Hepatic ischemia-reperfusion (I/R) injury

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1 Original Article / Liver Urinary trypsin inhibitor attenuates hepatic ischemia-reperfusion injury by reducing nuclear factor-kappa B activation Yi-Jun Wu, Qi Ling, Xin-Hui Zhou, Yan Wang, Hai-Yang Xie, Ji-Ren Yu and Shu-Sen Zheng Hangzhou, China BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemiareperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-κB) and its related target genes and products such as vascular endothelial cell adhesion molecule and CXC chemokines. We aimed to assess the roles of those mediators in a UTI-treated mouse model of hepatic I/R injury. METHODS: Treatment group 1 (UTI given 5 minutes prior to liver ischemia), treatment group 2 (UTI given 5 minutes after the anhepatic phase) and a control group were investigated. Blood and liver samples were obtained and compared at 1, 3, 6 and 24 hours after reperfusion. RESULTS: Attenuation of pathological hepatocellular damage was greater in the treatment groups than in the control group (P<0.05). Compared with the control group, the UTI treatment groups showed significantly lower serum alanine aminotransferase and aspartate aminotransferase levels, decreased myeloperoxidase activity, and reduced NFκB activation. Also downregulated was the expression of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2 at the mrna level. P-selectin protein and intercellular adhesion molecule-1 protein expression were also downregulated. In addition, the treatment group 1 showed a better protective effect against I/R injury than the treatment group 2. Author Affiliations: Department of General Surgery (Wu YJ, Ling Q, Wang Y, Xie HY, Yu JR and Zheng SS) and Department of Gynecology (Zhou XH), First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou , China Corresponding Author: Shu-Sen Zheng, MD, PhD, FACS, Department of General Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou , China (Tel: ; zyzss@ zju.edu.cn) 2009, Hepatobiliary Pancreat Dis Int. All rights reserved. CONCLUSIONS: UTI reduces NF-κB activation and downregulates the expression of its related mediators, followed by the inhibition of neutrophil aggregation and infiltration in hepatic I/R injury. The protective role of UTI is more effective in prevention than in treatment. (Hepatobiliary Pancreat Dis Int 2009; 8: 53-58) KEY WORDS: ischemia-reperfusion injury; nuclear factor-kappa B; tumor necrosis factor-alpha; urinary trypsin inhibitor Introduction Hepatic ischemia-reperfusion (I/R) injury is a consequence of hepatic sugar, liver transplantation, or hemorrhagic shock. It can lead to liver failure, systemic inflammatory syndrome, and even multiple organ failure, which are associated with high mortality. Thus it is essential to clarify the molecular mechanisms of hepatic I/R injury and diminish the related damage. Increasing evidence suggests that the acute inflammatory response plays a key role in I/R injury, and nuclear factor-kappa B (NF-κB) is a central transcriptional factor in the regulation of inflammatory factors. [1] The primary form of NF-κB consists of a heterodimer of p50 and p65, which are localized in the cytoplasm and bind to inhibitory proteins of the IκB family. In response to inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IκB is phosphorylated and degraded, which unmasks the nuclear localization sequence of NF-κB subunits, allowing translocation to the nucleus where NF-κB binds to DNA and induces target gene transcription. [2] The activation of NF-κB can up-regulate vascular endothelial cell adhesion molecules such as selectins, β2-integrins and intercellular adhesion molecule (ICAM)-1 and increase the production of neutrophil chemoattractants, including CXC chemokines. [3, 4] The Hepatobiliary Pancreat Dis Int,Vol 8,No 1 February 15,

2 Hepatobiliary & Pancreatic Diseases International coordinated effect of cytokines, vascular endothelial cell adhesion molecules and chemokines results in the adhesion and migration of neutrophils, ultimately leading to liver injury. Urinary trypsin inhibitor (UTI), a protease inhibitor purified from human urine, inhibits the functions of various enzymes simultaneously, including trypsin, alpha-chymotrypsin, alidase, elastase and fibrinolysin. It has been found that UTI attenuates the inflammatory response and is protective against I/R injury. [5-7] However, the molecular mechanism of the protective effect of UTI has not yet been clearly elucidated in hepatic I/R injury. The purposes of this study were to assess the effect of UTI on hepatic I/R injury, and evaluate the role of NF-κB and its related genes and products (TNF-α, cytokine-induced neutrophil chemoattractant [KC] and macrophage inflammatory protein-2 [MIP-2], ICAM-1, P-selectin and E-selectin) in UTI-treated hepatic I/R injury. Methods Animals Balb/c (H-2d) mice, aged from 8 to 12 weeks, weighing g, were purchased from the Central Animal Facility, Zhejiang University School of Medicine, Hangzhou, China. All animals were kept under specific pathogen-free conditions with controlled light/dark cycles and free access to water and standard chow, and were fasted for 24 hours before surgical procedures. The investigations were conducted in compliance with the policies of the animal care committee of the local government. Surgical procedures The animals were operated on under intraperitoneal anaesthesia with 2% ketamine hydrochloride (0.2 ml) in a clean environment. After opening the abdominal cavity, we dissociated the infrahepatic vena cava, hepatic artery, pyloric vein and portal vein, and clamped the former three. The portal vein was cannulated with a 30G puncture pin and the liver was perfused through the portal vein with 4 saline (1 ml). After 30 seconds, a microvascular clamp was used to occlude the portal vein at the porta hepatis. Ringer's frazil was used to cover the liver surface after 20 minutes anhepatic phase, then we declamped the vena cava, liver artery and pyloric vein. In the experimental group, we collected blood samples via the vena cava at 1, 3, 6 and 24 hours after reperfusion, and animals were killed to acquire the liver tissue. Experimental groups All animals were divided into three groups as [5, 7] follows: treatment group 1 (n=24, U/kg UTI administered via the tail vein, 5 minutes prior to liver ischemia), treatment group 2 (n=24, U/kg UTI administered via the tail vein, 5 minutes after the anhepatic phase) and control group (n=24). Blood and liver samples were obtained from 6 mice in each group at 1, 3, 6 and 24 hours after reperfusion. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by an automatic analyzer. Histochemistry The pathological changes of liver tissues were assessed by light microscopy. Samples were fixed with 10% formalin, embedded in paraffin, cut into 6 mm sections, and stained with hematoxylin-eosin. All microscopic slides were analyzed by a pathologist blinded to the group assignments of the animals. The expression of P-selectin, E-selectin, ICAM-1 and IκB-α in the liver tissue were detected by immunohistochemistry with the streptavidinperoxidase method. The sections were then ranked for staining intensity (-, +, ++, +++), a higher rank indicating stronger expression. Myeloperoxidase (MPO) activity test MPO activity was assayed by spectrophotometrically measuring the H 2 O 2 -dependent oxidation of 3, 3', 5, 5'-tetramethylbenzidine at 650 nm. All reagents in the assay were provided in a MPO kit (Jiancheng Biotechnology, Nanjing, China). NF-κB assay Nuclear extracts were prepared using a nuclear extract kit (Active Motif) and the binding activity of NF-κB/p65 with DNA in nuclei was detected by a TransAM TM NF-κB/p65 Transcription Factor assay kit (Active Motif) according to the manufacturer's protocols. RNA extraction and reverse transcriptionpolymerase chain reaction (RT-PCR) RNA was extracted by Trizol kit from each mg sample of snap-frozen liver tissue. The concentration and A260/A280 (>1.8) was determined by ultraviolet photospectrometry. Total RNA (4 μg) 54 Hepatobiliary Pancreat Dis Int,Vol 8,No 1 February 15,2009

3 UTI attenuates hepatic I/R injury by reducing NF-κB activation was reverse-transcribed to c-dna and expanded by PCR. The primers of TNF-α, MIP-2, KC and β-actin were as follows: TNF-α sense 5'-AGC CCA CGT AGC AAA CCA CCA A-3', anti-sense 5'-ACA CCC ATT CCC TTC ACA GAG CAA T-3'; MIP-2 sense 5'-GAA CAA AGG CAA GGC TAA CTG A-3', anti-sense 5'-AAC ATA ACA ACA TCT GGG CAA T-3'; KC sense 5'-CGG AAT TCG CCA CCA GCC GCC TG-3', anti-sense 5'-ACC ATT CCT GTT CAA AAG CAG-3'; β-actin sense 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3', anti-sense 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'. Statistical analysis Quantitative variables were expressed as mean± SD. Categorical variables were presented as values or percentages. One-way ANOVA was used to compare quantitative variables. The Chi-square test was used to compare categorical variables. SPSS for Windows version 11.0 (SPSS Inc., Chicago, IL) was used to ALT (U/L) AST (U/L) Control group Treatment group 1 Treatment group Time (h) Control group Treatment group 1 Treatment group Time (h) Fig. 1. Dynamic changes of ALT and AST after reperfusion. complete all the analyses, and a P value of less than 0.05 was considered statistically significant. Results Dynamic change of serum ALT and AST levels The serum levels of ALT and AST were greatly elevated after reperfusion in all three groups, and peaked at the 6-hour point. Compared with the control group, both treatment groups 1 and 2 showed markedly lower ALT and AST levels at 6 and 24 hours after reperfusion (P< 0.05). The levels of ALT at 6 and 24 hours, and AST at 6 hours after reperfusion in the treatment group 1 were lower than those in the treatment group 2 (P<0.05) (Fig. 1). Pathological changes Pathological changes were more remarkable, and hepatocellular injury (hepatocyte vacuolization, swelling of the hepatocytes, discontinuity of the endothelial lining bordering the sinusoids and disrupted hepatic trabecula) was more severe in the control group than in the treatment groups (Fig. 2). MPO activity (U/g) Control group Treatment group 1 Treatment group Time (h) Fig. 3. Comparison of MPO activity in the control group, treatment group 1, and treatment group 2. Fig. 2. Effects of ulinastatin on mouse liver I/R injury (6 hours after reperfusion) (light microscope, HE stain, original magnification 400). A: control group; B: treatment group 1; C: treatment group 2. Hepatobiliary Pancreat Dis Int,Vol 8,No 1 February 15,

Hepatobiliary & Pancreatic Diseases International Group Table. NF-κB levels in experimental groups Time after reperfusion 1 hour 3 hours 6 hours 24 hours Control 0.039±0.013 0.056±0.030 0.122±0.040 0.

4 Hepatobiliary & Pancreatic Diseases International Group Table. NF-κB levels in experimental groups Time after reperfusion 1 hour 3 hours 6 hours 24 hours Control 0.039± ± ± ±0.040 Treatment group 1 Treatment group ± ± ± ± ± ± ± ±0.036 Fig. 4. Expression of TNF-α mrna at 3 hours after reperfusion (A), and KC mrna (B) and MIP-2 mrna (C) at 6 hours after reperfusion in the three groups. Fig. 5. Expression of P-selectin (at 6 hours after reperfusion) in the control group (A) and treatment group 1 (B); ICAM-1 (at 24 hours after reperfusion) in the control group (C) and treatment group 1 (D); and IκB-α at 3 hours after reperfusion (E) and at 6 hours after reperfusion in the control group (F). MPO activity The MPO activity of liver tissue in the different groups and time-points are shown in Fig. 3. The MPO levels at 1, 6 and 24 hours in the treatment group 1 and at 6 hours in the treatment group 2 were lower than those in the control group (0.0961±0.134 U/g vs ±0.349 U/g, ±0.261 U/g vs ±0.400 U/g, 0.116±0.109 U/g vs ±0.222 U/g, and 0.399± U/g vs ±0.400 U/g, P<0.05, respectively). NF-κB activity The NF-κB level, which increased after reperfusion and peaked at 6 hours, was lower in the treatment group 1 than in the control group at 3 and 6 hours after reperfusion, and also lower than that in the treatment group 2 at 3 hours after reperfusion (Table). Expression of TNF-α, KC and MIP-2 mrna In the control group, expression of TNF-α, KC and MIP-2 mrna increased after reperfusion and reached a maximum at 3, 6, and 6 hours, respectively. The treatment group 1 showed lower expression of TNF-α, KC and MIP-2 mrna than the control group (P<0.05). There was no statistically significant difference in TNF-α, KC and MIP-2 mrna between the treatment group 2 and the control group or between the treatment groups 1 and 2 (Fig. 4). Expression of P-selectin, E-selectin, ICAM-1 and IκB-α P-selectin, E-selectin and ICAM-1 were mainly expressed on endothelial cells of the sinusoids and hepatic vasculature. Their expression increased after reperfusion and peaked at 6, 6 and 24 hours, respectively. Expression of P-selectin at 6 hours and ICAM-1 at 24 hours was higher in the control group than in the treatment group 1 (P<0.05). E-selectin was expressed in all groups with no statistical difference. Cytoplasmic IκB-α level decreased after reperfusion in the control group. Higher levels of IκB-α protein were found in the treatment groups than in the control group. However, their difference was not significant in all groups (Fig. 5). Discussion Serum ALT and AST levels are well considered to be 56 Hepatobiliary Pancreat Dis Int,Vol 8,No 1 February 15,2009

5 UTI attenuates hepatic I/R injury by reducing NF-κB activation indicative of hepatocellular damage. This study showed that hepatic I/R caused notable hepatocellular damage, which was maximized at 6 hours after reperfusion. The degree of liver injury was remarkably reduced by UTI since the treated mice showed significantly lower levels of ALT and AST than untreated mice. The histopathological findings also revealed an apparent difference between UTI-treated animals and untreated ones. As the intracellular content of MPO is constant in neutrophils, their accumulation was evaluated by measurement of the MPO activity. We found that the MPO activity rapidly increased after reperfusion in paralle with the increase of ALT and AST levels. This elevation was also markedly inhibited by UTI treatment, indicating that UTI protects against I/R injury by inhibiting neutrophil accumulation. To determine the molecular mechanism of the protective effect of UTI in hepatic I/R injury, we assessed the level of I-κB protein, the activation of NF-κB, the expression of TNF-α and KC and MIP-2 mrna, and the levels of ICAM-1 and P-selectin and E-selectin proteins. Our results demonstrated that hepatic I/R caused significant elevation of TNF-α, which peaked at 3 hours after reperfusion. The increase of this pro-inflammatory cytokine was followed by the activation of NF-κB, which also increased in a time-dependent manner and peaked at 6 hours after reperfusion. The levels of IκB-α protein decreased simultaneously, indicating the important role of IκB-α [2, 8] in NF-κB activation. During hepatic I/R injury, a pattern similar to NF-κB was found in its target genes and protein products. The expression of MIP-2 and KC mrna in liver tissue increased after reperfusion and reached a maximum at 6 hours, as similar as the expression of E-selectin and P-selectin proteins. ICAM-1 protein in endothelial cells of the sinusoids was consistently elevated during the first 24 hours after reperfusion. It was reported that P-selectin and ICAM-1 mediate neutrophil interactions, neutrophil migration, and the infiltration of neutrophils into the ischemic liver. [9, 10] The CXC chemokines, KC and MIP-2, play a major role in the recruitment of neutrophils in a wide variety of inflammatory conditions including [11, 12] I/R injury. The changes of cytokines, vascular endothelial cell adhesion molecules and chemokines lead to the adhesion and migration of neutrophils in liver tissue, which was also shown by the rapidly increased activation of MPO at the same time-point. The TNF-α level in response to NF-κB activation was compared in the different groups in this study, the UTI-treated mice showed significantly lower TNF-α than untreated ones. The administration of UTI significantly down regulated NF-κB activation, followed by decreases of KC, MIP-2, P-selectin and ICAM-1. These data are consistent with serum ALT and AST levels and the MPO activity described above. The levels of IκB-α protein were higher in UTI-treated than untreated mice at any time point after reperfusion. However, the indistinct difference indicated that downregulation of NF-κB activation by UTI may have other [13, 14] pathways. UTI also inhibited the expression of vascular endothelial cell adhesion molecules and CXC chemokines. This phenomenon is consistent with other findings that UTI ameliorates I/R injury by decreasing the accumulation and adhesion of neutrophils. [5-7] As is well known, hepatic I/R injury occurs in two phases: the acute injury phase occurring within 1-6 hours after reperfusion, and the subacute-phase that peaks at 9-24 hours after reperfusion. [15] In contrast to the relatively long period of I/R injury, the serum half-life of UTI is short (35 minutes). When is the suitable time for administration of UTI to protect the liver, before ischemia or after reperfusion? In this study, pretreatment with UTI was better than 'later' treatment with UTI in hepatic I/R injury, suggesting that it would be better to administer UTI before liver ischemia. In summary, hepatic I/R injury triggers a cascade of responses, including the activation of NF-κB, the production of inflammatory cytokine TNF-α and the induction of adhesion molecule expression (P-selectin and ICAM-1) and neutrophil chemoattractant chemokine expression (KC and MIP-2). UTI protects hepatic cells against I/R injury by reducing the NF-κB activation, subsequently inhibiting the expression of TNF-α and vascular endothelial cell adhesion molecules and CXC chemokines. The protective role of UTI is more effective in prevention than in treatment. Funding: None. Ethical approval: Not needed. Contributors: WYJ proposed the study and wrote the first draft. LQ analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZSS is the guarantor. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. References 1 Fan H, Sun B, Gu Q, Lafond-Walker A, Cao S, Becker LC. Oxygen radicals trigger activation of NF-kappaB and AP-1 and upregulation of ICAM-1 in reperfused canine heart. Am J Physiol Heart Circ Physiol 2002;282:H Senftleben U, Karin M. The IKK/NF-kappa B pathway. Crit Care Med 2002;30:S Hepatobiliary Pancreat Dis Int,Vol 8,No 1 February 15,

6 Hepatobiliary & Pancreatic Diseases International 3 Jaeschke H, Hasegawa T. Role of neutrophils in acute inflammatory liver injury. Liver Int 2006;26: Lalor PF, Sun PJ, Weston CJ, Martin-Santos A, Wakelam MJ, Adams DH. Activation of vascular adhesion protein-1 on liver endothelium results in an NF-kappaB-dependent increase in lymphocyte adhesion. Hepatology 2007;45: Okuhama Y, Shiraishi M, Miyaguni T, Higa T, Tomori H, Hiroyasu S, et al. Evidence of neutrophil involvement in the protective effects of urinary trypsin inhibitor against ischemia reperfusion in vitro. Transplant Proc 1998;30: Ren B, Wu H, Zhu J, Li D, Shen Y, Ying R, et al. Ulinastatin attenuates lung ischemia-reperfusion injury in rats by inhibiting tumor necrosis factor alpha. Transplant Proc 2006;38: Zhang XQ, Sun JL, Chen YJ, Ma R, Fan XH, Sun JZ. Amelioration of ischemia-reperfusion injury of transplanted small intestine by ulinastatin: effects on accumulation and adhesion of neutrophil. Transplant Proc 2005;37: Sun Z, Andersson R. NF-kappaB activation and inhibition: a review. Shock 2002;18: Young CS, Palma JM, Mosher BD, Harkema J, Naylor DF, Dean RE, et al. Hepatic ischemia/reperfusion injury in P-selectin and intercellular adhesion molecule-1 doublemutant mice. Am Surg 2001;67: Roebuck KA. Oxidant stress regulation of IL-8 and ICAM-1 gene expression: differential activation and binding of the transcription factors AP-1 and NF-kappaB. Int J Mol Med 1999;4: Lukacs NW, Hogaboam C, Campbell E, Kunkel SL. Chemokines: function, regulation and alteration of inflammatory responses. Chem Immunol 1999;72: Mosher B, Dean R, Harkema J, Remick D, Palma J, Crockett E. Inhibition of Kupffer cells reduced CXC chemokine production and liver injury. J Surg Res 2001;99: Joseph JD, Yeh ES, Swenson KI, Means AR, Winkler. The peptidyl-prolyl isomerase Pin1. Prog Cell Cycle Res 2003;5: Okaya T, Lentsch AB. Hepatic expression of S32A/S36A IkappaBalpha does not reduce postischemic liver injury. J Surg Res 2005;124: Colletti LM, Kunkel SL, Walz A, Burdick MD, Kunkel RG, Wilke CA, et al. The role of cytokine networks in the local liver injury following hepatic ischemia/reperfusion in the rat. Hepatology 1996;23: Received June 24, 2008 Accepted after revision September 26, Hepatobiliary Pancreat Dis Int,Vol 8,No 1 February 15,2009

CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'

CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3' Table S1. The primer sets used for real-time RT-PCR analysis. Gene Forward Reverse VEGF PDGFB TGF-β MCP-1 5'-GTT GCA GCA TGA ATC TGA GG-3' 5'-GGA GAC TCT TCG AGG AGC ACT T-3' 5'-GAA TCA GGC ATC GAG AGA