Hepatic ischemia-reperfusion (I/R) injury is encountered during hepatic transplantation, trauma, hypovolemic shock, and elective liver resection and is the result of both ischemia and inflammation induced by I/R insult (1). Hepatic I/R injury leads to the release of multiple proinflammatory mediators, such as high-mobility group box 1 protein (HMGB1), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α), through the activation of both immune cells and parenchymal cells in a Toll-like receptor 4 (TLR4)–dependent manner (2).
High-mobility group box 1 protein is a DNA-binding protein participating in chromatin structure and transcriptional regulation. It can also exhibit proinflammatory cytokine-like activity when actively secreted by monocytes and macrophages as well as numerous other cell types, including hepatocytes (3, 4). Damaged and necrotic nonimmune cells, but not apoptotic cells, passively release HMGB1 (5). In hepatic I/R, hepatocytes have been shown to release HMGB1 in a TLR4-dependent manner (4). High-mobility group box 1 protein contributes to the activation of TLR4 in neighboring macrophages (6). High-mobility group box 1 protein exerts its effects through a number of receptors, such as receptor for advanced glycation end products (RAGE), TLR2, and TLR4 (7). The status of cysteine 106 in the B-box of HMGB1 is critical to defining the activity of HMGB1; when cysteine 106 is in the thiol configuration, HMGB1 can trigger TLR4 (8). In macrophages exposed to lipopolysaccharides (LPSs), the release of HMGB1 is an active process involving hyperacetylation, phosphorylation, translocation from the nucleus to secretory lysosomes, and eventually the extracellular release of the molecule (9). The number of inflammatory diseases associated with HMGB1 has expanded dramatically since the original description of the role of HMGB1 in sepsis (10) and includes hemorrhagic shock, lupus, rheumatoid arthritis, and heat stroke as well as trauma (10–14).
Recently, Tracey (15) discovered a novel anti-inflammatory mechanism mediated through vagal efferent activity with acetylcholine (ACh) acting at the α7 nicotinic ACh receptor (α7nAChR). The pathway, termed the cholinergic anti-inflammatory pathway (CAP), has been shown to affect sepsis, trauma, I/R injury, and pancreatitis (16). The α7nAChR agonist, PNU-282987, prevented end-organ damage and suppressed tissue levels of proinflammatory cytokines and activation of nuclear factor κB (NF-κB) in hypertension (17). Recent experiments demonstrated that α7nAChR agonists significantly attenuate the inflammatory response, preventing the activation of the NF-κB pathway and inhibiting HMGB1 secretion (18, 19). Because hepatic I/R injury is known to involve HMGB1-induced NF-κB activation (6), we tested the protective effects of an α7nAChR agonist in a murine liver I/R model.
MATERIALS AND METHODS
All procedures were approved by the Institutional Animal Care and Use Committee of Tongji University. Male pathogen-free C57BL/6 mice (8–10 weeks old) were obtained from the Laboratory Animal Research Center of Shanghai. Animals were housed in cages in an air-conditioned room (20°C ± 1°C) with controlled 12-h light/dark cycles and had ad libitum access to water and rat chow (Global Diet; Shanghai, China). All animals received human care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.
We used a mouse model of 70% partial hepatic ischemia for 60 min as previously reported (20). Forty-five C57BL/6 mice were randomly assigned into 3 groups: sham group, vehicle plus I/R group, and PNU-282987 plus I/R group. Described briefly, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg; Sigma, St. Louis, Mo). An atraumatic clip was used to interrupt the arterial and portal venous blood supply to the left and middle liver lobes. After 60 min of partial warm ischemia, the clamp was removed, initiating hepatic reperfusion. This method of partial hepatic ischemia prevents mesenteric venous congestion by permitting portal decompression through the right and caudate lobes. The belly was covered with analexipharmic plastic wrap to minimize evaporative loss. During continuous ischemia, evidence of ischemia was confirmed by visualizing the pale blanching of the ischemic lobes. The clamp was removed, gross evidence of reperfusion was based on immediate color change, and the abdomen was closed with continuous 4-0 polypropylene suture. Each mouse’s temperature was maintained at 37°C by means of a warming pad and heat lamp. Mice were pretreated 30 to 40 min before surgery with α7nAChR agonist PNU-282987 (1 mg/kg; Sigma) or with an equal volume of the vehicle (0.9% NaCl solution). Blood and hepatic tissue samples were collected at 3, 6, and 12 h after reperfusion. At the end of the observation period following reperfusion, the mice were anesthetized with sodium pentobarbital and killed by exsanguinations at each sampling time point.
Inflammatory cytokines, such as HMGB1, TNF-α, and IL-1β, were shown to play key roles in the pathophysiology of hepatic I/R injury. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR), we measured steady-state mRNA levels for these cytokines in the liver after I/R. Hepatic tissue samples were subjected to hematoxylin-eosin (H&E) staining and immunohistochemical staining for HMGB1. Otherwise, HMGB1 and NF-κB activity in hepatic tissue was assessed by Western blot.
Determination of blood biochemistry
Blood samples were centrifuged for 10 min at 3,000 revolutions/min (rpm) at 4°C, divided into aliquots, and stored at −80°C until assayed. The blood serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined in the Shanghai Tenth People’s Hospital clinical laboratory.
For histological analysis, hepatic tissue samples were fixed in 4% paraformaldehyde phosphate-buffered saline overnight at 4°C. The samples were dehydrated, embedded in paraffin, and cut into 5-μm sections. After deparaffinization, the tissues were stained with H&E for histological study.
Isolation of nuclear and cytoplasmic proteins
Hepatic tissue was diced into very small pieces using a clean razor blade. The pieces were suspended in a buffer that contained 10 mM Tris, pH 7.5, 1.5 mMMgCl2, 10 mM KCl, and 0.1% Triton X-100 and lysed by homogenization. Homogenates were incubated on ice (15 min) with gentle agitation. Nuclei were collected by low-speed centrifugation at 7,500 rpm for 5 min. The supernatant that contained cytoplasmic and membrane protein was collected and stored at −80°C for Western blot analysis. Nuclear proteins were extracted at 4°C by mildly resuspending the nuclei pellet in buffer that contained 20 mM Tris, pH 7.5, 20% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 0.1% Triton X-100, followed by 1-h incubation at 4°C with occasional vortexing. After centrifugation at 14,000 rpm for 15 min at 4°C, the supernatant that contained nuclear protein was collected. Protein concentration was quantitated with bicinchoninic acid protein assay reagent (Pierce Chemical Co, Rockford, Ill).
Western blotting analysis
Western blot analyses for HMGB1 and NF-κB were performed as described (21). The cytoplasmic protein (30 μg/sample) and nuclear protein (25 μg/sample) were boiled in protein loading buffer for 5 min and separated on 12% sodium dodecyl sulfate–polyacrylamide electrophoresis gel. After electrophoresis, separated proteins were transferred onto a polyvinylidene fluoride membrane and blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature. Then, the membrane was incubated overnight at 4°C with primary polyclonal rabbit antibody to HMGB1 (1:1,000; Abcam, Cambridge, UK), primary monoclonal rabbit antibody to p65 (1:1,000; CST, USA), primary monoclonal rabbit antibody to β-actin (1:1,000∼2,000; Abcam), and primary polyclonal rabbit antibody to proliferating cell nuclear antigen (1:500–1,000; Bioworld, St. Louis, Mo), respectively. The membranes were washed in TBST 3 times, incubated with chemiluminescence-labeled secondary antibody, and determined by using an Odyssey (LI-COR Biosciences, Lincoln, Neb) image analysis system.
Immunohistochemistry for HMGB1
To determine changes in the HMGB1 expression in nuclei in a semiquantitative manner, immunohistochemical staining for HMGB1 was performed using a rabbit polyclonal antibody. The fresh liver tissue specimens were fixed in 4% paraformaldehyde phosphate-buffered saline, dehydrated, and embedded in paraffin. The samples were cut into 5-μm sections using a machine (Leica, Wetzlar, Germany) and placed onto slides. After blocking the endogenous peroxidase activity, sections were preincubated with 5% normal fetal bovine serum for 20 min. The sections were incubated with a 1:1,000 dilution of the primary antibody HMGB1 overnight at 4°C. Then, the sections were incubated with EnVision + reagent (EnVision + System; DAKO, Carpinteria, Calif) for 30 min. 3,3-Diaminobenzidine was used as the substrate, and the sections were counterstained with Mayer’s hematoxylin and mounted.
SYBR green real-time RT-PCR
Total RNA was extracted from the liver using the TRIzol reagent (Takara, Japan TaKaRa BIO INC., Shiga, Japan) according to the manufacturer’s instructions. The mRNA for TNF-α, IL-1β, HMGB1, and 18s was quantified in duplicate by SYBR green two-step, real-time RT-PCR. One microgram of total RNA from each sample was used for RT with an oligo-dT (Takara) and a Superscript II (Takara) to generate first-strand cDNA. Polymerase chain reaction mixture was prepared using SYBR Green PCR Master Mix (Takara, Applied Biosystems, Japan) using the primers. The expression of each gene was normalized with 18s mRNA content. The following primers were used:
18SrRNA forward: 5′-CCTGGATACCGCAGCTAGGA-3′
18SrRNA reverse: 5′-GCGGCGCAATACGAATGCCCC-3′ (expected product 112 base pairs [bp])
HMGB1 forward: 5′- TGGGCAAAGGAGATCCTAAA-3′
HMGB1 reverse: 5′- GCAGACATGGTCTTCCACCT-3′ (expected product 160 bp)
TNF-α forward: 5′-TAGCAAACCACCAAGTG-3′
TNF-α reverse: 5′-ACAAGGTACAACCCATCG-3′ (expected product 124 bp)
IL-1β forward: 5′-GCTGAAAGCTCTCCACCTCAA-3′
IL-1β reverse: 5′-TCGTTGCTTGGTTCTCCTTGTA-3′ (expected product 87 bp)
SPSS software was used to analyze data, which are presented as mean (SD). Comparisons between groups were performed using one-way analysis of variance and a post hoc Tukey test. A significant difference was presumed at a probability value less than 0.05.
PNU-282987 pretreatment prevents I/R-induced liver injury
To evaluate the effects of PNU-282987 on hepatic I/R injury, serum AST and ALT levels were measured. As shown in Figure 1, serum AST and ALT levels were significantly increased after hepatic I/R injury compared with the sham group at 3, 6, and 12 h after reperfusion (P < 0.05). In contrast to hepatic I/R injury alone, pretreatment with PNU-282987 significantly prevented increases in serum levels of AST or ALT at all 3 time points. (P < 0.05).
The effects of PNU-282987 on liver damage were confirmed by assessing histological changes
Representative histological H&E slides from hepatic tissues from vehicle-pretreated or PNU-282987–pretreated mice subjected to I/R are shown in Figure 2. Ischemia-reperfusion in vehicle-pretreated mice produced large necrotic areas after reperfusion, which increased over time. Additional morphological manifestations of hepatocyte necrosis, such as increased swelling, vacuolization, and blebbing, as well as polymorphonuclear cell infiltration, were seen in the livers of the I/R group. Pretreatment with PNU-282987 markedly reduced all of these I/R-induced changes.
Analysis of hepatic tissue levels of HMGB1 by Western blotting
High-mobility group box 1 protein levels increase in the liver during I/R, and HMGB1 contributes to inflammation-associated liver injury following I/R. To determine whether PNU-282987 influences HMGB1 expression in hepatic I/R injury, Western blot analysis was performed on livers from animals that were subjected to hepatic I/R. High-mobility group box 1 protein expression was upregulated as early as 3 h after reperfusion and reached its maximal level at 6 h after reperfusion, then decreased by 12 h after reperfusion. High-mobility group box 1 protein levels were significantly lower in the PNU-282987–treated group at all time points (Figs. 3A, B).
Effect of PNU-282987 on hepatic tissue levels of HMGB1 by immunohistochemistry
The expression of HMGB1 in the liver was also assessed by immunohistochemistry (Fig. 4). Low but detectable levels of HMGB1 were observed in the nuclei of hepatocytes in the sham operation group. In contrast, intense HMGB1 staining was seen in the nuclei of hepatocytes in the vehicle-pretreated mice subjected to hepatic I/R. High-mobility group box 1 protein–specific staining was markedly reduced in liver slices from PNU-282987–pretreated mice compared with vehicle-pretreated mice.
HMGB1 and cytokine gene expression
Ischemia-reperfusion results in local upregulation of proinflammatory cytokines in addition to HMGB1. Therefore, to explore the mechanism for the protective action of PNU-282987 on hepatic I/R injury, we measured HMGB1, IL-1β, and TNF-α mRNA levels at 3, 6, and 12 h after reperfusion (Fig. 5). Control animals constitutively expressed low levels of HMGB1, IL-1β, and TNF-α mRNA. Levels of HMGB1 mRNA were enhanced markedly at 3, 6, and 12 h after reperfusion compared with the sham-operated group. Interleukin 1β and TNF-α mRNA levels were significantly increased at 3 and 6 h (P < 0.05). Pretreatment with PNU-282987 prevented the increases in HMGB1, IL-1B, and TNF-α mRNA in the liver following I/R (P < 0.05).
Effect of PNU-282987 on activation of the transcription factor NF-κB in the liver
To investigate the mechanisms involved in the inhibitory effects of PNU-282987 on the induction of HMGB1 and cytokines in hepatic I/R injury, we examined the effect of PNU-282987 on the nuclear translocation of NF-κB (Fig. 6). Western blotting analysis demonstrated that liver NF-κB activation was increased in the vehicle plus I/R group compared with the sham-operated group at 3, 6, and 12 h after reperfusion (P < 0.05). Pretreatment with PNU-282987 significantly reduced hepatic NF-κB activation compared with the vehicle plus I/R group at all time points studied (P < 0.05).
In this study, we evaluated the protective effect of PNU-282987, a α7nAChR agonist, on hepatic I/R injury in mice. Mice that underwent hepatic ischemia followed by reperfusion showed characteristic signs of liver damage and inflammation. These signs include elevated serum transaminase, histological changes consistent with severe liver damage, and increased expression of inflammation cytokines. Pretreatment with PNU-282987 decreased levels of the hepatic injury markers (Fig. 2) and significantly decreased the levels of cytokine mRNA, while inhibiting NF-κB signaling in the liver (Figs. 3–6). Thus, activation of α7nAChR could be a drug target to limit I/R in the clinical setting.
Hepatic I/R injury can occur in the setting of liver resection and always occurs during transplantation and hemorrhagic shock. Liver I/R may lead to local and remote organ failure with significant rates of morbidity and mortality (1, 22). Although a great variety of mechanisms have been proposed to explain remote organ injury induced by liver I/R, it is likely that inflammatory mediators released by the inflamed liver are to blame.
High-mobility group box 1 protein has a number of effects on the immune system when released from cells. This release can be passive in the setting of cell necrosis or active from cells activated by LPSs or hypoxia. Tsung and colleagues (4, 6) have reported that HMGB1 expressed and released by ischemic hepatocytes can activate neighboring immune cells via a TLR4-dependent mechanism. This triggers an inflammatory response manifested by the release of proinflammatory cytokines such as HMGB1, TNF-α, and IL-1β by Kupffer cells (23). In this study, we observed that TNF-α and IL-1β mRNA levels were significantly increased as early as 3 h after liver I/R (Fig. 5). Cytokine neutralization by antibodies significantly attenuates liver injury induced by hepatic I/R, suggesting that suppression of cytokine expression and release can serve as an important target to limit inflammatory I/R injury (6).
Although Wang et al. (10) suggested that HMGB1 was a late mediator of systemic inflammation in sepsis, our current and previous findings support the role of HMGB1 as an early mediator following acute hepatic I/R injury. Blockade of HMGB1 protects against warm hepatic I/R injury (6, 24). Thus, HMGB1 represents a proximal mediator of I/R-induced inflammation in the liver. We show here that hepatic HMGB1 expression increased as early as 3 h after reperfusion and in a time-dependent manner up to 6 h after reperfusion, then decreased thereafter. Pretreatment with PNU-282987 significantly inhibited HMGB1 expression (Figs. 3–5), as well as upregulated cytokine mRNA levels. Therefore, PNU-282987 appears to target the proximal event in bolt hepatocytes and Kupffer cells in hepatic I/R.
Our finding that PNU-282987 can suppress hepatic I/R-induced inflammation indicates that these events can be regulated by peripheral nicotinic ACh receptors. In recent years, CAP has been acknowledged as a potential anti-inflammatory therapy, by downregulating immune function and controlling inflammatory responses (15, 25). In the α7nAChR wild-type mice, decreased levels of TNF and HMGB1 after choline administration were observed; however, α7nAChR knockout mice did not respond to choline administration (26). Electrical stimulation of the vagus nerve in the I/R model decreased inflammation and serum TNF-α levels (27). In addition, several studies demonstrate that nicotine and specific agonists of α7nAChRs such as GTS-21 ameliorate lethal sepsis (18), pancreatitis (28), and renal (29) and myocardial I/R injury (19). PNU-282987 was effective in ameliorating indomethacin-induced small intestinal ulceration (30). Recent studies have reported that both HMGB1 and heme oxygenase 1 (HO-1) expression can be induced in the hepatic I/R model and sepsis (31, 32). Tsoyi et al. (31) found that nicotine increased HO-1 expression in RAW 246.7 cells through α7nAChR and increased the survival rate in LPS- and cecal ligation and puncture-induced sepsis. They concluded that a possible mechanism for eliciting CAP by nicotine (through α7nAChR) was HO-1–mediated inhibition of HMGB1 release both in vitro and in vivo. In our study, however, we did not explore local expression of HO-1 within the liver. Instead, our study confirms that α7nAChR stimulation can act proximally to prevent HMGB1 upregulation.
To address the mechanisms by which PNU-282987 exerts anti-inflammatory effects on hepatic I/R injury in mice, we examined hepatic NF-κB activation and found a significant inhibition of NF-κB activity by PNU-282987–pretreated mice (Fig. 6). Indeed, most inflammatory scenarios and a broad range of cytokines converge at the activation of the NF-κB pathway. Wang et al. (33) discovered that nicotine suppressed HMGB1 release through α7nAChR and inhibited NF-κB signaling during endotoxemia. Previous studies have established that the nuclear transcription factor NF-κB plays critical roles in the transcriptional regulation of proinflammatory genes (34, 35). In addition, NF-κB cis-elements have been identified in the promoter region of the HMGB1 gene (35). Therefore, we hypothesized that activation of α7nAChR by PNU-282987 may inhibit HMGB1 expression by interfering with NF-κB signaling. Given that HMGB1 acts as an early upstream inflammatory signal in hepatic I/R injury, it could induce the expression of downstream inflammatory cytokines. As predicted, we demonstrated that pretreatment with PNU-282987 inhibited HMGB1 and other cytokine expression and NF-κB activation.
In conclusion, we confirmed that HMGB1 expression levels increased in hepatic tissue in a mouse hepatic I/R injury model. Pretreatment with PNU-282987 inhibits HMGB1 expression, reduces necrosis, suppresses inflammatory cytokine production, and prevents NF-κB activation, exerting a protective role in hepatic I/R injury in mice.
The authors also thank Fuming Shen for supplying the drug PNU-282987 and Ji Ma, Weifan Xiao, Wang He, Yue Zhang, Xu Zhou, Hui Zeng, Suhong Xie, et al., for their valuable discussion and technical assistance.
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