the control group, and ^# p < 0.05 and ^## p < 0.01 compared with the t-BHP group.

Figure 3

Measurements of serum ALT and serum AST levels. Groups are as described in “Methods.” Values are mean ± SE of n = 6 rats/group. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 compared with the control group and ^## p < 0.01 and ^### p < 0.001 compared with the CCl₄ group.

Figure 4

Effect of oligonol on ROS and MDA levels in rat liver intoxicated with CCl₄. ROS generation was measured by DCF formation with a fluorescent probe, DCFDA. Effect of oligonol on CCl₄-induced lipid peroxidation activity from the liver was measured by the method of Buege and Aust. Values are mean ± SE of n = 6 rats/group. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 compared with the control group. ^### p < 0.001 compared with the CCl₄ group.

Figure 5

Effects of oligonol on CCl₄-induced histopathological changes in rat livers. Representative H&E stained sections of livers of rats treated as described in methods. (a) Liver section of control group shows normal liver architecture, intact hepatocytes, and radiating hepatic cords from the central vein (CV); (b) CCl₄-induced damage is indicated by distortion of the tissue architecture, submassive necrosis (thick arrows), fatty changes (thin arrows) in hepatocytes, and aggregations of inflammatory cells (arrowheads); (c) oligonol (10 mg/kg) plus CCl₄; and (d) oligonol (50 mg/kg) plus CCl₄; oligonol treatment reduced the pathological alterations induced by CCl₄. All images are original magnification ×400.

Figure 6

Effects of oligonol on TNF-α and IL-1β mRNA expression after CCl₄ administration. RT-PCR was performed to measure TNF-α and IL-1β mRNA expression in the liver tissues. Values are mean ± SE of n = 6. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 compared with the control group and ^## p < 0.01 and ^### p < 0.001 compared with the CCl₄ group.

Figure 7

Effects of oligonol on COX-2 and iNOS mRNA expression after CCl₄ administration. RT-PCR was performed to measure COX-2 and iNOS mRNA expression in the liver tissues. Values are mean ± SE of n = 6. ^∗∗ p < 0.01 compared with the control group and ^# p < 0.05 and ^## p < 0.01 compared with the CCl₄ group.

Figure 8

Effects of oligonol on CCl₄-induced NF-κB p65 activation. Western blotting was performed to detect nuclear and cytoplasmic localization of NF-κB p65 in the livers tissues. The relative level of NF-κB p65 in the nuclear and cytosol compared with β-actin and histon, respectively, and quantified by image analysis. Values are mean ± SE of n = 6. ^∗ p < 0.05 compared with the control group and ^# p < 0.05 compared with the CCl₄ group.

Figure 9

Effects of oligonol on CCl₄-induced MAPKs and Akt activation. Western blotting was performed to detect ERK, JNK, and p38 MAPK, as well as Akt, in liver tissues. Activation of these kinases was detected using their specific phosphorylated antibody. The band intensity of phosphorylated forms of MAPKs and Akt was quantified by densitometry and normalized to total forms of MAPKs and Akt, respectively. Values are mean ± SE of n = 6. ^∗ p < 0.05 and ^∗∗ p < 0.01 compared with the control group and ^# p < 0.05 and ^## p < 0.01 compared with the CCl₄ group.