Ischemia/reperfusion (I/R) injury is triggered when the liver is transiently deprived of oxygen and subsequently reoxygenated. Injury to the liver caused by I/R can occur in the procedure of liver transplantation, liver resection, and trauma (1–3). Liver I/R injury is implicated as a potent contributor in increasing the rate of acute liver failure and chronic liver dysfunction after liver surgery, therefore, the reduction of liver I/R injury can contribute to minimizing postoperative injury to the liver.
The mechanisms of liver damage in I/R is associated with leukocyte infiltration and subsequent release of cytokines, free radicals, and proteolytic enzymes (4, 5). Several studies have demonstrated that blocking post-I/R leukocyte recruitment protects against liver I/R injury (6–8). The process of leukocyte recruitment across endothelial and extracellular matrix barriers involves complex cascades of tethering, rolling, adhesion to the endothelium, and transmigration from the vasculature. Leukocyte adhesion is mediated through interaction with adhesive molecules, such as L-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1. Vascular endothelial (VE) cadherin is one of the key molecules of mediating endothelial cell-cell contacts at intercellular junctions (9). Vascular endothelial cadherin has binding sites for a part of the amino terminus of the β-chain of fibrin. Binding VE cadherin to the β-chain of fibrin leads to transmigration of leukocytes across endothelial cell monolayers (10, 11).
The peptide Bβ15-42 is a 28–amino acid corresponding to the N-terminal sequence of the β-chain of fibrin. Because of lack of a leukocyte binding site, Bβ15-42 competes with fibrin for binding to VE cadherin, thereby inhibiting leukocyte transmigration across endothelial junctions and reducing the inflammatory response (11–13). Recently, results from Groger et al. (12) showed that Bβ15-42 prevents stress-induced RhoA activation, leading to endothelial stabilization and preservation of the endothelial barrier in shock.
It has been shown that Bβ15-42 peptide reduces myocardial inflammation and infarct size in rodent and pig models of myocardial I/R (13–15), and the results have been transferred successfully in a subsequent clinical phase II trial in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention (16). The protective effect of Bβ15-42 could be further confirmed in kidney I/R injury, hemorrhagic shock and reperfusion, lipopolysaccaride-induced shock, and sepsis animal models (12, 17–20).
Prompted by these results, we hypothesized that Bβ15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury, which is characterized as an inflammatory injury. To test this hypothesis, we investigated the effect of Bβ15-42 in a rat model of liver I/R.
MATERIALS AND METHODS
The experiments were designed to investigate whether Bβ15-42 treatment could protect from liver I/R injury. Rats were treated with Bβ15-42 (2.4 mg/kg) or control random peptide (2.4 mg/kg) at initiation of reperfusion, followed by a second injection 2 h after the onset of reperfusion. The dose of 2.4 mg/kg Bβ15-42 was based on dose titrations in rodent models of myocardial I/R injury (13, 15). Rats were killed at 0.5, 6, 24, and 48 h of reperfusion. Liver injury, inflammatory cytokines, neutrophil infiltration, hepatic high-mobility group box 1 (HMGB1) expression and release, and mitogen-activated protein kinase (MAPK) activation were analyzed.
Synthesis of peptides
Endotoxin-free Bβ15-42 (GHRPLDKKREEAPSLRPAPPPISGGGYR) control random peptide (DRGAPAHRPPRGPISGRSTPEKEKLLPG) were produced by solid-phase peptide synthesis and purified with reverse-phase HPLC as described previously (13).
Male inbred Lewis rats, purchased from Vital River Cooperation (Beijing, China), weighing within 270 to 320 g, were used in this study. All animals were housed under standard animal care conditions and had free to access to water and rat chow ad libitum. All procedures were carried out according to the ethical guidelines of the Animal Care and Use Committee of Huazhong University of Science and Technology.
Partial hepatic warm I/R
Partial hepatic warm I/R model was performed as described previously (21). In brief, after opening the abdomen and dissecting interlobular ligaments, a microvascular clamp was used to interrupt the arterial and portal venous blood supply to the left lateral and median liver lobes for 60 min.
Liver tissue was fixed in 4.5% buffered formalin for at least 24 h. Paraffin embedding was performed using standard techniques. Sections (4 μm) were stained with hematoxylin-eosin and assessed for inflammation and tissue damage.
Naphthol-AS-D-chloroacetate esterase staining
Neutrophil infiltration into liver tissue was evaluated by naphthol-AS-D-chloroacetate esterase (ASDCL) staining, as reported previously (22–24). Each sample was evaluated for ASDCL staining–positive neutrophils per high-power field (HPF) of five random sections with magnification of 200×. Naphthol-AS-D-chloroacetate esterase staining–positive neutrophils were counted manually.
Gel electrophoresis and Western blotting
Western blotting was performed as described previously (25). In brief, proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinyldifluoride membranes. Antibodies against rabbit anti-HMGB1 (1:1,000; Abcam, Cambridge, UK), rabbit anti–phosphor–extracellular signal–regulated kinase (ERK) (Thr202/Tyr204, 1:1,000; Cell Signaling Technology, Beverly, Mass), rabbit anti-ERK (1:1,000; Cell Signaling Technology), rabbit anti–phosphor–c-Jun N-terminal kinase (JNK) (Thr183/Tyr185, 1:1,000; Cell Signaling Technology), rabbit anti-JNK (1:1,000; Cell Signaling Technology), rabbit anti–phospho-p38 (Thr180/Tyr182, 1:1,000; Cell Signaling Technology), rabbit anti-p38 (1:1,000; Cell Signaling Technology), and rabbit anti–glyceraldehyde 3-phosphate dehydrogenase (1:20,000; Sigma-Aldrich, St. Louis, Mo) were used for Western blot analysis. The gray value of bands was calculated by ImageJ 1.43 G (National Institutes of Health, Bethesda, Md).
Enzyme-linked immunosorbent assay
Serum HMGB1 was determined with a commercial enzyme-linked immunosorbent assay ([ELISA] Shino-Test, Kanagawa, Japan) according to the manufacturer’s instruction. Serum tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels were analyzed using commercially available ELISA kits (R&D Systems, Minneapolis, Minn).
Quantitative polymerase chain reaction
Total RNA was isolated by TRIzol Reagent (Invitrogen, Carlsbad, Calif) according to the manufacturer’s instruction. Complementary DNA synthesis was performed using the First-Strand cDNA synthesis kit (Invitrogen). The levels of mRNA expression were determined by real-time polymerase chain reaction (PCR) using a Roch Light cycle system (Roche, Rotkreuz, Switzerland) with SYBR green master mix (Qiagen, Hilden, Germany) and primers (Table 1). Thermal cycling conditions consisted of a 10-min template denaturation step at 95°C, followed by 50 cycles of 95°C for 30 s, 50 °C for 30 s and 72 °C for 20 s. Normal liver tissue was used as reference sample to generate the standard curve. Relative quantification of target mRNA expression was calculated and further normalized to hypoxanthine guanine phosphoribosyltransferase.
Data were expressed as mean ± SD. Differences between groups were evaluated for significance by one-way analysis of variance combined with Bonferroni post hoc test. A value of P < 0.05 was considered to indicate statistical significance.
Fgα, Fgβ, Fgγ mRNA expression is increased in the liver after warm I/R injury
It has been reported that expression of Fg is increased in a rat model of kidney I/R (20). To determine whether liver warm I/R injury also could upregulate Fg expression, hepatic mRNA levels for Fgα, Fgβ, and Fgγ were measured by quantitative PCR. As shown in Figure 1, after 60 min of warm ischemia, hepatic Fgα, Fgβ, and Fgγ mRNA expression levels were significantly increased as early as 6 h, reached a peak at 24 h, and remained higher at 48 h after reperfusion, accounting for a 5-, 10-, 5-fold increase at 24 h of reperfusion, respectively.
Bβ15-42 treatment protects against liver I/R injury
Bβ15-42 has been demonstrated to protect against inflammation and injury after I/R in heart and kidney (13–15, 17, 20, 26). To determine whether Bβ15-42 treatment could attenuate liver I/R injury, a total dose of Bβ15-42 or control random peptide was administered to rats at initiation of reperfusion and 2 h thereafter. As shown in Figure 2A, liver warm I/R resulted in significant increases in serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). After 60 min of warm ischemia injury, serum ALT was significantly increased at 6 and 24 h of reperfusion (6 h, 890 ± 119 IU/L; 24 h, 329 ± 75 IU/L vs. normal control, 58 ± 7 IU/L; P < 0.001). In contrast, Bβ15-42 treatment significantly decreased ALT and AST levels when compared with random peptide control group. The serum levels of ALT were drastically decreased after reperfusion to 487 ± 85 IU/L and 201 ± 55 IU/L at 6 and 24 h, respectively. Similar results were obtained for AST.
As shown in Figure 2B, liver histology confirmed the serum aminotransferase estimation of liver injury. Hepatocellular necrosis and cytoplasmic vacuolization of hepatocytes were observed 6 h after reperfusion in the control group. In contrast, minimal damage was noted in Bβ15-42–treated rats.
Bβ15-42 treatment decreases the production of inflammatory mediators
Hepatic TNF-α, IL-6, IL-1β, and inducible nitric oxide synthase (iNOS) mRNA expression levels were measured by quantitative PCR. As shown in Figure 3, A to D, after 60 min of warm ischemia and 6 h of reperfusion, hepatic TNF-α, IL-6, IL-1β, and iNOS mRNA expression levels were significantly increased in both random peptide control group and Bβ15-42 treatment group. However, Bβ15-42–injected rats expressed significantly less hepatic TNF-α, IL-6, IL-1β, and iNOS mRNA compared with random peptide-treated rats. Furthermore, serum TNF-α and IL-6 levels in Bβ15-42–treated rats were significantly less than those in control random peptide–treated rats (Fig. 3, E and F).
Bβ15-42 decreases hepatic neutrophil infiltration
For quantification of the inflammatory response in livers subjected to 60 min of warm ischemia and 24 h of reperfusion, the number of ASDCL staining–positive neutrophils infiltrating the liver was determined. The number of ASDCL staining–positive neutrophils was increased after I/R injury. Neutrophil infiltration was significantly less in Bβ15-42–treated rats than in control random peptide–injected rats, indicating a profound abrogation of leukocyte infiltration into the liver (Fig. 4).
Bβ15-42 treatment decreases HMGB1 translocation and release
Warm liver I/R injury resulted in upregulation of HMGB1 expression and release in hepatic I/R (21, 27). As shown in Figure 5, I/R increased hepatic HMGB1 expression when compared with normal rats. Of note, Bβ15-42–treated rats exhibited a minimal increase in HMGB1 protein compared with random peptide–injected rats. To assess the extracellular release of HMGB1 after warm liver I/R injury, serum HMGB1 concentration was quantified using an ELISA. We previously demonstrated that serum levels of HMGB1 were highest at 0.5 h after reperfusion and rapidly decreased thereafter (21). Therefore, we determined the serum HMGB1 levels at 0.5 h after reperfusion. Serum HMGB1 levels were significantly increased in both groups when compared with normal rats. However, Bβ15-42–treated rats showed lower serum HMGB1 levels when compared with control random peptide–injected rats (10.7 ± 3.1 ng/mL vs. 5.9 ± 2.0 ng/mL; P < 0.05).
Bβ15-42 treatment modulates liver I/R–induced inflammatory signaling pathways
Among the most proximal events in I/R, the best known is the activation of mitogen-activated protein kinases (MAPKs) (28). To determine if Bβ15-42 treatment could influence MAPK activation, we assessed phosphorylation of ERK, JNK, and p38. After I/R, phosphorylation of ERK was increased at 0.5 h after reperfusion, and then decreased thereafter. However, phosphorylations of ERK levels were higher in Bβ15-42–treated rats than that in control random peptide–treated rats. In addition, liver I/R–induced JNK and p38 activation was decreased in Bβ15-42–treated rats. Treatment with Bβ15-42 did not affect total cellular levels of ERK, JNK, and p38. Thus, these data suggest that Bβ15-42 treatment could modulate liver I/R–induced inflammatory signaling pathways (Fig. 6).
Hepatic I/R related to leukocyte-dependent inflammatory response remains a significant complication in liver surgery. Recent studies suggest that Bβ15-42 has protective properties in I/R injury in heart and kidney (13–15, 17, 20, 26). However, its role in the pathogenesis of liver I/R injury has not been explored. In the present study, we documented that hepatic Fgα, Fgβ, and Fgγ mRNA expression levels were increased after liver I/R injury. Furthermore, we demonstrated that rats treated with Fgβ-derived Bβ15-42 peptide presented reduced liver enzymes, reduced hepatic inflammatory cytokine levels, and neutrophil infiltration. The protective mechanism of Bβ15-42 on hepatic I/R injury seems to involve its ability to reduce HMGB1 release and modulate liver I/R–induced MAPK activation.
Fibrinogen, a major blood plasma clotting protein, is made up of two sets of three different polypeptide chains (Fgα, Fgβ, and Fgγ). In addition to its role in blood coagulation, Fg has been recognized as an important regulator of tissue repair. Drew et al. (29) demonstrated that Fg seems to be important in tissue repair, and Fg-deficient mice showed a delayed wound closure and reduced tensile strength. Fibrinogen has also been shown to regulate the tissue repair process in neurons and lung epithelial cells (30). Furthermore, Krishnamoorthy et al. (20) found that Fg was increased in the kidney as a protective mechanism to facilitate cell proliferation and tissue repair in kidney I/R injury. Here, we showed that hepatic Fg was upregulated, suggesting that Fg may function as a protective mechanism after hepatic I/R injury.
Currently, there is only indirect evidence for the role of Fg-derived products in liver I/R injury. For example, Harada et al. (31) demonstrated that serum levels of fragment E of fibrin and Fg degradation products were increased after liver I/R injury in rats. In fact, recent studies have shown a pathogenic role for Fg-derived products in other organ systems. Fibrinogen-β–derived Bβ15-42 peptide presented reduced organ injury by inhibition of infiltrating leukocytes in I/R in heart and kidney (13–15, 17, 26). We showed that Bβ15-42 administration substantially reduced inflammation and injury in liver after I/R. These findings provide direct evidence that fibrin(ogen)-derived Bβ15-42 are involved in the pathogenesis of liver I/R injury.
The mechanism of protective action of Bβ15-42 involves its ability to inhibit leukocyte-endothelial interaction, which plays an important role in I/R injury. By binding to VE cadherin, Bβ15-42 blocks potential binding sites for E fragments, which is derived from the N-terminal segments of fibrin. Fragment E carries a binding site for VE cadherin and also CD11c and can function as a bridge molecule between endothelial cells and leukocytes (32). Bβ15-42 competes for the VE cadherin binding site, thereby preventing transmigration of leukocytes across endothelial cell monolayers (13). Furthermore, Groger et al. (12) demonstrated that the molecular key for the protective effect of Bβ15-42 is the src kinase Fyn, which associates with VE cadherin–containing junctions. Bβ15-42 binding to VE cadherin leads to the dissociation of Fyn from VE cadherin. Fyn association with p190RhoGAP results in the inactivation of RhoA, causing endothelial stabilization and preservation of the endothelial barrier in shock (12). More recently, Krishnamoorthy et al. (20) documented that Bβ15-42 administration protected mice from I/R-induced kidney injury by aiding in epithelial cell proliferation and tissue repair. It should be noted that the peptide Bβ15-42 is a natural degradation product occurring after fibrin inactivation. Under normal physiological conditions, Bβ15-42 does not inhibit fibrin fragment interactions with VE cadherin because of the low affinity. When Bβ15-42 was given at a supraphysiological dose, it protected against I/R injury in the heart and kidney (13–15, 20). Consistent with these observations, we found that pharmacologic blocking of interaction by Bβ15-42 reduced leukocyte infiltration and liver I/R injury. To study the effects of Bβ15-42 on proinflammatory cytokine gene expression, rats were preconditioned with Bβ15-42 after reperfusion. We demonstrated that after 60 min of warm ischemia, Bβ15-42 treatment reduced TNF-α, IL-6, IL-1β, and iNOS gene expression after reperfusion. These cytokines are known to play a pivotal role in the pathophysiology of hepatic I/R injury.
Because recent studies suggest that extracellular HMGB1, released from damaged liver cells or secreted by immune cells, causes a proinflammatory intrahepatic microenvironment in the postischemic liver (21, 27), we investigated HMGB1 expression and release. High-mobility group box 1 was initially defined as a nuclear protein that loosely binds to chromatin and plays a pivotal role for bending DNA and regulating transcription (33–35). It has become apparent in recent years that HMGB1 mediates a response to inflammation and tissue damage (35, 36). High-mobility group box 1 in the normal rat liver is mainly present in the nucleus of hepatocytes, and it is translocated from nucleus to cytoplasm and release into circulation in liver I/R injury (21, 25). Furthermore, blocking HMGB1 using a neutralizing antibody to HMGB1 protected against liver I/R injury (27). Our results showed that Bβ15-42 treatment led to a lower hepatic HMGB1 expression and serum levels, suggesting that the mechanism of protective action of Bβ15-42 seems to involve its ability to reduce I/R-induced HMGB1 expression and release.
Because Bβ15-42 treatment led to lower serum HMGB1 levels, which could modulate liver I/R–induced MAPK activation (27), we next examined MAPK activation. Activation of MAPK contributes to the inflammatory response after hepatic I/R (28). Activation of ERK occurred early in ischemic livers, and ERK activation after redox stress is protective in hepatocytes (37). Our results showed that Bβ15-42 pretreatment partially increased I/R-induced ERK1/2 activation when compared with control random peptide–injected rats. Furthermore, we demonstrated that Bβ15-42 decreased I/R-induced JNK and p38 activation. Conversely, JNK and p38 MAPK are phosphorylated and activated after reperfusion, and their activation is associated with the induction of both necrosis and apoptosis in liver I/R. Inhibition of JNK or p38 MAPK activation could decrease hepatic I/R injury in the liver (38, 39). Taken together, these data suggest that modulating MAPK activation is also involved in the hepatic protective effects of Bβ15-42. It remains unclear whether Bβ15-42 could directly alter MAPK activation or whether this is secondary to the Bβ15-42–mediated less inflammatory mediator release, such as HMGB1, which could modulate MAPK activation in liver I/R injury.
In summary, we documented that Bβ15-42 treatment could ameliorate liver I/R injury. The mechanism of protective action of Bβ15-42 seemed to involve its ability to reduce the hepatic inflammatory response by preventing HMGB1 release and altering MAPK activation. Therefore, the protective effect of Bβ15-42 on hepatic warm I/R injury might extend its potential clinical applications in liver surgery.
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