Xanthohumol suppresses inflammatory response to warm ischemia–reperfusion induced liver injury

https://doi.org/10.1016/j.yexmp.2012.05.003Get rights and content

Abstract

Liver ischemia/reperfusion (I/R) leads to formation of reactive oxygen species (ROS), which cause hepatic injury and initiate an inflammatory response, which is a critical problem after liver surgery and transplantation. Xanthohumol, the major prenylated chalcone found in hops, has been discussed for its anti-inflammatory and ROS-scavenging properties, and thus, we aimed to investigate the effect of xanthohumol in a model of warm I/R liver injury. Xanthohumol was applied to BALB/c mice orally at a dose of 1 mg/g body weight for 5 days before I/R-injury was induced by clamping the vascular blood supply to the median and left lateral liver lobe for 1 h followed by a 6 h period of reperfusion. At this time, HPLC analysis revealed hepatic xanthohumol levels of approximately 2 μM, a concentration which has been shown to inhibit inflammatory effects in vitro. Assessment of hepatic HMOX1 expression, hepatic glutathione content and immunohistochemical analysis for proteins conjugated with the reactive aldehyde 4-hydroxynonenal indicated that I/R-induced oxidative stress was significantly inhibited in xanthohumol-fed compared to control mice. Histological analysis, TUNEL staining and determination of transaminase serum levels revealed no significant effects of xanthohumol on acute hepatocellular injury. However, at the same time point, pretreatment with xanthohumol almost completely blunted the I/R-induced AKT and NFκB activation and the expression of the proinflammatory genes IL-1alpha, IL-6, MCP-1 and ICAM-1, which are known to play a crucial role in the subacute phase of I/R-induced liver damage. In conclusion, these data indicate the potential of xanthohumol application to prevent adverse inflammatory responses to I/R-induced liver damage such as after surgical liver resection or transplantation.

Highlights

► Xanthohumol reduces oxidative stress in hepatic ischemia/reperfusion (I/R) injury. ► Xanthohumol (XN) ameliorates I/R induced NFkappaB activation in the liver. ► XN inhibits expression of genes, which affect I/R-induced liver damage.

Introduction

Hepatic ischemia/reperfusion (I/R) injury occurs in a variety of clinical scenarios, including transplantation, liver resection, trauma, and hypovolemic shock. The molecular mechanisms causing liver injury after I/R have been subject of several studies, which revealed a series of complex interactions of various participant inflammatory pathways (Walsh et al., 2009, Zwacka et al., 1998a). The process of hepatic I/R-injury can be divided into two phases; an acute phase (the first 6 h after reperfusion) and the following subacute phase (Fan et al., 1999). The acute phase is characterized by acute generation of reactive oxygen species (ROS) subsequent to reoxygenation of the liver leading to marked hepatocellular damage, measurable by an increase of serum alanine transaminase levels peaking 3 to 6 h after reperfusion (Parks and Granger, 1988, Rauen et al., 1994). The secondary subacute phase is associated with vigorous inflammatory responses and the progression of necrotic processes (Gujral et al., 2001, Zwacka et al., 1998a). This can lead to chronic liver inflammation, a decline in liver function, and eventually complete organ failure.

Whereas earlier studies suggested that hepatic I/R‐damage was mainly inflicted in the acute phase by ROS directly, more recent data indicate that oxidative stress may injure the tissue more indirectly by initiating a cascade of adverse cellular responses leading to inflammation, e.g. by activating redox-sensitive transcription factors such as NFκB (Jaeschke, 1995, Shin et al., 2008, Zwacka et al., 1998b). Antioxidant as well as anti-inflammatory therapies depict two practicable and medically sensible treatment options for preventing or ameliorating I/R-induced liver injury. Antioxidant substances could reduce I/R-induced tissue damage directly caused by ROS and, more importantly, attenuate the ROS-triggered inflammatory response which could be further suppressed by additional anti-inflammatory therapies.

Xanthohumol (XN), the principal prenylated chalcone of the hop plant (Humulus lupulus L.), has been discussed for its antioxidant (Gerhauser et al., 2002, Miranda et al., 2000, Yamaguchi et al., 2009) as well as anti-inflammatory properties (Dorn et al., 2010b, Lupinacci et al., 2009, Stevens and Page, 2004) suggesting this natural substance as a potential candidate for an I/R-damage-preventing treatment of the liver. In addition, XN is known as an inhibitor of the transcription factor NFκB (Albini et al., 2006, Colgate et al., 2007, Dorn et al., 2010c) whose activity increases upon hepatic I/R (Suetsugu et al., 2005, Tacke et al., 2009). Whereas it has been unclear for a long time whether NFκB-dependent signaling withholds a protective or damaging role in I/R, more recent data show that the NFκB pathway does not serve as a survival pathway in hepatic I/R, but instead can aggravate hepatocellular death and liver damage (Luedde et al., 2005, Suetsugu et al., 2005). NFκB-mediated expression of adhesion molecules like ICAM-1 (Inter-Cellular Adhesion Molecule-1) may be responsible for increased tissue damage inflicted by infiltrating neutrophils in the late phase of hepatic I/R-injury (Jaeschke, 2006, Jaeschke et al., 1996). However, as shown by Beraza et al. (2007), complete abolishment of NFκB activation in conditional NEMO-knockout mice resulted in massive hepatic inflammation and apoptosis after I/R indicating that suppression of NFκB induction is preferable to complete inhibition of basal NFκB activity for preventing I/R-induced liver injury.

The aim of this study was to investigate the effects of XN on oxidative stress, hepatocellular damage and inflammation in the acute phase of a murine model of warm I/R-injury.

Section snippets

Animal model of warm ischemia/reperfusion injury of the liver

Male BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany) at 8 weeks of age and housed in a 22 °C controlled room under a 12 h light–dark cycle with free access to food and water. After one week of acclimatization mice were fed either with standard diet (control) or standard diet supplemented with 0.5% (w/w) XN. XN was obtained from the Nookandeh Institute for Natural Chemicals (Homburg/Saar, Germany) with a purity ≥ 98% determined by HPLC. All chows were prepared by

Effect of xanthohumol on oxidative stress induced during the acute phase of hepatic ischemia–reperfusion

To assess the effect of XN on ischemia/reperfusion (I/R) injury, XN was applied orally to mice by supplementation of the standard diet with XN at a concentration of 5 mg/g chow starting 5 days prior to warm I/R. Previously we have shown that this XN-concentration in the chow did not affect food consumption of mice and led to a daily uptake of approximately 1 mg/g body weight (Dorn et al., 2010a). Since we aimed to assess the XN effect on the acute phase of hepatic I/R-injury mice were sacrificed 6 

Discussion

The aim of this study was to investigate the effect of the prenylated chalcone xanthohumol (XN) on hepatic ischemia–reperfusion (I/R) injury, which is an important problem after liver transplantation and surgical resection (Bahde and Spiegel, 2010, Klune and Tsung, 2010). Based on the known pharmacological properties of XN we focused our analysis on the early phase of I/R-induced liver damage, which is characterized by ROS‐formation following reoxygenation (Parks and Granger, 1988, Rauen et

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We want to thank Ruth Schewior and Marina Fink (Department of Internal Medicine I, University Hospital Regensburg) for the excellent technical assistance.

This project was supported in part by an unrestricted research grant from the Joh. Barth & Sohn GmbH (Nuremberg, Germany).

Financial relationships of the authors with Joh. Barth & Sohn GmbH are as follows: C.H. is a consultant, and C.D. is working in the laboratory of C.H.

S.M. and A.W. are employed by a Joh. Barth & Sohn subsidiary.

J.H. does

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      The rats were divided into four groups each of 10 rats and classified as follows: (1) Sham group: Pre-treated with normal saline for 28 consecutive days and then exposed to the surgical procedure only as that in I/R exposed rats but without clamping the renal arteries; (2) Sham group + XN: Pre-treated with XN (1 mg/kg b.w) for 28 consecutive 28 and then exposed to surgical procedure only as in group 1; (3) I/R group: received normal saline for 28 days and then exposed to renal I/R as discussed below; (4): Xanthohumol (XN) pre-treated group (XH + I/R): pre-treated with XN (1 mg/kg b.w) for 28 days and then exposed to renal I/R on day 28. However, selection of this dose was based on Dorn's study [13] that showed an antioxidant and anti-inflammatory protective role of XN against hepatic I/R injury at a similar dose. However, it is important to mention here that XN has been well tolerated and is considered safe, even at very high doses (up to 1000 mg/kg), as this dose did not affect the major organ functions, homoeostasis, or the protein, lipid, and carbohydrate metabolism in rats [14].

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