Protective effect of post-ischemic treatment with trans-resveratrol on cytokine production and neutrophil recruitment by rat liver

Biochimie 92 (2010) 405e410

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Biochimie journal homepage: www.elsevier.com/locate/biochi

Research paper

Protective effect of post-ischemic treatment with trans-resveratrol on cytokine production and neutrophil recruitment by rat liver Sahar Hassan-Khabbar a,1, Michel Vamy h,1, Charles-Henry Cottart a, b, Dominique Wendum c, d, Françoise Vibert a, Jean-François Savouret e, Patrice Thérond a, f, Jean-Pierre Clot a, Anne-Judith Waligora g, Valérie Nivet-Antoine a, b, * a

Equipe d'Accueil 3617, Faculté de Pharmacie, Université Paris Descartes, 4 avenue de l'Observatoire, 75006 Paris, France Département de Biochimie, Hôpital Charles-Foix, Assistance Publique-Hôpitaux de Paris, Paris, France Université Pierre et Marie Curie-Paris 6, Paris, France d Service d'Anatomie Pathologique, Hôpital Saint-Antoine, Assistance Publique-Hôpitaux de Paris, Paris, France e INSERM, Unité Mixte de Recherche S-747, Unité de Formation et de Recherche Biomédicale, Université Paris Descartes, Paris, France f Service de Biochimie, Centre Hospitalier de Versailles, Le Chesnay, France g Equipe d'Accueil 4065, Faculté de Pharmacie, Université Paris Descartes, Paris, France h Equipe d'Accueil 2510, Faculté de Pharmacie, Université Paris Descartes, 4 avenue de l'Observatoire, Paris, France b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 September 2009 Accepted 15 December 2009 Available online 24 December 2009

Oxidative and inflammatory processes are elicited during hepatic post-ischemic reperfusion and generate liver damage. This study investigated the early anti-inflammatory effect of trans-resveratrol (Tres) and its consequences on the late self-aggravating inflammatory process in liver ischemia-reperfusion (I/R). Partial hepatic ischemia was initiated in rats for 1 h and T-res (0.02 and 0.2 mg/kg) was administered intravenously 5 min before starting reperfusion for 3 h. Plasma levels of aminotransferases and cytokines (tumour necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6) and hepatic neutrophil recruitment were assessed. Hepatic expression of stress protein (heat-shock protein (HSP-70), heme oxygenase-1 (HO-1)) and cytokine (TNF-a, IL-1b, keratinocyte chemoattractant (KC)) mRNA was investigated. I/R caused an increase in aminotransferase levels and increased polymorphonuclear cell infiltration. Postischemic treatment with T-res (0.02 and 0.2 mg/kg) resulted in a significant decrease in aminotransferase, IL-1b and IL-6 plasma levels by about 40%, 60% and 40%, respectively, compared to the vehicle I/R group. Post-ischemic treatment with T-res (0.02 mg/kg) also significantly decreased hepatic neutrophil recruitment. TNF-a, IL-1b, KC and HO-1 hepatic mRNA expression was reduced by T-res without any change in HSP-70 mRNA. This T-res mediated decrease in early release of cytokines and neutrophil recruitment led to a reduction in the late inflammatory process. T-resveratrol might be useful in the prevention of inflammation secondary to hepatic surgery or liver transplantation. Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Heat-shock protein TNF-a IL-1b KC Oxidative stress

1. Introduction

Abbreviations: ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; HO-1, heme oxygenase-1; HSP, heat-shock protein; IL, interleukin; I/R, ischemia-reperfusion; KC, keratinocyte chemoattractant; MPO, myeloperoxidase; PCR, polymerase chain reaction; ROS, reactive oxygen species; RT, reverse transcription; SE, standard error; TNF, tumour necrosis factor; T-res, trans-resveratrol. * Corresponding author at: Equipe d'Accueil 3617, Faculté de Pharmacie, Université Paris Descartes, 4 avenue de l'Observatoire, 75006 Paris, France. Tel.: þ33 1 53 73 97 96; fax: þ33 1 53 73 97 99. E-mail address: [email protected] (V. Nivet-Antoine). 1 These authors contributed equally to this work. 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.12.009

Although ischemia induces liver injury, the damage induced during reperfusion is more severe. Both hepatic and extrahepatic mechanisms are involved in a complex process leading to hepatic ischemia-reperfusion (I/R) injury. It has been established that the initial phase of reperfusion (before 4 h of reperfusion) is fundamental in initiating events leading to the deleterious effects observed in the late phase (after 6 h of reperfusion). It is essential to use a molecule able to interfere with these initial events to limit reperfusion damage and boost the defence mechanisms of target cells [1]. Trans-resveratrol (T-res) is a polyphenol compound which has a protective effect on hepatocytes, increasing hepatic allograft

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survival after transplantation [2]. This molecule is known to act in several ways [3e5]. Its rapid and acute effects are mainly due to its anti-oxidant and anti-inflammatory activities, which are essential for modulation of cell signalling. In a previous study, a single dose of T-res administered post-ischemia was shown to reduce liver damage after 3 h of reperfusion [6]. This beneficial effect was due to replacement of the depleted anti-oxidant defence system in hepatic I/R injury. T-res has been suggested to change the nitric oxide/ superoxide (NO/O2) ratio, improving NO bioavailability and NOinduced protection [7]. Liver reperfusion leads to a damaging inflammatory process. Resveratrol has been shown to have anti-inflammatory properties in other organs, decreasing proinflammatory cytokine and heatshock protein (HSP) gene expression [8]. HSPs are markers of cellular stress and are induced during hepatic I/R. Tumour necrosis factor (TNF)-a and interleukin (IL)-1b are proinflammatory cytokines expressed after hepatic I/R involved in early events leading to the expression of chemokines such as keratinocyte chemoattractant (KC, also called CXCL1). Furthermore, these proinflammatory cytokines and KC are also involved in the recruitment of neutrophils, which are mainly responsive to hepatocyte damage in the late phase [9]. The aim of this study was to determine whether T-res could modify the inflammatory process and neutrophil recruitment initiated after reperfusion, and reduce self-aggravating inflammatory injury.

Liver injury was assessed by measuring the activity of cytolytic enzymes (aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT)) in plasma using automated techniques (Vitros 250). Plasma IL-6, IL-1b and TNF-a levels were assessed using a Quantikine kit (R&D systems, Abingdon, UK). The minimum detectable concentrations were 21 pg/ml, 5 pg/ml and 5 pg/ml, respectively.

2. Materials and methods

2.7. Histological analyses

2.1. Chemicals

A blind histological analysis was performed with quantitative evaluation of neutrophil infiltration. Three groups consisting of four rats each were analyzed (vehicle I/R group, 0.02 mg/kg T-res I/R group, 0.2 mg/kg T-res I/R group). Liver tissue samples from the left lateral lobe were immersed in 10% buffered formalin for 24 h and embedded in paraffin. Ultra-thin sections (4 mm thick) were then prepared and deparaffinized. Polymorphonuclear cell infiltration was quantified after myeloperoxidase (MPO) immunostaining. Briefly, endogenous peroxidase activity was blocked with 0.1% hydrogen peroxide in methanol for 30 min. Microwave antigen retrieval was performed before immunostaining in citrate buffer (10 mmol/l, pH 6). Tissue sections were incubated with anti-MPO antibody (1/100; DakoCytomation) for 60 min at room temperature. Immunolabelling was achieved using a super sensitive link-label immunohistochemistry detection system (Biogenex, San Ramon, CA, USA); the tissue sections were incubated successively with biotinylated anti-immunoglobulins followed by horseradish peroxidase-labelled streptavidin. Peroxidase activity was revealed with 3 amino-ethyl carbazole solution and tissue sections were counterstained with haematoxylin. For each rat, polymorphonuclear cells were counted in five different perivenular (centrilobular) fields (0.38 mm2/field) and the mean  standard error (SE) was calculated.

T-res (trans-3,5,40 -trihydroxystilbene) was purchased from Cayman Chemical Co. (Ann Arbor, MI). All other chemicals were obtained from Sigma (St Louis, MO). 2.2. Animals Male SpragueeDawley rats (280e320 g) were obtained from Charles-River (l'Arbresle, France) and were treated as recommended in the Guide for the Care and use of Laboratory Animals drawn up by the Institute of Laboratory Animal Resources. Rats were housed in a temperature- and humidity-controlled room with a 12 h/12 h light/dark cycle and free access to water and food. 2.3. Ischemia-reperfusion Rats were anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital. Body temperature, recorded through a probe inserted 1.5 cm into the rectum, was maintained at 37  0.5  C by placing the animals on a thermostatically controlled heating mat (Harvard Apparatus, Holliston, Massachusetts, USA). After midline incision of the abdomen, partial ischemia was achieved by clamping the portal vein and hepatic artery supplying the median and left lateral lobes (about 70% of the hepatic parenchyma) for 1 h. Occlusion induced an immediate change in liver colour to a paler shade. The clamp was then removed, allowing tissue reperfusion. Muscle and cutaneous tissues were closed with sutures during reperfusion. Animals were sacrificed after 3 h of reperfusion. The left and median lobes of the liver were removed, immediately immersed in liquid nitrogen and conserved at 80  C. 2.4. Administration of trans-resveratrol T-res was dissolved at different concentrations in a solution of ethanol: physiological saline (3:2) and administered by injection

via the penis vein 5 min before reperfusion. Rats were subjected to 1 h of ischemia and sacrificed after 3 h of reperfusion (I/R). Rats were randomly divided into four groups: (1) vehicle I/R: rats were vehicle-treated and sacrificed at the end of reperfusion (n ¼ 11); (2) 0.02 mg/kg T-res I/R (n ¼ 8); (3) 0.2 mg/kg T-res I/R (n ¼ 9): rats were treated with different doses of T-res and sacrificed at the end of reperfusion; (4) sham group: rats were laparotomized, vehicletreated after 1 h, and then sacrificed 3 h later (n ¼ 11). 2.5. Blood sampling Blood samples were collected by section of the vena cava and aorta after 3 h of reperfusion. Samples were centrifuged at 3000 rpm for 15 min at 4  C and plasma samples were stored at 20  C. 2.6. Plasma analyses

2.8. Quantitative real-time polymerase chain reaction Frozen fragments (100 mg) of the hepatic median lobe were crushed with an Ultra-turrax J25 instrument (Fisher-Bioblock) for 40 s and total mRNA was extracted using the Trizol reagent method (Invitrogen, Illkirch, France) according to the manufacturer's instructions. One microgram of total liver RNA was treated with Dnase I (Invitrogen) and converted into complementary DNA (cDNA) using Superscript II reverse transcriptase, oligo(dT)12e18 primers, and Rnase OUT Recombinant Ribonuclease Inhibitor (Invitrogen). cDNA products were subjected to real-time polymerase chain reaction (PCR) using a Smart Cycler (Cepheid,

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Sunnyvale, Canada). Quantitect SYBR Green PCR and Quantitect primer assay (Qiagen, Courtaboeuf, France) kits were used to quantify HSP-70, HO-1, IL-1b, KC and TNF-a gene expression. All reactions were done in a total volume of 25 ml in triplicate according to the manufacturer's instructions. GAPDH was used as the housekeeping gene after a validation step to verify equal loading of RNA and cDNA in the reverse transcription (RT) and PCR reactions. Data analysis was performed by the 2-DDCt method as described by Livak and Schmittgen [10].

407

Table 2 Effect of trans-resveratrol on plasma concentrations of IL-1b and IL-6 after 1 h of ischemia and 3 h of reperfusion. Group

IL-1b (pg/ml)

Sham (n ¼ 7) Vehicle (I/R) (n ¼ 9) 0.02 mg/kg T-res (I/R) (n ¼ 8) 0.2 mg/kg T-res (I/R) (n ¼ 9)

61.43 63.45 21.88 25.7

   

3.45 6.04 3.73### 4.43###

IL-6 (pg/ml) 313.75 558.43 335.25 301

   

37.46 63.59* 11.61# 64.54##

T-res: trans-resveratrol; IL: interleukin; I/R: ischemia-reperfusion. *p  0.01 vs. sham group; #p  0.05, ##p  0.01 and ###p  0.001 vs. vehicle (I/R) group.

2.9. Statistical analysis All data are presented as mean  SE. Groups were compared by non-parametric analysis of variance, followed by the ManneWhitney U test. p  0.05 was considered statistically significant. 3. Results

the centrilobular areas of I/R rats treated with T-res (0.02 and 0.2 mg/kg) decreased by 60% and 40%, respectively, compared to the vehicle I/R group (I/R ¼ 27.65  4.62; 0.02 mg/kg T-res I/R ¼ 11.05  2.97#; 0.2 mg/kg T-res I/R ¼ 16.65  2.19#; #p  0.05 vs. I/R).

3.1. Effect of trans-resveratrol on liver injury caused by hepatic ischemia-reperfusion evaluated with the aminotransferase assay

3.4. Effect of trans-resveratrol on HO-1 and HSP-70 mRNA expression after ischemia-reperfusion

The release of ALAT and ASAT into plasma reflects hepatic injury. Three hours after reperfusion, ALAT and ASAT levels in the vehicle I/ R group were 14-times and 18-times higher, respectively, than those in the sham group (Table 1). Post-ischemic treatment with low doses of T-res (0.02 and 0.2 mg/kg) significantly decreased ALAT levels by about 40% compared to the vehicle I/R group. At these doses, ASAT levels were decreased by 45% and 36%, respectively, compared with the vehicle I/R group.

I/R induced a significant increase in HO-1 and HSP-70 mRNA levels by 10- and 1000-fold, respectively, when compared to the sham group (Fig. 2A and B). With 0.02 and 0.2 mg/kg T-res, HO-1 transcript levels decreased significantly by about 3-fold when compared to the I/R group. T-res had no effect on HSP-70 mRNA expression.

3.2. Effect of trans-resveratrol on TNF-a, IL-1b and IL-6 plasma concentrations after ischemia-reperfusion Plasma concentrations of TNF-a were below the limit of detection at all times in all groups. After 3 h of reperfusion, plasma concentrations of IL-1b in the vehicle group were similar to those in the sham group (Table 2). Administration of T-res at 0.02 and 0.2 mg/kg resulted in a significant decrease in IL-1b plasma concentrations by about 60% when compared to the vehicle I/R group. After 3 h of reperfusion, plasma concentrations of IL-6 in the vehicle group were significantly increased by about 80% when compared to the sham group. T-res at 0.02 and 0.2 mg/kg significantly decreased IL-6 plasma concentrations by about 40% when compared to the vehicle I/R group, to levels comparable to those in sham rats. 3.3. Effect of trans-resveratrol on neutrophil recruitment after 3 h of reperfusion Post-ischemic treatment with T-res (0.02 mg/kg) induced a clear decrease in liver polymorphonuclear cell infiltration, as estimated by MPO immunostaining (Fig. 1). Polymorphonuclear cells in

Table 1 Effect of post-ischemic treatment with trans-resveratrol on plasma alanine aminotransferase and aspartate aminotransferase concentrations after 1 h of ischemia and 3 h of reperfusion.

ALAT (IU/l) ASAT (IU/l)

Sham (n ¼ 9)

Vehicle (I/R) (n ¼ 11)

0.02 mg/kg T-res (I/R) (n ¼ 8)

0.2 mg/kg T-res (I/R) (n ¼ 9)

213  13 153  15

3047  450* 2740  292*

1809  127# 1517  116##

1722  143# 1746  141#

T-res: trans-resveratrol; ALAT: alanine aminotransferase; ASAT: aspartate aminotransferase; I/R: ischemia-reperfusion. *p  0.001 vs. sham group; #p  0.05, ##p  0.01 vs. vehicle (I/R) group.

3.5. Effect of trans-resveratrol on TNF-a, IL-1b and KC mRNA expression after ischemia-reperfusion I/R induced a significant increase in TNF-a, IL-1b and KC mRNA expression when compared to the sham group (Fig. 3). After administration of T-res (0.02 and 0.2 mg/kg), transcript levels of TNF-a, IL-1b and KC decreased significantly when compared to the vehicle I/R group (Fig. 3), resulting in transcript levels similar to those of sham rats. 4. Discussion I/R-induced tissue damage is a serious post-operative complication after liver surgery. The mechanism of injury is closely related to the inflammatory response, which results in microcirculatory failure followed by necrosis and cell death. Liver injury has a biphasic pattern, with an initial phase conditioning the late phase response. In the initial phase, 0.5e4 h after the onset of reperfusion, reactive oxygen species (ROS) are released and Kupffer cells are activated. This leads to an inflammatory process with cytokine production followed by chemokine expression and enhanced neutrophil sequestration. The late phase corresponds to selfaggravating inflammatory injury and is characterized by neutrophil adhesion to the hepatic vascular endothelium and migration into the hepatic parenchyma. The ensuing massive release of oxidants and proteases directly damage hepatocytes. Among cytokines involved during the initial phase of reperfusion, TNF-a and IL-1b are known to play an important role in neutrophil accumulation [11,12]. KC, a neutrophil selective chemokine, has been shown to mediate neutrophil infiltration [13,14]. On the other hand, IL-6 is also expressed during I/R, limiting hepatocellular injury and promoting hepatocyte regeneration [15]. During the reperfusion phase, several HSPs are also induced which probably interfere in the inflammatory process [16,17]. T-res, a polyphenol compound, has pleiotropic anti-inflammatory and anti-oxidant properties, probably by interfering with

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Fig. 1. Effect of post-ischemic treatment with trans-resveratrol (T-res) on polymorphonuclear cell infiltration of hepatic tissue. Polymorphonuclear cell infiltration was visualized by myeloperoxidase immunostaining of the centrilobular area of a rat from: (A) the vehicle I/R group; and (B) the 0.02 mg/kg T-res I/R group. Original magnification, 200.

transcription factors such as NF-kB [3]. In a previous study, postischemic T-res treatment induced a cytoprotective effect after 3 h of reperfusion by reducing aminotransferase release and hepatic sinusoidal dilation [6]. This beneficial effect was observed with low doses of T-res (0.2 and 0.02 mg/kg) and was related to its antioxidant capacity by glutathione preservation. In the current study, it was hypothesized that T-res might also prevent the inflammatory process secondary to I/R, thereby reducing the late process that leads to endothelial dysfunction.

fold increased compared with sham

fold increased compared with sham

A

HO-1

15

*

10

#

#

R 0.02

R 0.2

5

0 Sh am

I/R

B

H s p7 0

*

1500

1000

500

0 Sh am

I/R

R 0.02

R 0.2

Fig. 2. Effect of post-ischemic treatment with trans-resveratrol (T-res) on hepatic mRNA expression of: (A) HO-1; and (B) HSP-70. Rats were subjected to 1 h of ischemia and 3 h of reperfusion, and injected with vehicle (I/R), 0.02 (R 0.02) or 0.2 (R 0.2) mg/kg T-res, 5 min before reperfusion (n ¼ 11, 8 and 9, respectively). Sham group: rats were laparotomized, vehicle-treated after 1 h, and then sacrificed 3 h later (n ¼ 9). *p  0.05 vs. sham group; #p  0.05 vs. I/R group.

Hepatic expression of TNF-a, IL-1b, IL-6 and KC mRNA was investigated after 3 h of reperfusion to study this potential antiinflammatory effect of T-res in the early phase of reperfusion and project the consequences of the anti-inflammatory effect of T-res to the late phase of reperfusion. Very low levels (<5 pg/ml) of TNF-a were detected in plasma from sham rats after 3 h of reperfusion. In a comparable model, low levels were observed after 3 h of reperfusion [12], whereas other authors reported an early increase in TNF-a within minutes of reperfusion [18,19]. Conversely, significantly elevated IL-6 concentrations were observed after 3 h of reperfusion, while concentrations of IL-1b were similar to sham controls, suggesting that 3 h of reperfusion is too late to observe TNF-a and IL-1b release induced by I/R. An elevation of IL-6 levels has been observed previously after 2 h of reperfusion [18] and these results are in accordance with reported findings that IL-6 release is delayed compared to TNFa and IL-1b [18,20]. In our study, T-res treatment significantly decreased plasma concentrations of IL-6 and IL-1b. This effect of Tres on IL-6 concentrations has also been reported in a hepatic trauma/haemorrhagic shock/resuscitation model and in peritoneal mice macrophages after stimulation [21,22]. Concerning the decrease in IL-1b concentrations by T-res, alpha-lipoic acid, which has anti-oxidant properties, has been shown to diminish IL-1b levels after 1 h of reperfusion in a model similar to ours, leading to hepatoprotection by glutathione preservation [23]. Our data suggest that post-ischemic treatment with T-res could exert an early anti-inflammatory effect in hepatic I/R, by decreasing cytokine levels. To support this idea, a clear decrease in neutrophil recruitment, known to depend on cytokine production, was also observed in the centrilobular zone of treated rats. These effects are in accordance with results observed in a liver transplantation model where cytokine production decreased secondary to inhibition of NF-kB binding activity by T-res [2]. Furthermore, NF-kB inactivation improves hepatic cytolysis after I/R, by diminishing neutrophil infiltration, TNF-a mRNA and protein expression [24]. Anti-inflammatory properties of T-res have also been linked to its ability to inhibit the activation of the Aryl hydrocarbon Receptor (AhR) at mM concentrations and above [5]. This mechanism of action appears unlikely. In the present work, the peak plasmatic concentration elicited by 0.2 mg/kg T-res never attained this level (data not shown). Moreover, the rats were sacrificed 3 h after T-res injection although expression of AhR dependent genes is increased after 4e8 h. To understand the consequences of post-ischemic T-res treatment on the self-aggravating inflammatory process, hepatic cytokine and chemokine mRNAs were analyzed by quantitative RT-PCR. Low levels of TNF-a mRNA were present in I/R liver after 3 h of reperfusion. This result is not surprising because expression of

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expression after 3 h of reperfusion and then reduce the selfaggravating inflammatory process. The effect of T-res treatment on stress protein expression was also investigated. HSP-70 and HO-1 are induced by hepatic I/R and exhibit cytoprotective effects. HSP-70 is considered to be highly protective against oxidative stress [27], independently from hepatic recruitment of neutrophils [16]. HO-1 regulation seems to depend on ROS formation [28] and HO-1 is reported to exert an indirect anti-oxidant and anti-inflammatory effect, with a role in neutrophil sequestration [17,29]. In parallel, other authors have reported that T-res might exert a cytoprotective effect in cell lines via HO-1 or HSP-70 induction [30e32]. In the present study, induction of HSP70 and HO-1 mRNA was observed after 3 h of reperfusion. These results are in accordance with those of Yamaguchi et al. [33] and Ito et al. [34]. In our model, treatment with T-res significantly decreased the concentration of HO-1 transcripts. This decrease, observed after T-res administration just before reperfusion, could reflect a decrease in oxidative stress as shown in a previous study [6]. Conversely, HSP-70 mRNA expression was not modified by T-res treatment. The different response of the two HSPs observed in our study might be linked to the differential involvement of transcription factors: heat-shock factor-1 for HSP-70 [35] and NF-kB for HO-1 [31]. In conclusion, T-res treatment elicited a decrease in IL-1b, TNFa and KC mRNA expression. This could be due, at least in part, to a decrease in oxidative stress [6]. T-res reduces an excessive inflammatory response and neutrophil-induced cell damage in the liver during I/R. Moreover, T-res also plays a crucial role by attenuating the inflammatory process in hepatic I/R, as observed with IL1 receptor antagonists [36]. These results suggest that T-res might be useful in preventing hepatic inflammation secondary to liver surgery and/or the prevention of hepatic allograft rejection. T-res might therefore be a viable therapeutic option to selectively break the cycle of self-aggravating inflammatory injury. Acknowledgements We thank R. Djelidi for technical assistance in HPLC and S. Dumont (Pierre et Marie Curie Université, Paris 6) for technical assistance in histology. References [1]

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Fig. 3. Effect of post-ischemic treatment with trans-resveratrol (T-res) on hepatic expression of: (A) interleukin-1b mRNA; (B) tumour necrosis factor-a mRNA; and (C) keratinocyte chemoattractant mRNA. Rats were subjected to 1 h of ischemia and 3 h of reperfusion, and injected with vehicle (I/R), 0.02 (R 0.02) or 0.2 (R 0.2) mg/kg T-res, 5 min before reperfusion (n ¼ 11, 8 and 9, respectively). Sham group: rats were laparotomized, vehicle-treated after 1 h, and then sacrificed 3 h later (n ¼ 11). *p  0.05 vs. sham group; #p  0.05 vs. I/R group; ##p  0.005 vs. I/R group.

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TNF-a mRNA in this model is biphasic. Indeed, TNF-a mRNA can be detected after 60 min and 210 min but not at the intermediate time of 120 min [25]. Concerning IL-1b, mRNA levels after hepatic I/R have also been reported to be increased after 1 h of reperfusion [11], whereas levels of the KC transcript rose 1e2 h after IL-1 [26]. In our study, a significant increase in IL-1b, TNF-a and KC mRNA expression was observed in the I/R group. Post-ischemic T-res treatment decreased TNF-a, IL-1b and KC mRNA levels. These results show that T-res could diminish cytokine and chemokine mRNA

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