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High-mobility group box 1 exacerbates CCl4-induced acute liver injury in mice
Clinical Immunology (2014) 153, 56–63
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
High-mobility group box 1 exacerbates CCl4-induced acute liver injury in mice☆ Maojian Chen a,1 , Wenjian Huang a,1 , Chao Wang a , Hao Nie a , Gang Li a , Ting Sun a , Fei Yang a , Yanxiang Zhang a , Kegang Shu a , Congyi Wang a,b , Quan Gong a,⁎ a
Department of Immunology, School of Medicine, Yangtze University, Jingzhou 434023, China The Center for Biomedical Research, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
b
Received 12 November 2013; accepted with revision 30 March 2014 KEYWORDS Acute liver injury; Carbon tetrachloride; Lipid peroxidation; HMGB1
Abstract High-mobility group box 1 (HMGB1) is a nuclear factor that can also serve as an imflammatory mediator once released into extracellular milieu. Therefore, HMGB1 has been recognized to play a pivotal role in inflammatory diseases such as sepsis, acute lung injury, ischemia reperfusion injury and type 1 diabetes. Nevertheless, its impact on carbon tetrachloride (CCl4)-induced hepatic injury is yet to be elucidated. In the present report, we demonstrated evidence indicating that high levels of HMGB1 were not only present in the necrotic area of liver but also in the serum after CCl4 challenge. In line with these observations, administration of exogenous recombinant HMGB1 exacerbated CCl4-induced hepatic injury, while HMGB1 blocking antibody provided protection for mice against CCl4-induced acute liver injury as evidenced by the decrease of serum transaminase and reduction of hepatic tissues necrosis. Mechanistic studies revealed that blockade of HMGB1 attenuated CCl4-induced MDA accumulation along with improved SOD and GSH activity. Treatment of mice with HMGB1 neutralizing antibody also significantly inhibited the production of proinflammatory mediators TNF-α and IL-6 along with attenuated HMGB1 expression and its extracellular release. Together, our data suggest an essential role for HMGB1 in CCl4-induced acute liver injury, while HMGB1 neutralizing antibody could be served as an effective regimen for preventing CCl4-induced acute liver injury. © 2014 Elsevier Inc. All rights reserved.
☆ This work was supported by the foundation of National Natural Science (Grant 81271872), the foundation of Hubei Provincial Department of Education (Grant D20121206), the foundation of Health Department of Hubei Province (Grant XF2012-5) and the College Student Innovation Experiment Program of Yangtze University (Grant 201210489335). ⁎ Corresponding author at: Department of Immunology, School of Medicine, Yangtze University, Jingzhou, Hubei 434023, China. E-mail address: [email protected] (Q. Gong). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.clim.2014.03.021 1521-6616/© 2014 Elsevier Inc. All rights reserved.
HMGB1 exacerbates CCl4-induced acute liver injury in mice
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1. Introduction
2.2. Materials
Liver diseases such as viral hepatitis, alcoholic liver injury, drug-induced liver injury, liver fibrosis and autoimmune hepatitis represent major threats to human health worldwide [1]. The hepatotoxin carbon tetrachloride (CCl4), an industrial solvent, is frequently used to induce acute liver injury in animals, which serves as a well-established murine model that resembles acute chemical liver injury in humans [2]. This type of acute liver injury is characterized by the marked increase of serum levels for transaminase and typical centrilobular necrosis. During the course of liver injury, CCl4 is metabolized by cytochrome P450 to form reactive intermediates such as trichloromethyl free radicals (CCl+3) and peroxyl radical (OOCCl3), which then initiate lipid peroxidation associated with cellular damage [3,4]. Of note, the subsequent activation of Kupffer cells along with increased production of proinflammatory cytokines such as TNF-α, IL-6 and IL-8 is also a contributing factor to the development of CCl4-induced acute liver injury [5]. High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding protein, and by which it modulates chromatin structure, facilitates interaction of proteins, and acts as a transcription factor to regulate gene expressions [6,7]. HMGB1 contains two domains (A box and B box), the cytokine activity of HMGB1 is localized to B box, while A box serves as an antagonist, in which it functions as a competitor for the full-length HMGB1 but without capability to generate pro-inflammatory effect [7]. Interestingly, intracellular HMGB1 can be either secreted by the activated immune cells or passively released by the necrotic or damaged cells [8,9], and extracellular HMGB1 binds to the cell surface receptors such as receptor for advanced glycation end products (RAGE) and toll-like receptor (TLR) 4 and TLR 2, and through which it behaves as a critical mediator of innate immune response to infection and injury [10–12]. Given the importance of HMGB1 in the initiation and progression of proinflammatory processes, we thus conducted studies to explore the role of HMGB1 in CCl4-induced acute liver injury. We demonstrated evidence that CCl4 injection induced HMGB1 expression and its extracellular release, and blockade of HMGB1 provided protection for mice against CCl4-induced hepatocellular damage, suggesting that HMGB1 could be a viable target for prevention of chemical induced liver injury.
Recombinant HMGB1 was purchased from Uscn Life Science Inc. (Wuhan, China). A polyclonal HMGB1 neutralizing antibody was generated as previously reported [13]. Control rabbit IgG was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Carbon tetrachloride (CCl4) was purchased from Beijing Chemical Reagent Company (Beijing, China). Alanine transferase (ALT) and aspartate transferase (AST) were obtained from Sichuan Maker Science and Technology Co., Ltd. (Chengdu, China). Total superoxide dismutase (T-SOD), malondialdehyde (MDA), glutathione (GSH) and Coomassie Brilliant Blue protein assay kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). TNF-α ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). IL-6 ELISA kit was purchased from NeoBioscience Technology Co., Ltd. (Shenzhen, China). HMGB1 ELISA kit was purchased from Elaborate Biotechnology Co., Ltd. (Wuhan, China). RNA simple Total RNA kit was purchased from TIANGEN (TIANGEN Biotech, China). RNA PCR kit was obtained from Takara (Takara Biotechnology, Shanghai, China).
2. Materials and methods 2.1. Animals Male BALB/c mice (6–8 week old, 20–25 g) were purchased from the Center for Animal Experiment of Wuhan University (Wuhan, China). The mice were raised in a laboratory with standard food and water and were exposed to a 12 h light/12 h dark cycles. The room temperature is 25 ± 1 °C and humidity is 50 ± 5%. All of the animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Yangtze University.
2.3. Animal treatment CCl4 was dissolved in olive oil at a concentration of 0.3% and intraperitoneally injected at a dose of 10 ml/kg body weight to induce acute liver injury [14]. The mice were administered intravenously with anti-HMGB1 Ab (600 μg/mouse) or the same amount of normal rabbit IgG (as controls) 30 min before or after CCl4 administration [13]; solvent control group was intraperitoneally injected with olive oil alone. Serum samples and livers were collected at the indicated time points after CCl4 injection.
2.4. Evaluation of liver injury Serum aminotransferase activities were analyzed using an automated chemistry analyzer (Olympus AU, Japan). Liver tissues fixed in 10% formalin were embedded in paraffin, sectioned at 4 μm and stained with hematoxylin–eosin for light microscopic examination.
2.5. Measurement of lipid peroxidation and antioxidant enzymes Liver tissues were subjected to preparation of cell lysates with cold Tris–HCl (5 mmol/l containing 2 mmol/l EDTA, pH 7.4), and the homogenates were then centrifuged at 4000 rpm (4 °C) for 20 min to collect supernatants for determination of MDA, SOD and GSH concentrations. MDA was estimated by measuring thiobarbituric acid (TBA) assay using a MDA assay kit, and expressed as nmol/mg protein. SOD activity was detected by determining the reduction of nitroblue tetrazolium (NBT) with a SOD assay kit, and expressed as U/mg protein. GSH was assayed by the method of Griffith (1980) using a GSH assay kit, and expressed as mg/g protein. Coomassie Brilliant Blue protein assay kits were used for the measurement of liver tissue protein.
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2.6. Immunohistochemical staining After deparaffin and rehydration, the embedded liver sections were treated with 3% H2O2 for 15 min, followed by microwave antigen retrieval for another 15 min in citrate buffer. The nonspecific proteins were blocked with 10% goat serum or rabbit serum for 30 min. For HMGB1 staining, the specimens were incubated with a rabbit anti-mouse HMGB1 polyclonal antibody (R&D, Minneapolis, MN) at 4 °C overnight, followed by a 30-min incubation with a horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology CO. LTD, China). The sections were finally incubated with diaminobenzidine (DAB) as a chromogenic substrate and counterstained with hematoxylin, dehydrated, cleared in xylenes, and mounted using the permount mounting medium (Upstate, Lake Placid, NY).
2.7. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from livers at the indicated time points using the TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) as instructed, and cDNA synthesis was carried out using 2 μg RNA with a PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Co., Ltd., Da Lian, LChina). PCR amplifications were performed with standard methods using following specific primers: β-action (F) 5′-CTG TCC CTG TAT GCC TCT G-3′, (R) 5′-CAT CGT ACT CCT GCT TGC T-3′; and HMGB1 (F) 5′-GAT GGG CAA AGG AGA TCC TAA G-3′, (R) 5′-TCA CTT TTT TGT CTC CCC TTT GGG-3′.
2.8. ELISA analysis of cytokine production Serum samples were subjected to ELISA analysis of levels for TNF-α, IL-6 and HMGB1 using commercial kits according to the manufacturers' instructions.
2.9. Statistical analysis All data are present as means ± SD. Statistical differences were determined by Student's t test or one-way ANOVA. All data analyses were conducted using the SPSS v17.0 statistical analysis software (SPSS Inc, Chicago, USA). In any case, a p-value less than 0.05 was considered to be statistically significant.
3. Results 3.1. CCl4 is potent to induce hepatic HMGB1 expression We first sought to address the impact of CCl4 challenge on liver expression of HMGB1. To this end, we did immunostaining of liver sections from CCl4-treated mice. In general, HMGB1 was noted to be localized predominantly in the nucleus of hepatocytes in all control mice. In sharp contrast, a large amount of HMGB1 was translocated into the cytoplasm of hepatocytes, and extracellular HMGB1 was detected in the necrotic area in mice after 16 h of CCl4 treatment (Fig. 1 A). To confirm HMGB1 release, we then conducted ELISA analysis
M. Chen et al. of serum samples. Indeed, serum levels for HMGB1 were significantly up-regulated after 16 h of CCl4 challenge (Fig. 1 B). Next, we examined HMGB1 mRNA levels in mice after CCl4 challenge. In line with the above results, CCl4 was found time-dependently to induce HMGB1 expression, and a significant up-regulation of HMGB1 mRNA was noted as early as 4 h of CCl4 challenge (Fig. 1 C). Together, these data suggest that CCl4 is potent to induce hepatic HMGB1 expression and extracellular release.
3.2. HMGB1 blocking antibody protects mice against CCl4-induced acute liver injury The above results prompted us to explore the impact of extracellular HMGB1 on CCl4-induced acute liver injury. For this purpose, the mice were preinjected intravenously with an HMGB1 neutralizing antibody (600 μg/mouse) 30 min before CCl4 challenge (0.3%, 10 ml/kg body weight), and the mice were next subjected to ALT and AST analysis 4 h and 16 h after CCl4 administration, respectively. Similar with previous studies [15], no significant difference was characterized in mice between each group at 4 h after CCl4 challenge for ALT (olive oil vs. IgG, vs. HMGB1 Ab 74.35 ± 4.07 vs. 79.45 ± 5.02 vs.78.34 ± 4.81; IgG/CCl4 vs. HMGB1 Ab/CCl4, 83 ± 6.02 vs. 82 ± 4.23), and AST (olive oil vs. IgG vs. HMGB1 Ab, 78.43 ± 4.57 vs. 80.34 ± 5.42 vs. 77.87 ± 4.76; IgG/CCl4 vs. HMGB1 Ab/CCl4, 79 ± 5.02 vs. 78.9 ± 4.31). However, a significant increase for both ALT and AST was identified in all control mice 16 h after CCl4 administration. Interestingly, pretreatment of animals with an HMGB1 neutralizing antibody significantly attenuated CCl4-induced increase for ALT and AST (Fig. 2 A-B). Next, we did histological analysis and massive necrosis was present in the liver of mice treated with control IgG, while necrosis was almost absent in animals treated with an HMGB1 neutralizing Ab (Fig. 3). To assess the feasibility of therapeutic role for our HMGB1 neutralizing Ab, we next treated the animals with our neutralizing Ab 30 min after CCl4 injection. Surprisingly, delayed administration of HMGB1 blocking Ab also provided protection for mice against CCl4-induced acute liver injury (Figs. 2 C–D, 3).
3.3. Administration of recombinant HMGB1 (rHMGB1) exacerbates CCl4-induced acute liver injury To further demonstrate the role of HMGB1 in CCl4induced acute liver injury, a nonlethal dose (20 μg) of rHMGB1 was injected into mice immediately after CCl4 administration (0.3%, 10 ml/kg body weight), and serum ALT and AST were assessed 16 h after CCl4 injection. Similar as previous studies, in general this low dose of rHMGB1 did not cause a perceptible liver injury [12]. However, administration of exogenous rHMGB1 significantly exacerbated CCl4-induced acute liver injury as manifested by the much higher levels of ALT and AST levels than that of animals treated with CCl4 alone (Fig. 4 A–B), suggesting a synergic action between HMGB1 and CCl4 during the course of acute liver injury.
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Figure 1 HMGB1 expression is up-regulated in hepatic tissues after CCl4 treatment. Mice were treated with olive oil or CCl4 (0.3%, 10 ml/kg). (A) A immunohistochemistry analysis for HMGB1 was performed for hepatocytes of liver tissues at 16 h after CCl4 injection. HMGB1 was enhanced in the cytoplasm of hepatocytes and in the necrotic area 16 h after CCl4 administration (original magnification ×400). (B) Serum HMGB1 content was detected at 4 h and 16 h after CCl4 treatment. Data are expressed as means ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. olive oil. (C) The mRNA expression of HMGB1 in the livers was determined by RT-PCR at 4 h and 16 h, respectively. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎P b 0.05 vs. olive oil, ⁎⁎P b 0.01 vs. olive oil.
3.4. Blockade of HMGB1 attenuates CCl4-induced oxidative stress in the liver To address the mechanisms by which blockade of HMGB1 alleviates CCl4-induced acute liver injury, we checked the impact of HMGB1 neutralizing Ab on CCl4-induced oxidative stress. We examined the concentration of MDA, SOD and GSH in the liver 16 h after CCl4 injection. In line with our expectation, administration of CCl4 induced a marked increase for hepatic MDA. In sharp contrast, administration of HMGB1 neutralizing Ab significantly attenuated CCl4-induced increase of MDA (Fig. 5 A). On the contrary, administration of CCl4 resulted in a significant reduction for SOD and GSH activities, while the reduction of their activities was significantly hampered by the pretreatment of HMGB1
neutralizing antibody (Fig. 5 B–C). Collectively, these data suggest that blockade of HMGB1 suppresses CCl4-induced hepatic oxidative stress.
3.5. Blockade of HMGB1 inhibits TNF-α and IL-6 production after CCl4 induction Given the role of proinflammatory cytokines especially TNF-α and IL-6 played in the pathoetiology of CCl4-induced acute liver injury [16,17], we next assessed the impact of HMGB1 blocking Ab on serum levels of TNF-α and IL-6. As expected, CCl4 time-dependently induced serum production of TNF-α (Fig. 6 A) and IL-6 (Fig. 6 B). However, much lower serum levels of TNF-α and IL-6 were noted in animals treated
Figure 2 HMGB1 blocking antibody treatment protects against CCl4-induced acute liver injury in mice. Mice were treated with HMGB1 neutralizing antibody (600 μg/mouse) or control IgG 30 min (A–B) before or (C–D) after CCl4 administration. The serum levels of ALT and AST were detected 16 h after CCl4 treatment. Data are expressed as means ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
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Figure 3 Hematoxylin–eosin staining of liver specimens from control IgG or anti-HMGB1 antibody animals 16 h post CCl4 administration (original magnification ×100 and ×400).
with HMGB1 neutralizing Ab as compared with that of animals treated with control IgG (Fig. 6). These data suggest that blockade of HMGB1 inhibits CCl4-induced proinflammatory response.
3.6. Blockade of HMGB1 signaling attenuates CCl4induced HMGB1 expression Finally, we investigated the effect of HMGB1 neutralizing Ab on HMGB1 expression and release by RT-PCR and ELISA analysis. As aforementioned, CCl4 time-dependently induces hepatic HMGB1 expression and enhances the production of serum HMGB1 (Fig. 1); we now noted that blockade of HMGB1 signaling by a neutralizing Ab significantly suppressed CCl4induced hepatic HMGB1 expression (Fig. 7 A–B), and more importantly, a 4.5-fold reduction for serum HMGB1 was also noted (Fig. 7C). Altogether, our data suggest that HMGB1 neutralizing antibody pretreatment may inhibit HMGB1 expression and release and thus attenuate CCl4-induced acute liver injury.
4. Discussion HMGB1 is originally recognized as a nuclear transcriptional co-factor, but once it is released into extracellular milieu, it also acts as strong inflammatory mediator not only triggering the production of proinflammatory cytokines, but also initiating adaptive immune response through regulation of DC function [18,19]. Therefore, extracellular HMGB1 is found to be implicated in the pathogenesis of disorders such as sepsis, arthritis, cancer and autoimmune diseases [12,20–22]. As a consequence, HMGB1-targeted therapy has become a research hotspot in recent years. Recently, we demonstrated that HMGB1 exacerbates Con A-induced hepatic injury and blockade of HMGB1 protects animals from T cell-mediated hepatitis [19]. In the present study, we further demonstrated the involvement of HMGB1 in CCl4-induced acute liver injury. Indeed, CCl4 treatment significantly enhanced HMGB1 expression in the liver (Fig. 1), and administration of exogenous rHMGB1 promoted CCl4-induced liver injury (Fig. 4). Particularly, blockade of HMGB1 by administration of a neutralization antibody markedly attenuated CCl4-induced abnormalities for
Figure 4 Administration of recombinant HMGB1 worsens CCl4-induced hepatic injury. Mice were treated with a nonlethal dose of recombinant HMGB1 (20 μg/mouse) or vehicle PBS alone, or along with CCl4 (0.3%, 10 ml/kg). Serum ALT and AST levels were analyzed after 16 h of CCl4 injection. Data are expressed as means ± SD; n = 6–8 mice per group. **P b 0.01 vs. mice CCl4.
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Figure 5 Anti-HMGB1 antibody pretreatment reduces CCl4 -caused oxidative stress in mice. Liver homogenate was analyzed at 16 h after CCl4 administration. (A) The activity of MDA, (B) the activity of SOD and (C) the activity of GSH were measured. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
serum ALT and AST and hepatic necrosis (Figs. 2–3). In line with our findings, Tu and colleagues also reported that injection of CCl4 significantly increases hepatic levels of HMGB1, and curcumin protects animals against hepatic injury and fibrosis by inhibition of HMGB1 expression [23]. Taken together, these studies provided additional evidence supporting that HMGB1 is implicated in the pathogenesis of CCl4-induced liver injury. It has been well known that CCl4 is a strong hepatotoxin, in which it induces excessive production of free radicals along with oxidative stress, and through which it causes cellular necrosis and inflammatory responses, and eventually leads to acute liver injury associated with functional impairment [24,25]. MDA is an end-product of lipid peroxidation, and therefore, altered MDA levels in the liver are a characteristic marker for abnormal peroxidation and impaired antioxidant-defense [26]. SOD is an enzyme that catalyzes the dismutation of superoxide anions into oxygen and hydrogen peroxide. It acts as an important antioxidant mechanism in cells against superoxide radical [27]. On the other hand, GSH is a major non-enzymatic antioxidant that neutralizes free radicals and converts harmful poisons within the body into harmless substances, which can be then eliminated from the body via an excretory mechanism [28]. Consistent with previous studies [24,25,29], our study showed that CCl4 administration induced oxidative stress in the liver as evidenced by the increased MDA levels and reduced SOD and GSH activities. Importantly, pretreatment of animals with HMGB1 blocking antibody significantly inhibited the MDA accumulation along with improved SOD and GSH activity after CCl4 induction. (Fig. 5), suggesting that blockade of HMGB1 could be an
effective approach for reducing oxidative burden during the course of CCl4-induced acute liver injury. A number of studies have consistently demonstrated that proinflammatory cytokines are also important players in the pathogenesis of CCl4-induced acute liver injury, in which TNF-α and IL-6 are thought to be the most important [5,30]. Although TNF-α plays a key role in host defensive responses against injury and infection, excessive and prolonged production of TNF-α, however, is thought to be involved in the pathology of liver damage and systematic toxicity [31]. Nevertheless, IL-6 is required for the regenerative process after liver injury [32]; excessive IL-6 production can also cause hepatocyte damage [33]. Of important note, TNF-α is found to synergize with IL-6 to induce other inflammatory cytokine and chemokine expressions, which then exacerbate CCl4-induced hepatic injury [5]. Interestingly, a recent study revealed that administration of HMGB1 blocking antibody significantly suppressed serum levels for TNF-α and IL-6 in a rat model of traumatic injury [34]. Consistent with this study, we demonstrated that pretreatment of animals with an HMGB1 neutralizing antibody inhibited the production of TNF-α and IL-6 induced by CCl4 injection (Fig. 6). Collectively, those data indicate that the protective effect elicited by blockade of HMGB1 is associated with attenuation of TNF-α and IL-6 production. Our present studies suggested a self feedback mechanism for blockade of HMGB1 signaling, in which administration of a HMGB1 neutralizing antibody not only suppresses TNF-α and IL-6 expression, but also attenuates its own expression and release (Fig. 7). Indeed, recombinant A box, an antagonist for the full-length HMGB1, also demonstrated feasibility to
Figure 6 Anti-HMGB1 antibody pretreatment inhibits the release of cytokines. Mice were pretreated with HMGB1 neutralization antibody or control IgG. TNF-α and IL-6 in serum were measured by ELISA at 0 h, 4 h and 16 h after CCl4 administration, respectively. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
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Figure 7 Anti-HMGB1 antibody pretreatment inhibits the expression and release of HMGB1. Mice were pretreated with HMGB1 neutralization antibody or control IgG before CCl4 administration. (A–B) The expression of HMGB1 mRNA was detected by RT-PCR at 4 h and 16 h, respectively. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎P b 0.05 vs. IgG/CCl4, ⁎⁎P b 0.01 vs. IgG/CCl4. (C) Serum HMGB1 content was detected 16 h after CCl4 treatment. Data are expressed as means ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
inhibit serum HMGB1 levels in a cecal ligation and puncture (CLP) model [20], and similar results were also noted in a murine cardiac allograft model [35]. However, the mechanisms through which HMGB1 antagonists modulate the expression and release of HMGB1 are yet to be fully elucidated; future studies with focus to address this issue would be necessary. Nevertheless, there is evidence that administration of HMGB1 neutralizing antibody represses the phosphorylation of c-Jun NH2-terminal kinase (JNK) but enhances nuclear factor-KB (NF-KB) activation in a model with liver ischemia reperfusion (I/R) injury [12]. It would be interesting to examine whether this mechanism also applies to our model in the present study. In summary, we demonstrated evidence supporting that extracellular HMGB1 contributes to CCl4-induced hepatic injury, and therefore, blockade of HMGB1 by administration of a neutralizing antibody significantly attenuated CCl4induced hepatic injury, which involves suppression of oxidative stress and inhibition of the production of inflammatory mediators such as TNF-α, IL-6 and HMGB1 itself.
Conflicts of interest The authors declare that there are no conflicts of interest.
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[27] K. Amirshahrokhi, S. Bohlooli, Effect of methylsulfonylmethane on paraquat-induced acute lung and liver injury in mice, Inflammation 36 (2013) 1111–1121. [28] A.M. Al-Dbass, S.K. Al-Daihan, R.S. Bhat, Agaricus blazei Murill as an efficient hepatoprotective and antioxidant agent against CCl4induced liver injury in rats, Saudi, J. Biol. Sci. 19 (2012) 303–309. [29] N. Tirkey, S. Pilkhwal, A. Kuhad, K. Chopra, Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney, BMC Pharmacol. 5 (2005) 2. [30] Y.L. Wu, L.H. Lian, Y. Wan, J.X. Nan, Baicalein inhibits nuclear factor-κB and apoptosis via c-FLIP and MAPK in D-GalN/LPS induced acute liver failure in murine models, Chem. Biol. Interact. 188 (2010) 526–534. [31] I. Bohlinger, M. Leist, J. Barsig, S. Uhlig, G. Tiegs, A. Wendel, Interleukin-1 and nitric oxide protect against tumor necrosis factor alpha-induced liver injury through distinct pathways, Hepatology 22 (1995) 1829–1837. [32] T. Sakamoto, Z. Liu, N. Murase, T. Ezure, S. Yokomuro, V. Poli, A.J. Demetris, Mitosis and apoptosis in the liver of interleukin6-deficient mice after partial hepatectomy, Hepatology 29 (1999) 403–411. [33] T. Wüstefeld, T. Rakemann, S. Kubicka, M.P. Manns, C. Trautwein, Hyperstimulation with interleukin 6 inhibits cell cycle progression after hepatectomy in mice, Hepatology 32 (2000) 514–522. [34] J. Shimazaki, N. Matsumoto, H. Ogura, T. Muroya, Y. Kuwagata, J. Nakagawa, K. Yamakawa, H. Hosotsubo, Y. Imamura, T. Shimazu, Systemic involvement of high-mobility group box 1 protein and therapeutic effect of anti-high-mobility group box 1 protein antibody in a rat model of crush injury, Shock 37 (2012) 634–638. [35] Y. Huang, H. Yin, J. Han, B. Huang, J. Xu, F. Zheng, Z. Tan, M. Fang, L. Rui, D. Chen, S. Wang, X. Zheng, C.Y. Wang, F. Gong, Extracellular HMGB1 functions as an innate immune-mediator implicated in murine cardiac allograft acute rejection, Am. J. Transplant. 7 (2007) 799–808.
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Clinical Immunology www.elsevier.com/locate/yclim
High-mobility group box 1 exacerbates CCl4-induced acute liver injury in mice☆ Maojian Chen a,1 , Wenjian Huang a,1 , Chao Wang a , Hao Nie a , Gang Li a , Ting Sun a , Fei Yang a , Yanxiang Zhang a , Kegang Shu a , Congyi Wang a,b , Quan Gong a,⁎ a
Department of Immunology, School of Medicine, Yangtze University, Jingzhou 434023, China The Center for Biomedical Research, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
b
Received 12 November 2013; accepted with revision 30 March 2014 KEYWORDS Acute liver injury; Carbon tetrachloride; Lipid peroxidation; HMGB1
Abstract High-mobility group box 1 (HMGB1) is a nuclear factor that can also serve as an imflammatory mediator once released into extracellular milieu. Therefore, HMGB1 has been recognized to play a pivotal role in inflammatory diseases such as sepsis, acute lung injury, ischemia reperfusion injury and type 1 diabetes. Nevertheless, its impact on carbon tetrachloride (CCl4)-induced hepatic injury is yet to be elucidated. In the present report, we demonstrated evidence indicating that high levels of HMGB1 were not only present in the necrotic area of liver but also in the serum after CCl4 challenge. In line with these observations, administration of exogenous recombinant HMGB1 exacerbated CCl4-induced hepatic injury, while HMGB1 blocking antibody provided protection for mice against CCl4-induced acute liver injury as evidenced by the decrease of serum transaminase and reduction of hepatic tissues necrosis. Mechanistic studies revealed that blockade of HMGB1 attenuated CCl4-induced MDA accumulation along with improved SOD and GSH activity. Treatment of mice with HMGB1 neutralizing antibody also significantly inhibited the production of proinflammatory mediators TNF-α and IL-6 along with attenuated HMGB1 expression and its extracellular release. Together, our data suggest an essential role for HMGB1 in CCl4-induced acute liver injury, while HMGB1 neutralizing antibody could be served as an effective regimen for preventing CCl4-induced acute liver injury. © 2014 Elsevier Inc. All rights reserved.
☆ This work was supported by the foundation of National Natural Science (Grant 81271872), the foundation of Hubei Provincial Department of Education (Grant D20121206), the foundation of Health Department of Hubei Province (Grant XF2012-5) and the College Student Innovation Experiment Program of Yangtze University (Grant 201210489335). ⁎ Corresponding author at: Department of Immunology, School of Medicine, Yangtze University, Jingzhou, Hubei 434023, China. E-mail address: [email protected] (Q. Gong). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.clim.2014.03.021 1521-6616/© 2014 Elsevier Inc. All rights reserved.
HMGB1 exacerbates CCl4-induced acute liver injury in mice
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1. Introduction
2.2. Materials
Liver diseases such as viral hepatitis, alcoholic liver injury, drug-induced liver injury, liver fibrosis and autoimmune hepatitis represent major threats to human health worldwide [1]. The hepatotoxin carbon tetrachloride (CCl4), an industrial solvent, is frequently used to induce acute liver injury in animals, which serves as a well-established murine model that resembles acute chemical liver injury in humans [2]. This type of acute liver injury is characterized by the marked increase of serum levels for transaminase and typical centrilobular necrosis. During the course of liver injury, CCl4 is metabolized by cytochrome P450 to form reactive intermediates such as trichloromethyl free radicals (CCl+3) and peroxyl radical (OOCCl3), which then initiate lipid peroxidation associated with cellular damage [3,4]. Of note, the subsequent activation of Kupffer cells along with increased production of proinflammatory cytokines such as TNF-α, IL-6 and IL-8 is also a contributing factor to the development of CCl4-induced acute liver injury [5]. High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding protein, and by which it modulates chromatin structure, facilitates interaction of proteins, and acts as a transcription factor to regulate gene expressions [6,7]. HMGB1 contains two domains (A box and B box), the cytokine activity of HMGB1 is localized to B box, while A box serves as an antagonist, in which it functions as a competitor for the full-length HMGB1 but without capability to generate pro-inflammatory effect [7]. Interestingly, intracellular HMGB1 can be either secreted by the activated immune cells or passively released by the necrotic or damaged cells [8,9], and extracellular HMGB1 binds to the cell surface receptors such as receptor for advanced glycation end products (RAGE) and toll-like receptor (TLR) 4 and TLR 2, and through which it behaves as a critical mediator of innate immune response to infection and injury [10–12]. Given the importance of HMGB1 in the initiation and progression of proinflammatory processes, we thus conducted studies to explore the role of HMGB1 in CCl4-induced acute liver injury. We demonstrated evidence that CCl4 injection induced HMGB1 expression and its extracellular release, and blockade of HMGB1 provided protection for mice against CCl4-induced hepatocellular damage, suggesting that HMGB1 could be a viable target for prevention of chemical induced liver injury.
Recombinant HMGB1 was purchased from Uscn Life Science Inc. (Wuhan, China). A polyclonal HMGB1 neutralizing antibody was generated as previously reported [13]. Control rabbit IgG was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Carbon tetrachloride (CCl4) was purchased from Beijing Chemical Reagent Company (Beijing, China). Alanine transferase (ALT) and aspartate transferase (AST) were obtained from Sichuan Maker Science and Technology Co., Ltd. (Chengdu, China). Total superoxide dismutase (T-SOD), malondialdehyde (MDA), glutathione (GSH) and Coomassie Brilliant Blue protein assay kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). TNF-α ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). IL-6 ELISA kit was purchased from NeoBioscience Technology Co., Ltd. (Shenzhen, China). HMGB1 ELISA kit was purchased from Elaborate Biotechnology Co., Ltd. (Wuhan, China). RNA simple Total RNA kit was purchased from TIANGEN (TIANGEN Biotech, China). RNA PCR kit was obtained from Takara (Takara Biotechnology, Shanghai, China).
2. Materials and methods 2.1. Animals Male BALB/c mice (6–8 week old, 20–25 g) were purchased from the Center for Animal Experiment of Wuhan University (Wuhan, China). The mice were raised in a laboratory with standard food and water and were exposed to a 12 h light/12 h dark cycles. The room temperature is 25 ± 1 °C and humidity is 50 ± 5%. All of the animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Yangtze University.
2.3. Animal treatment CCl4 was dissolved in olive oil at a concentration of 0.3% and intraperitoneally injected at a dose of 10 ml/kg body weight to induce acute liver injury [14]. The mice were administered intravenously with anti-HMGB1 Ab (600 μg/mouse) or the same amount of normal rabbit IgG (as controls) 30 min before or after CCl4 administration [13]; solvent control group was intraperitoneally injected with olive oil alone. Serum samples and livers were collected at the indicated time points after CCl4 injection.
2.4. Evaluation of liver injury Serum aminotransferase activities were analyzed using an automated chemistry analyzer (Olympus AU, Japan). Liver tissues fixed in 10% formalin were embedded in paraffin, sectioned at 4 μm and stained with hematoxylin–eosin for light microscopic examination.
2.5. Measurement of lipid peroxidation and antioxidant enzymes Liver tissues were subjected to preparation of cell lysates with cold Tris–HCl (5 mmol/l containing 2 mmol/l EDTA, pH 7.4), and the homogenates were then centrifuged at 4000 rpm (4 °C) for 20 min to collect supernatants for determination of MDA, SOD and GSH concentrations. MDA was estimated by measuring thiobarbituric acid (TBA) assay using a MDA assay kit, and expressed as nmol/mg protein. SOD activity was detected by determining the reduction of nitroblue tetrazolium (NBT) with a SOD assay kit, and expressed as U/mg protein. GSH was assayed by the method of Griffith (1980) using a GSH assay kit, and expressed as mg/g protein. Coomassie Brilliant Blue protein assay kits were used for the measurement of liver tissue protein.
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2.6. Immunohistochemical staining After deparaffin and rehydration, the embedded liver sections were treated with 3% H2O2 for 15 min, followed by microwave antigen retrieval for another 15 min in citrate buffer. The nonspecific proteins were blocked with 10% goat serum or rabbit serum for 30 min. For HMGB1 staining, the specimens were incubated with a rabbit anti-mouse HMGB1 polyclonal antibody (R&D, Minneapolis, MN) at 4 °C overnight, followed by a 30-min incubation with a horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology CO. LTD, China). The sections were finally incubated with diaminobenzidine (DAB) as a chromogenic substrate and counterstained with hematoxylin, dehydrated, cleared in xylenes, and mounted using the permount mounting medium (Upstate, Lake Placid, NY).
2.7. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from livers at the indicated time points using the TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) as instructed, and cDNA synthesis was carried out using 2 μg RNA with a PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Co., Ltd., Da Lian, LChina). PCR amplifications were performed with standard methods using following specific primers: β-action (F) 5′-CTG TCC CTG TAT GCC TCT G-3′, (R) 5′-CAT CGT ACT CCT GCT TGC T-3′; and HMGB1 (F) 5′-GAT GGG CAA AGG AGA TCC TAA G-3′, (R) 5′-TCA CTT TTT TGT CTC CCC TTT GGG-3′.
2.8. ELISA analysis of cytokine production Serum samples were subjected to ELISA analysis of levels for TNF-α, IL-6 and HMGB1 using commercial kits according to the manufacturers' instructions.
2.9. Statistical analysis All data are present as means ± SD. Statistical differences were determined by Student's t test or one-way ANOVA. All data analyses were conducted using the SPSS v17.0 statistical analysis software (SPSS Inc, Chicago, USA). In any case, a p-value less than 0.05 was considered to be statistically significant.
3. Results 3.1. CCl4 is potent to induce hepatic HMGB1 expression We first sought to address the impact of CCl4 challenge on liver expression of HMGB1. To this end, we did immunostaining of liver sections from CCl4-treated mice. In general, HMGB1 was noted to be localized predominantly in the nucleus of hepatocytes in all control mice. In sharp contrast, a large amount of HMGB1 was translocated into the cytoplasm of hepatocytes, and extracellular HMGB1 was detected in the necrotic area in mice after 16 h of CCl4 treatment (Fig. 1 A). To confirm HMGB1 release, we then conducted ELISA analysis
M. Chen et al. of serum samples. Indeed, serum levels for HMGB1 were significantly up-regulated after 16 h of CCl4 challenge (Fig. 1 B). Next, we examined HMGB1 mRNA levels in mice after CCl4 challenge. In line with the above results, CCl4 was found time-dependently to induce HMGB1 expression, and a significant up-regulation of HMGB1 mRNA was noted as early as 4 h of CCl4 challenge (Fig. 1 C). Together, these data suggest that CCl4 is potent to induce hepatic HMGB1 expression and extracellular release.
3.2. HMGB1 blocking antibody protects mice against CCl4-induced acute liver injury The above results prompted us to explore the impact of extracellular HMGB1 on CCl4-induced acute liver injury. For this purpose, the mice were preinjected intravenously with an HMGB1 neutralizing antibody (600 μg/mouse) 30 min before CCl4 challenge (0.3%, 10 ml/kg body weight), and the mice were next subjected to ALT and AST analysis 4 h and 16 h after CCl4 administration, respectively. Similar with previous studies [15], no significant difference was characterized in mice between each group at 4 h after CCl4 challenge for ALT (olive oil vs. IgG, vs. HMGB1 Ab 74.35 ± 4.07 vs. 79.45 ± 5.02 vs.78.34 ± 4.81; IgG/CCl4 vs. HMGB1 Ab/CCl4, 83 ± 6.02 vs. 82 ± 4.23), and AST (olive oil vs. IgG vs. HMGB1 Ab, 78.43 ± 4.57 vs. 80.34 ± 5.42 vs. 77.87 ± 4.76; IgG/CCl4 vs. HMGB1 Ab/CCl4, 79 ± 5.02 vs. 78.9 ± 4.31). However, a significant increase for both ALT and AST was identified in all control mice 16 h after CCl4 administration. Interestingly, pretreatment of animals with an HMGB1 neutralizing antibody significantly attenuated CCl4-induced increase for ALT and AST (Fig. 2 A-B). Next, we did histological analysis and massive necrosis was present in the liver of mice treated with control IgG, while necrosis was almost absent in animals treated with an HMGB1 neutralizing Ab (Fig. 3). To assess the feasibility of therapeutic role for our HMGB1 neutralizing Ab, we next treated the animals with our neutralizing Ab 30 min after CCl4 injection. Surprisingly, delayed administration of HMGB1 blocking Ab also provided protection for mice against CCl4-induced acute liver injury (Figs. 2 C–D, 3).
3.3. Administration of recombinant HMGB1 (rHMGB1) exacerbates CCl4-induced acute liver injury To further demonstrate the role of HMGB1 in CCl4induced acute liver injury, a nonlethal dose (20 μg) of rHMGB1 was injected into mice immediately after CCl4 administration (0.3%, 10 ml/kg body weight), and serum ALT and AST were assessed 16 h after CCl4 injection. Similar as previous studies, in general this low dose of rHMGB1 did not cause a perceptible liver injury [12]. However, administration of exogenous rHMGB1 significantly exacerbated CCl4-induced acute liver injury as manifested by the much higher levels of ALT and AST levels than that of animals treated with CCl4 alone (Fig. 4 A–B), suggesting a synergic action between HMGB1 and CCl4 during the course of acute liver injury.
HMGB1 exacerbates CCl4-induced acute liver injury in mice
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Figure 1 HMGB1 expression is up-regulated in hepatic tissues after CCl4 treatment. Mice were treated with olive oil or CCl4 (0.3%, 10 ml/kg). (A) A immunohistochemistry analysis for HMGB1 was performed for hepatocytes of liver tissues at 16 h after CCl4 injection. HMGB1 was enhanced in the cytoplasm of hepatocytes and in the necrotic area 16 h after CCl4 administration (original magnification ×400). (B) Serum HMGB1 content was detected at 4 h and 16 h after CCl4 treatment. Data are expressed as means ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. olive oil. (C) The mRNA expression of HMGB1 in the livers was determined by RT-PCR at 4 h and 16 h, respectively. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎P b 0.05 vs. olive oil, ⁎⁎P b 0.01 vs. olive oil.
3.4. Blockade of HMGB1 attenuates CCl4-induced oxidative stress in the liver To address the mechanisms by which blockade of HMGB1 alleviates CCl4-induced acute liver injury, we checked the impact of HMGB1 neutralizing Ab on CCl4-induced oxidative stress. We examined the concentration of MDA, SOD and GSH in the liver 16 h after CCl4 injection. In line with our expectation, administration of CCl4 induced a marked increase for hepatic MDA. In sharp contrast, administration of HMGB1 neutralizing Ab significantly attenuated CCl4-induced increase of MDA (Fig. 5 A). On the contrary, administration of CCl4 resulted in a significant reduction for SOD and GSH activities, while the reduction of their activities was significantly hampered by the pretreatment of HMGB1
neutralizing antibody (Fig. 5 B–C). Collectively, these data suggest that blockade of HMGB1 suppresses CCl4-induced hepatic oxidative stress.
3.5. Blockade of HMGB1 inhibits TNF-α and IL-6 production after CCl4 induction Given the role of proinflammatory cytokines especially TNF-α and IL-6 played in the pathoetiology of CCl4-induced acute liver injury [16,17], we next assessed the impact of HMGB1 blocking Ab on serum levels of TNF-α and IL-6. As expected, CCl4 time-dependently induced serum production of TNF-α (Fig. 6 A) and IL-6 (Fig. 6 B). However, much lower serum levels of TNF-α and IL-6 were noted in animals treated
Figure 2 HMGB1 blocking antibody treatment protects against CCl4-induced acute liver injury in mice. Mice were treated with HMGB1 neutralizing antibody (600 μg/mouse) or control IgG 30 min (A–B) before or (C–D) after CCl4 administration. The serum levels of ALT and AST were detected 16 h after CCl4 treatment. Data are expressed as means ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
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Figure 3 Hematoxylin–eosin staining of liver specimens from control IgG or anti-HMGB1 antibody animals 16 h post CCl4 administration (original magnification ×100 and ×400).
with HMGB1 neutralizing Ab as compared with that of animals treated with control IgG (Fig. 6). These data suggest that blockade of HMGB1 inhibits CCl4-induced proinflammatory response.
3.6. Blockade of HMGB1 signaling attenuates CCl4induced HMGB1 expression Finally, we investigated the effect of HMGB1 neutralizing Ab on HMGB1 expression and release by RT-PCR and ELISA analysis. As aforementioned, CCl4 time-dependently induces hepatic HMGB1 expression and enhances the production of serum HMGB1 (Fig. 1); we now noted that blockade of HMGB1 signaling by a neutralizing Ab significantly suppressed CCl4induced hepatic HMGB1 expression (Fig. 7 A–B), and more importantly, a 4.5-fold reduction for serum HMGB1 was also noted (Fig. 7C). Altogether, our data suggest that HMGB1 neutralizing antibody pretreatment may inhibit HMGB1 expression and release and thus attenuate CCl4-induced acute liver injury.
4. Discussion HMGB1 is originally recognized as a nuclear transcriptional co-factor, but once it is released into extracellular milieu, it also acts as strong inflammatory mediator not only triggering the production of proinflammatory cytokines, but also initiating adaptive immune response through regulation of DC function [18,19]. Therefore, extracellular HMGB1 is found to be implicated in the pathogenesis of disorders such as sepsis, arthritis, cancer and autoimmune diseases [12,20–22]. As a consequence, HMGB1-targeted therapy has become a research hotspot in recent years. Recently, we demonstrated that HMGB1 exacerbates Con A-induced hepatic injury and blockade of HMGB1 protects animals from T cell-mediated hepatitis [19]. In the present study, we further demonstrated the involvement of HMGB1 in CCl4-induced acute liver injury. Indeed, CCl4 treatment significantly enhanced HMGB1 expression in the liver (Fig. 1), and administration of exogenous rHMGB1 promoted CCl4-induced liver injury (Fig. 4). Particularly, blockade of HMGB1 by administration of a neutralization antibody markedly attenuated CCl4-induced abnormalities for
Figure 4 Administration of recombinant HMGB1 worsens CCl4-induced hepatic injury. Mice were treated with a nonlethal dose of recombinant HMGB1 (20 μg/mouse) or vehicle PBS alone, or along with CCl4 (0.3%, 10 ml/kg). Serum ALT and AST levels were analyzed after 16 h of CCl4 injection. Data are expressed as means ± SD; n = 6–8 mice per group. **P b 0.01 vs. mice CCl4.
HMGB1 exacerbates CCl4-induced acute liver injury in mice
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Figure 5 Anti-HMGB1 antibody pretreatment reduces CCl4 -caused oxidative stress in mice. Liver homogenate was analyzed at 16 h after CCl4 administration. (A) The activity of MDA, (B) the activity of SOD and (C) the activity of GSH were measured. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
serum ALT and AST and hepatic necrosis (Figs. 2–3). In line with our findings, Tu and colleagues also reported that injection of CCl4 significantly increases hepatic levels of HMGB1, and curcumin protects animals against hepatic injury and fibrosis by inhibition of HMGB1 expression [23]. Taken together, these studies provided additional evidence supporting that HMGB1 is implicated in the pathogenesis of CCl4-induced liver injury. It has been well known that CCl4 is a strong hepatotoxin, in which it induces excessive production of free radicals along with oxidative stress, and through which it causes cellular necrosis and inflammatory responses, and eventually leads to acute liver injury associated with functional impairment [24,25]. MDA is an end-product of lipid peroxidation, and therefore, altered MDA levels in the liver are a characteristic marker for abnormal peroxidation and impaired antioxidant-defense [26]. SOD is an enzyme that catalyzes the dismutation of superoxide anions into oxygen and hydrogen peroxide. It acts as an important antioxidant mechanism in cells against superoxide radical [27]. On the other hand, GSH is a major non-enzymatic antioxidant that neutralizes free radicals and converts harmful poisons within the body into harmless substances, which can be then eliminated from the body via an excretory mechanism [28]. Consistent with previous studies [24,25,29], our study showed that CCl4 administration induced oxidative stress in the liver as evidenced by the increased MDA levels and reduced SOD and GSH activities. Importantly, pretreatment of animals with HMGB1 blocking antibody significantly inhibited the MDA accumulation along with improved SOD and GSH activity after CCl4 induction. (Fig. 5), suggesting that blockade of HMGB1 could be an
effective approach for reducing oxidative burden during the course of CCl4-induced acute liver injury. A number of studies have consistently demonstrated that proinflammatory cytokines are also important players in the pathogenesis of CCl4-induced acute liver injury, in which TNF-α and IL-6 are thought to be the most important [5,30]. Although TNF-α plays a key role in host defensive responses against injury and infection, excessive and prolonged production of TNF-α, however, is thought to be involved in the pathology of liver damage and systematic toxicity [31]. Nevertheless, IL-6 is required for the regenerative process after liver injury [32]; excessive IL-6 production can also cause hepatocyte damage [33]. Of important note, TNF-α is found to synergize with IL-6 to induce other inflammatory cytokine and chemokine expressions, which then exacerbate CCl4-induced hepatic injury [5]. Interestingly, a recent study revealed that administration of HMGB1 blocking antibody significantly suppressed serum levels for TNF-α and IL-6 in a rat model of traumatic injury [34]. Consistent with this study, we demonstrated that pretreatment of animals with an HMGB1 neutralizing antibody inhibited the production of TNF-α and IL-6 induced by CCl4 injection (Fig. 6). Collectively, those data indicate that the protective effect elicited by blockade of HMGB1 is associated with attenuation of TNF-α and IL-6 production. Our present studies suggested a self feedback mechanism for blockade of HMGB1 signaling, in which administration of a HMGB1 neutralizing antibody not only suppresses TNF-α and IL-6 expression, but also attenuates its own expression and release (Fig. 7). Indeed, recombinant A box, an antagonist for the full-length HMGB1, also demonstrated feasibility to
Figure 6 Anti-HMGB1 antibody pretreatment inhibits the release of cytokines. Mice were pretreated with HMGB1 neutralization antibody or control IgG. TNF-α and IL-6 in serum were measured by ELISA at 0 h, 4 h and 16 h after CCl4 administration, respectively. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
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Figure 7 Anti-HMGB1 antibody pretreatment inhibits the expression and release of HMGB1. Mice were pretreated with HMGB1 neutralization antibody or control IgG before CCl4 administration. (A–B) The expression of HMGB1 mRNA was detected by RT-PCR at 4 h and 16 h, respectively. Data were expressed as mean ± SD; n = 6–8 mice per group. ⁎P b 0.05 vs. IgG/CCl4, ⁎⁎P b 0.01 vs. IgG/CCl4. (C) Serum HMGB1 content was detected 16 h after CCl4 treatment. Data are expressed as means ± SD; n = 6–8 mice per group. ⁎⁎P b 0.01 vs. IgG/CCl4.
inhibit serum HMGB1 levels in a cecal ligation and puncture (CLP) model [20], and similar results were also noted in a murine cardiac allograft model [35]. However, the mechanisms through which HMGB1 antagonists modulate the expression and release of HMGB1 are yet to be fully elucidated; future studies with focus to address this issue would be necessary. Nevertheless, there is evidence that administration of HMGB1 neutralizing antibody represses the phosphorylation of c-Jun NH2-terminal kinase (JNK) but enhances nuclear factor-KB (NF-KB) activation in a model with liver ischemia reperfusion (I/R) injury [12]. It would be interesting to examine whether this mechanism also applies to our model in the present study. In summary, we demonstrated evidence supporting that extracellular HMGB1 contributes to CCl4-induced hepatic injury, and therefore, blockade of HMGB1 by administration of a neutralizing antibody significantly attenuated CCl4induced hepatic injury, which involves suppression of oxidative stress and inhibition of the production of inflammatory mediators such as TNF-α, IL-6 and HMGB1 itself.
Conflicts of interest The authors declare that there are no conflicts of interest.
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