The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

PLOS ONE, Oct 2013

The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371/journal.pone.0076293&representation=PDF

The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

et al. (2013) The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice. PLoS ONE 8(10): e76293. doi:10.1371/journal.pone.0076293 The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS- Activated RAW 264.7 Cells and CLP-Induced Septic Mice Eun Jung Park. 0 Hwa Jin Jang. 0 Konstantin Tsoyi. 0 Young Min Kim 0 Sang Won Park 0 Hye Jung Kim 0 Jae 0 Heun Lee 0 Ki Churl Chang 0 Jane C. Deng, University of California Los Angeles, United States of America 0 Department of Pharmacology, School of Medicine Gyeongsang National University and Institute of Health Sciences , Jinju , Korea The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/ heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1. - Funding: This work was supported by Ministry of Health and Welfare (A111425). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Sepsis is defined as a systemic inflammatory response syndrome resulting from a microbial infection. Despite recent advances in antibiotic therapy and intensive care, sepsis is still the most common cause of death in intensive care units [1]. The pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory responses characterized by the excessive accumulation of various proinflammatory mediators, such as tumor necrosis factor (TNF) or interleukin (IL)-1 [2], interferon-gamma (IFN-c) [3], and nitric oxide (NO) [4]. Recently, it has been demonstrated that a ubiquitous protein, high mobility group box 1 (HMGB1), is released by activated macrophages/ monocytes and functions as a late mediator of lethal endotoxemia and sepsis [58]. Heme oxygenase-1 (HO-1), a stress-responsive protein that can be induced by stimulants such as inflammatory cytokines, heat shock, heavy metals and oxidants, degrades heme into three products: Fe++, biliverdin, and carbon monoxide (CO). Biliverdin is subsequently converted into bilirubin by biliverdin reductase. The increased level of Fe2+ stimulates the synthesis of ferritin (an iron-binding compound), which is known to be a cytoprotective antioxidant protein. The HO-1 system has anti-apoptotic, antioxidant and immunomodulatory functions under various conditions [911]. Much attention has been paid to CO and COreleasing molecules (CORMs) due to their ability to attenuate inflammatory responses in many different experimental models [1217]. Recently, we and others demonstrated that the HO-1/ CO system can play a very important role in sepsis through the negative regulation of HMGB1 [1819]. Thus, it seems plausible that the identification of HO-1-inducing agents can lead to the development of therapeutic interventions for inflammatory disorders such as sepsis. Tetrahydroisoquinoline (THI) alkaloids are of special interest due to their pharmacological effects on inflammation and related disorders [2027]. Previously, we reported that THI alkaloids can induce HO-1 expression in many cells, including RAW 264.7 cells [20,2526]. Herein, we report that a novel compound, (S)-6,7dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline (THI-56, Fig. 1), induces HO-1 protein expression in RAW 264.7 cells and in the tissues of cecal ligation and puncture (CLP)induced septic mice. This increased HO-1 expression significantly attenuated the proinflammatory response and reduced the level of circulating HMGB1. Materials and Methods Materials The anti-HMGB1 antibody was purchased from Abcam (Cambridge, MA), and the anti-iNOS antibody was obtained from Transduction Laboratories (Lexington, KY). The anti-bactin, anti-Nrf-2 and anti-heme oxygenase-1 antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) western blotting detection reagent was purchased from Amersham (Buckinghamshire, UK). All other chemicals, including, LPS (Escherichia coli O111:B4) and zinc protoporphyrin IX (ZnPPIX), were purchased from SigmaAldrich (St. Louis, MO). The HMGB1 detection kit was purchased from Shino Test Corp. (Tokyo, Japan). The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) assay kits were obtained from IVD Lab Corp. (Uiwang, Korea). The enzymatic creatinine reagent kit was purchased from Sekisui Medical (Tokyo, Japan). Cell Culture and Stimulation RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were grown in RPMI-1640 medium supplemented with 25 mM N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid (HEPES), 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal calf serum. RAW 264.7 cells were plated at a density of 16107 cells per 100 mm dish. The cells were rinsed with fresh medium and stimulated with LPS (1 mg/ml) in the presence or absence of different concentrations of THI-56 (1, 10 and 25 mM). THI-56 was dissolved in sterile distilled water, and the solution was sterilized using a 0.2 mm filter. Cell Viability Cell viability was assessed colorimetrically using the MTT assay. Cells in the exponential phase were seeded at 16104 cells/well in 24-well plates. After different treatments, 20 ml of 5 mg/ml MTT solution was added to each well (0.1 mg/well), and the plates were incubated for 4 h. The supernatants were aspirated, the formazan crystals in each well were dissolved in 200 ml of dimethyl sulfoxide for 30 min at 37uC, and the optical density at 570 nm was read on a microplate reader (Bio-Rad, Hercules, CA). Assay for NOx Production The NO level was determined by measuring the level of its stable oxidative metabolite, nitrite (NOx), with Griess reagent as described previously [21]. At the end of the incubation, 100 ml of the culture medium was mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). The absorbance at 550 nm was measured, and the nitrite concentration was determined using a calibration curve constructed with sodium nitrite standards. Western Blot Analysis The cytoplasmic/nuclear fractionation was performed using a nuclear/cytosol fractionation kit (Cat # K266-25, BioVision, Mountain view, CA) according to the manufacturers manual. Total protein was obtained using lysis buffer containing 0.5% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl (pH 7.5), and protease inhibitors. The protein concentration of each sample was determined using a BCA protein Figure 4. The anti-inflammatory effect of THI-56 was reversed in HO-1-knockdown cells. Cells were transfected with ssiRNA or siHO-1 and transfection efficiency was checked (A). Then, the cells were treated with THI-56 (25 mM) for 8 h. After incubation, the cells were lysed, and western blot analysis was performed. Cells were transfected with ssiRNA or siHO-1 as described above. Next, cells were stimulated with LPS (1 mg/ml) for 16 h in the presence or absence of THI-56 (25 mM). After incubation, the cells were lysed for iNOS detection (B), and culture medium was collected for HMGB1 analysis (C). The data are presented as the means 6 SEM of three independent experiments. One-way analysis of variance followed by the Newman-Keuls test was used to compare the means of multiple groups (significant compared with the control, **P , 0.01; significant compared with LPS, {{P , 0.01, significant compared with ssiRNA, 1P,0.05, 11P,0.01). doi:10.1371/journal.pone.0076293.g004 assay kit (Pierce, Rockford, IL). To detect iNOS and HO-1, 20 mg aliquots of the total protein were electrophoresed on 8% and 10% polyacrylamide gels, respectively. The electrophoresed proteins were transferred to polyvinylidene difluoride (PVDF) membranes by semidry electrophoretic transfer at 15 V for 6075 min. The PVDF membranes were blocked overnight at 4uC in 5% bovine serum albumin (BSA). The membranes were incubated with primary antibodies diluted 1:500 in Tris-buffered saline/Tween 20 (TBS-T) containing 5% BSA for 2 h and then incubated with the secondary antibody at room temperature for 1 h. Anti-rabbit IgG was used as the secondary antibody (1:5000 dilution in TBST siRNA Technique HO-1 small interfering RNA (siRNA) was purchased from Invitrogen, and siNrf-2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The sequence of the mouse HO-1 siRNA (59 to 39) was as follows: (RNA) UUA CAU GGC AUA AAU UCC CAC UGC C. The siRNAs were transfected into RAW 264.7 cells according to the manufacturers protocol using the SuperFect transfection reagent from QIAGEN. The cells were incubated with the HO-1 and Nrf-2 siRNAs at 100 nM for 16 h in serum-free media. After incubation, the transfected cells were subjected to treatment as described in the figure legends. HMGB1 Analysis HMGB1 was analyzed as described previously [19]. Culture medium samples were briefly centrifuged to remove cellular debris. Equal volumes of samples were then concentrated 40-fold with Amicon Ultra-4-10000 NMWL filter units (Millipore). The centrifugation conditions were a fixed angle (35 degree) and 7500 g for 20 min at 4uC. Then, the concentrated samples were mixed with 2x loading dye and boiled at 95uC for 5 min. The proteins were separated on 12% SDS-polyacrylamide gels and transferred to immunoblot membranes. The membranes were blocked with 5% BSA overnight at 4uC. After blocking, the membranes were washed with Tris-buffered saline/Tween 20 (TBS-T) buffer for 1 h at room temperature. Next, the membranes were incubated with an anti-HMGB1 antibody (Abcam, 1:1000) at 4uC for 16 h. After incubation, the membranes were washed with TBS-T for 1 h at RT and incubated with a goat anti-rabbit IgGHRP secondary antibody (1:5000 dilution in TBST containing 1% BSA). The signals were detected by ECL (Amersham, Piscataway, NJ). HO-1 Activity Measurment of HO-1 activity was done as previously reported [28]. In brief, the harvested cells were conduced to three times of freeze-thawing and then addition to a reaction mixture consisting of phosphate buffer (1 ml final volume, pH 7.4) containing MgCl2 (2 mM), glucose-6-phosphate (2 mM), glucose-6-phosphate dehydrogenease (0.2 units), NADPH (0.8 mM), the substrate hemin (20 mM), and rat liver cytosol as a source of biliverdin reductase. The reaction mixture was incubated at 37uC for 1 h and completed by the addition of 1 ml of chloroform. After vortexed and centrifuged, the bilirubin of chloroform layer was examined by the contrast in absorbance between 464 and 530 nm (e = 40 mM21 cm21). Ethics Statement Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) and were treated ethically. The protocol was approved in advance by the Animal Research Committee of the Gyeongsang National University. Animal Model of Endotoxemia and CLP-induced Sepsis Endotoxemia was induced in BALB/c mice (male, 78 wk, 20 25 g, Koatech, Korea) by the i.p. injection of bacterial endotoxin (LPS, 15 mg/kg). To induce CLP-induced sepsis, BALB/c mice were anesthetized with ketamine (30 mg/kg) and xylazine (6 mg/ kg). Next, a 2-cm midline incision was made to allow exposure of the cecum with the adjoining intestine. The cecum was tightly ligated with a 3.0-silk suture 5.0 mm from the cecal tip and punctured once with a 22-gauge needle. The cecum was then gently squeezed to extrude a small amount of feces from the perforation site and returned to the peritoneal cavity. The laparotomy site was then closed with 4.0 silk. In sham control animals, the cecum was exposed but not ligated or punctured and then returned to the abdominal cavity. Histological Examination Twenty-four hours after CLP, all animals were sacrificed under ketamine (30 mg/kg, i.p.) anesthesia. The kidneys and the superior lobe of the right lung were excised for histopathological examination, fixed with 4% paraformaldehyde in PBS and embedded in paraffin wax. Four-micron-thick sections were cut using a microtome, and the deparaffinized tissue sections were Figure 6. HO-1-dependent organ protective effect of THI-56 in CLP mice. BALB/c mice were treated with THI-56 (10 mg/kg, i.p., n = 5), THI56 (10 mg/kg, i.p)+ZnPPIX (5 mg/kg, i.p., n = 5) or saline (i.p., n = 3) 2 h prior to CLP. After 24 h, the lungs, liver, and kidneys of each mouse were excised and processed as described in the Methods. The lung and kidney tissues were H&E-stained (A) and measured lung injury score as described in the Methods (B). Western blot analysis of iNOS and HO-1 protein expression in lung tissues was performed for the lungs of each group (C). The infiltration of macrophages in the livers of each group was assessed by staining with a macrophage marker (F4/80) (D). As marked by arrow, the histology of kidney demonstrated that CLP caused tubular injury, most prominent in the cortex, which was significantly attenuated by THI-56 in a ZnPPIX-sensitive manner (E). The data are presented as the means 6 SEM. One-way analysis of variance followed by the Newman-Keuls test was used to compare the means of multiple groups (significant compared with the control, *P , 0.05; significant compared with CLP, {P , 0.05, significant compared with THI-56+CLP, 1P,0.05). doi:10.1371/journal.pone.0076293.g006 subjected to hematoxylin and eosin (H&E) staining for histological examination. The lung injury scores were measured in section using neutrophil infiltration, hemorrhage, necrosis, congestion and edema [29]. Each injury was scored according to the following system (0 = normal; 1#25%; 2 = 2550%; 3 = 5075%; 4$75%). The sections were conducted by an investigator blinded to the treatment. To assess the infiltration of macrophages in the liver, deparaffinized liver sections were placed in a solution of 1% H2O2 for 10 min. After washing, the sections were incubated in 5% blocking serum for 1 h at room temperature. The slides were incubated overnight at 4uC in a humidified chamber with antirabbit-F4/80 (1:50; Santa Cruz Biotechnology). After being washed three times with 0.1 M PBS, the sections were incubated for 1 h at room temperature with a secondary biotinylated antibody (1:100). After washing, the sections were incubated in an avidin-biotin-peroxidase complex solution (Vector Laboratories, Burlingame, CA). The sections were developed with 0.05% diaminobenzidine (Sigma-Aldrich) containing 0.05% H2O2, dehydrated, and coverslipped with Permount (Sigma-Aldrich). CLP+THI56 (5 mg/kg)+ZnPP (10 mg/kg) CLP+THI56 (10 mg/kg)+ZnPP (10 mg/kg) Mice were intraperitoneally administered 5 mg/kg or 10 mg/kg THI56 2 h prior to CLP. Twenty four h after CLP, blood was withdrawn under anesthesia by heart puncture from each group of animals. The results are presented as mean 6 SEM (n = 36). Significantly different (*P,0.05, **P,0.01) from sham. Significantly different (#P,0.05, ##P,0.01) from CLP. Significantly different ($P,0.05) from CLP+THI56 (5 mg/kg). Significantly different ({P,0.05) from CLP+THI56 (10 mg/kg). doi:10.1371/journal.pone.0076293.t001 Statistical Evaluation The data are expressed as the mean 6 SEM of the results obtained from the specified number of replicate treatments. Differences between data sets were assessed by one-way analysis of variance followed by the Newman-Keuls tests. The Kaplan-Meier method was used to compare the differences in the mortality rates between groups. P,0.05 and P,0.01 were accepted as statistically significant. THI-56 Significantly Attenuated iNOS/NO and HMGB1 Release in Macrophages Induced by LPS We first assessed the effect of THI-56 on cell viability. Figure 2A shows that there was a significant loss of cell viability at concentrations of 50 mM and above, whereas no effect was found on cell viability up to 25 mM. Thus, for the subsequent experiments, we used concentrations less than 50 mM. We next were interested whether THI-56 can affect the proinflammatory responses of activated macrophages. As shown in Fig. 2B, C, and D, THI-56 significantly inhibited the production of iNOS/NO and the release of HMGB1 induced by LPS, suggesting that THI56 has potent anti-inflammatory effects on LPS-activated macrophages. THI-56 Induces HO-1 Expression Through the Nrf-2 Transcription Factor in Macrophages We next asked whether THI-56 can upregulate the expression of the HO-1 protein. It was clearly demonstrated that HO-1 expression and HO-1 activity is significantly induced by THI-56 treatment in a concentration- and time-dependent manner in macrophages (Fig. 3A, B, and C). Most of the genes encoding phase II detoxifying and antioxidant enzymes have an antioxidant redox element (ARE) sequence in their promoter regions. NE-F2regulated factor 2 (Nrf-2) is an important transcription factor that regulates ARE-driven HO-1 gene expression [30]. We therefore asked whether the HO-1-inducing effect of THI-56 is mediated by Nrf-2. THI-56 induced the translocation of Nrf-2 from the cytosol into the nucleus (Fig. 3D), and the knockdown of Nrf-2 using a specific siRNA abolished the HO-1 expression induced by THI-56 (Fig. 3E). According to these data, we can conclude that THI-56 induces HO-1 expression through Nrf-2 activation. HO-1 siRNA Abrogated the Anti-inflammatory Effect of THI-56 on LPS-activated Macrophages Because THI-56 significantly upregulated HO-1 expression in macrophages, we were interested in whether the HO-1-induction effect has an important role in the THI-56-mediated antiinflammatory effect. As shown in Fig. 4A, transfection efficiency of siHO-1 was more than 80%. THI-56 significantly inhibited the LPS-induced increases in the iNOS (Fig. 4B) and HMGB1 (Fig. 4C) levels in scramble siRNA-transfected cells. However, THI-56 failed to do elicit this response in siHO-1-transfected cells, suggesting that the beneficial anti-inflammatory effects of THI-56 are mediated through HO-1 induction. THI-56 Improves Survival in Experimental Sepsis Models Through HO-1 In light of the ability of THI-56 to attenuate LPS-induced HMGB1 release, we explored its efficacy in an animal model of lethal endotoxemia. The administration of THI-56 at 10 mg/kg significantly improved survival; however, the protective effect THI-56 was completely abrogated by co-treatment with ZnPPIX (an HO-1 inhibitor) (Fig. 5A). Although endotoxemia is a useful animal model to investigate complex cytokine cascades, a more clinically relevant animal model for sepsis should also be used. One well-characterized, standardized animal model of sepsis is the CLP model. It has been shown that the administration of antiHMGB1 rescues animals from septic death in this model [8]. We found that within a few hours after CLP, the animals demonstrated overt signs of illness, including piloerection, hunched posture, and diarrhea. Although these features were also shown in some mice of lower dose (5 mg/kg) of THI-56-treated group, the magnitude and frequency of these characters were much less in higher dose (10 mg/kg) of THI-56-treated mice. We therefore asked whether THI-56 can affect the death rate in this model. As expected, the administration of THI-56 significantly improved survival in the CLP-induced sepsis model (Fig. 5B). This increase in survival caused by THI-56 may be related to reductions of the levels of circulating proinflammatory cytokines, such as TNF-a and IL-1b. When the TNF-a and IL-1b levels were measured 6 h after CLP, increases in the levels of TNF-a and IL1b of approximately 8-fold and 31-fold, respectively, compared with the levels in the sham animals were observed. However, treatment with THI-56 (10 mg/kg, i.p.) 2 h prior to CLP significantly reduced the circulating TNF-a levels (Fig. 5C). The level of IL-1b tended to decrease with THI-56 administration, but this change was not statistically significant (Fig. 5C). Not only TNF-a but also HMGB1 plays a crucial role in lethality during sepsis. To further confirm that the increased survival induced by THI-56 is associated with both the blood HMGB1 levels and HO1-dependent phenomena, the serum HMGB1 levels in CLP-mice were compared in the presence and absence of either THI-56 or ZnPPIX. The elevated serum HMGB1 level in the CLP group was significantly reduced by THI-56 according to the western blot analysis. However, THI-56 failed to reduce the serum HMGB1 level in ZnPPIX-treated mice (Fig. 5D). More clear results were obtained for the circulating concentration of HMGB1. As shown in Fig. 5E, the circulating HMGB1 levels were clearly reduced by the administration of THI-56 to CLP-induced septic mice in a concentration-dependent manner, and this reduction was inhibited by ZnPPIX. In addition, we measured the changes in the blood levels of HMGB1 after CLP. As shown in Fig. 5F, the HMGB1 concentration started to increase after 12 h and continued to rise until 24 h after CLP. We therefore were curious whether the blood HMGB1 levels can be reduced by THI-56 when administered after CLP. Mice were subjected to CLP and then treated with THI-56 (10 mg/kg, i.p) at 0, 8, and 12 h after CLP. At 24 h after the CLP operation, blood was collected by cardiac puncture from ketamine-anesthetized (30 mg/kg, i.m.) mice. Fig. 5G clearly shows that the administration of THI-56 after CLP significantly reduced the circulating HMGB1 level. Even when given 12 h after CLP, THI-56 still significantly reduced the blood HMGB1 levels in CLP septic mice. THI-56 Protects Against Multiple Organ Failure in Sepsis Models in a Dose-dependent Manner Organ damage is one of leading causes of deaths in patients with sepsis. Thus, we investigated whether the protection against lung injury by THI-56 in CLP-induced septic mice was related to HO1 activity. As shown in Fig. 6A and B, histopathological changes and lung injury were evident in lung and kidney tissues from mice with CLP-induced sepsis. It is clear that THI-56 reduced lung injury by reducing iNOS expression and upregulating HO-1 expression in lung tissues (Fig. 6C). In addition, the infiltration of macrophages into the liver also declined by THI-56 in a concentration-dependent and ZnPPIX-sensitive manner (Fig. 6D). Histopathological changes of kidney injury were clearly shown in CLP mice, whereas THI-56 reduced this injury in a ZnPPIXsensitive manner (Fig. 6E). The data in Table 1 show that CLP increased levels of ALT, AST, BUN, and creatinine and that the administration of THI-56 significantly reduced the levels of markers of liver and kidney damage in a concentration-dependent manner. The proinflammatory mediators expressed during sepsis can be roughly divided into two groups. The first group is the early proinflammatory mediators, such as TNF-a and IL-1b, which are induced within hours after the induction of sepsis. Initial experimental observations have suggested that the inhibition of these cytokines with neutralizing antibodies is beneficial in the treatment of sepsis [31]. However, clinical trials and more elegant animal studies have shown that the neutralization of early cytokines does not protect against sepsis [32]. Currently, it is suggested that early cytokines may be even protective against infection [32]. The second group of proinflammatory mediators is late-phase cytokines such as HMGB1, which are induced within 1624 h after the initiation of sepsis. One interesting point is that increases in the late-phase cytokine HMGB1 are tightly associated with increased mortality in animal models of sepsis [7], whereas the administration of neutralizing antibodies to HMGB1 protects animals from death in the LPS and CLP models [78]. It has been proposed that the attenuation of the circulating levels of HMGB1 has a potential therapeutic uses for the treatment of sepsis. We showed that THI-56 significantly inhibits HMGB1 release, both in vitro and in in vivo sepsis models, indicating that THI-56 is a potential candidate for the treatment of sepsis. HMGB1 is predominantly localized in the nucleus in resting macrophages. However, HMGB1 is translocated from the nucleus to the cytosol and subsequently to the extracellular milieu in response to different stimuli [5]. It has been suggested that several signaling proteins, such as C-jun N-terminal kinase (JNK), classical protein kinase C proteins (PKCs) and calcium/calmodulindependent protein kinase (CaMK) IV, are involved in HMGB1 shuttling in response to LPS stimulation in macrophages [3335]. Moreover, it has also been claimed that the IFN-b/Janus kinase-2 (JAK-2)/signal transduction and activator of transcription-1 (STAT-1) signaling cascade is responsible for HMGB1 release in LPS-activated macrophages [36]. Interestingly, in our previous study, we showed that another THI alkaloid (CKD712) differentially regulates iNOS and COX-2 in LPS-treated cells through the negative regulation of the JAK-2/STAT-1 pathway [26]. It is possible that THI-56 can also interfere with this cascade. However, further study is needed to prove or disprove this hypothesis. In this study, we demonstrated that the inhibitory effect of THI-56 on HMGB1 release is mediated through HO-1 induction. These data strongly support our previous observations that the pharmacological or genetic upregulation of HO-1 greatly inhibits LPS-induced HMGB1 release both in vitro and in vivo [19]. In addition, the HMGB1 level is higher in HO-12/2 mice than in HO-1+/+ mice when animals are subjected to either LPS injection or CLP-induced polymicrobial sepsis, indicating that HO-1 negatively regulates HMGB1 under septic conditions [1819]. The present results indicate that the specific knockdown of the HO-1 gene using HO-1 siRNA greatly reversed the inhibitory effect of THI-56 on iNOS expression and HMGB1 release in LPSstimulated macrophages. Thus, it might be important to provide evidence that the anti-inflammatory effect of THI-56 in vivo is mediated through HO-1. We therefore used the CLP-induced mouse model of sepsis. It is clear that the anti-inflammatory effects of THI-56 in vivo were dependent on HO-1 activity because the protective effect against organ damage was reversed by the administration of an HO-1 inhibitor. Another important finding is that the blood HMGB1 levels were significantly reduced by THI56 in CLP-mice, and this effect was also significantly reversed by an HO-1 inhibitor, ZnPPIX, providing further evidence that HO1 activity negatively regulates HMGB1 under systemic inflammatory conditions. Although the prevention of some of the complications of sepsis is important, whether THI-56 is therapeutically effective when administered after CLP is more clinically relevant. We found that even when THI-56 was administered 12 h after CLP, it still reduced the circulating HMGB1 levels, suggesting that this drug can potentially be beneficial in treating sepsis. THI-56 induced the translocation of Nrf-2 from the cytosol to the nucleus in a concentration-dependent manner and the deletion of Nrf-2 significantly diminished HO-1 expression in RAW 264.7 cells; therefore, the anti-inflammatory effects of THI56 are mediated through the up-regulation of HO-1 expression via Nrf2 activation. In summary, this study demonstrated that THI-56 improves survival in lethal endotoxemia and CLP-induced sepsis models. THI-56 significantly inhibited LPS-induced iNOS/NO and HMGB1 release in macrophages and reduced organ damage in CLP-induced septic mice. The beneficial effects of THI-56 were dependent on its ability to induce HO-1 expression through the activation of the Nrf-2 transcription factor. Because increased HMGB1 expression is tightly associated with increased mortality in animal models of sepsis, it should be noted that the administration of THI-56 even 12 h after CLP decreased the circulating HMGB1 levels in CLP mice. Therefore, THI-56 may be useful in the treatment of sepsis. Conceived and designed the experiments: KCC. Performed the experiments: HJJ EJP KT YMK. Analyzed the data: SWP HJK. Contributed reagents/materials/analysis tools: KCC JHL. Wrote the paper: KCC KT. 1. Oberholzer A , Oberholzer C , Moldawer LL ( 2001 ) Sepsis syndromes: understanding the role of innate and acquired immunity . Shock 16 ( 2 ): 83 - 96 . 2. Dinarello CA , Thompson RC ( 1991 ) Blocking IL-1: interleukin 1 receptor antagonist in vivo and in vitro . Immunol Today 12 ( 11 ): 404 - 410 . 3. Heinzel FP ( 1990 ) The role of IFN-gamma in the pathology of experimental endotoxemia . J Immunol 145 ( 9 ): 2920 - 2924 . 4. Dinapoli MR , Calderon CL , Lopez DM ( 1996 ) The altered tumoricidal capacity of macrophages isolated from tumor-bearing mice is related to reduce expression of the inducible nitric oxide synthase gene . J Exp Med 183 ( 4 ): 1323 - 1329 . 5. Lotze MT , Tracey KJ ( 2005 ) High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal . Nat Rev Immunol 5 ( 4 ): 331 - 342 . 6. Qin S , Wang H , Yuan R , Li H , Ochani M , et al. ( 2006 ) Role of HMGB1 in apoptosis-mediated sepsis lethality . J Exp Med 203 ( 7 ): 1637 - 642 . 7. Wang H , Bloom O , Zhang M , Vishnubhakat JM , Ombrellino M , et al. ( 1999 ) HMG-1 as a late mediator of endotoxin lethality in mice . Science 285 ( 5425 ): 248 - 251 . 8. Yang H , Ochani M , Li J , Qiang X , Tanovic M , et al. ( 2004 ) Reversing established sepsis with antagonists of endogenous high-mobility group box 1 . Proc Natl Acad Sci U S A 101 ( 1 ): 296 - 301 . 9. Ryter SW , Choi AM ( 2009 ) Heme oxygenase-1/carbon monoxide: from metabolism to molecular therapy . Am J Respir Cell Mol Biol 41 ( 3 ): 251 - 260 . 10. Morse D , Choi AM ( 2002 ) Heme oxygenase-1: the ''emerging molecule'' has arrived . Am J Respir Cell Mol Biol 27 ( 1 ): 8 - 16 . 11. Horvath I , MacNee W , Kelly FJ , Dekhuijzen PN , Phillips M , et al. ( 2001 ) ''Haemoxygenase-1 induction and exhaled markers of oxidative stress in lung diseases'', summary of the ERS Research Seminar in Budapest, Hungary, September, 1999 . Eur Respir J 18 ( 2 ): 420 - 430 . 12. Nakahira K , Kim HP , Geng XH , Nakao A , Wang X , et al. ( 2006 ) Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROSinduced trafficking of TLRs to lipid rafts . J Exp Med 203 ( 10 ): 2377 - 2389 . 13. Sato K , Balla J , Otterbein L , Smith RN , Brouard S , et al. ( 2001 ) Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-torat cardiac transplants . J Immunol 166 ( 6 ): 4185 - 4194 . 14. Remy S , Blancou P , Tesson L , Tardif V , Brion R , et al. ( 2009 ) Carbon monoxide inhibits TLR-induced dendritic cell immunogenicity . J Immunol 182 ( 4 ): 1877 - 1884 . 15. Bani-Hani MG , Greenstein D , Mann BE , Green CJ , Motterlini R ( 2006 ) A carbon monoxide-releasing molecule (CORM-3) attenuates lipopolysaccharideand interferon-gamma-induced inflammation in microglia . Pharmacol Rep 58 Suppl : 132 - 44 . 16. Sawle P , Foresti R , Mann BE , Johnson TR , Green CJ , et al. ( 2005 ) Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages . Br J Pharmacol 145 ( 6 ): 800 - 810 . 17. Tsoyi K , Ha YM , Kim YM , Lee YS , Kim HJ , et al. ( 2009 ) Activation of PPARgamma by carbon monoxide from CORM-2 leads to the inhibition of iNOS but not COX-2 expression in LPS-stimulated macrophages . Inflammation 32 ( 6 ): 364 - 371 . 18. Takamiya R , Hung CC , Hall SR , Fukunaga K , Nagaishi T , et al. ( 2009 ) Highmobility group box 1 contributes to lethality of endotoxemia in heme oxygenase1-deficient mice . Am J Respir Cell Mol Biol 41 ( 2 ): 129 - 35 . 19. Tsoyi K , Lee TY , Lee YS , Kim HJ , Seo HG , et al. ( 2009 ) Heme-oxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo . Mol Pharmacol 76 ( 1 ): 173 - 82 . 20. Heo JM , Kim HJ , Ha YM , Park MK , Kang YJ , et al. ( 2007 ) YS 51, 1 - ( betanaphtylmethyl)-6 , 7 - dihydroxy - 1 , 2 , 3 , 4 ,-tetrahydroisoquinoline, protects endothelial cells against hydrogen peroxide-induced injury via carbon monoxide derived from heme oxygenase-1 . Biochem Pharmacol 74 ( 9 ): 1361 - 70 . 21. Kang YJ , Koo EB , Lee YS , Yun-Choi HS , Chang KC ( 1999 ) Prevention of the expression of inducible nitric oxide synthase by a novel positive inotropic agent, YS 49, in rat vascular smooth muscle and RAW 264.7 macrophages . Br J Pharmacol 128 ( 2 ): 357 - 64 . 22. Kang YJ , Seo SJ , Yun-Choi HS , Lee DH , Kim YM , et al. ( 2002 ) A synthetic isoquinoline alkaloid, 1-(beta-naphthylmethyl) -6 , 7 - dihydroxy - 1 , 2 , 3 , 4 - tetrahydroisoquinoline (YS 51) , reduces inducible nitric oxide synthase expression and improves survival in a rodent model of endotoxic shock . J Pharmacol Exp Ther 301 ( 2 ): 561 - 7 . 23. Kim HJ , Tsoyi K , Heo JM , Kang YJ , Park MK , et al. ( 2007 ) Regulation of lipopolysaccharide-induced inducible nitric-oxide synthase expression through the nuclear factor-kappaB pathway and interferon-beta/tyrosine kinase 2/Janus tyrosine kinase 2-signal transducer and activator of transcription-1 signaling cascades by 2-naphthylethyl- 6 , 7 - dihydroxy - 1 , 2 , 3 , 4 -tetrahydroisoquinoline ( THI 53), a new synthetic isoquinoline alkaloid . J Pharmacol Exp Ther 320 ( 2 ): 782 - 789 . 24. Park JE , Kang YJ , Park MK , Lee YS , Kim HJ , et al. ( 2006 ) Enantiomers of higenamine inhibit LPS-induced iNOS in a macrophage cell line and improve the survival of mice with experimental endotoxemia . Int Immunopharmacol 6 ( 2 ): 226 - 233 . 25. Sun JJ , Kim HJ , Seo HG , Lee JH , Yun-Choi HS , et al. ( 2008 ) YS 49, 1 - ( alphanaphtylmethyl)-6 , 7 - dihydroxy - 1 , 2 , 3 , 4 -tetrahydroisoquinoline, regulates angiotensin II-stimulated ROS production, JNK phosphorylation and vascular smooth muscle cell proliferation via the induction of heme oxygenase-1 . Life Sci 82 ( 11 - 12 ): 600 - 607 . 26. Tsoyi K , Kim HJ , Shin JS , Kim DH , Cho HJ , et al. ( 2008 ) HO-1 and JAK-2/ STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride . Cell Signal 20 ( 10 ): 1839 - 1847 . 27. Tsoyi K , Kim WS , Kim YM , Kim HJ , Seo HG , et al. ( 2009 ) Upregulation of PTEN by CKD712, a synthetic tetrahydroisoquinoline alkaloid, selectively inhibits lipopolysaccharide-induced VCAM-1 but not ICAM-1 expression in human endothelial cells . Atherosclerosis 207 ( 2 ): 412 - 419 . 28. Park EJ , Kim YM , Park SW , Kim HJ , Lee JH , et al ( 2013 ) Induction of HO-1 through p38 MAPK/Nrf2 signaling pathway by ethanol extract of Inula helenium L. reduces inflammation in LPS-activated RAW 264.7 cells and CLPinduced septicmice . Food Chem Toxicol . 55 : 386 - 395 . 29. Alam J , Stewart D , Touchard C , Boinapally S , Choi AM , et al. ( 1999 ) Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene . J Biol Chem 274 ( 37 ): 26071 - 26078 . 30. Su X , Wang L , Song Y , Bai C ( 2004 ) Inhibition of inflammatory responses by ambroxol, a mucolytic agent, in a murine model of acute lung injury induced by lipopolysaccharide . Intensive Care Med 30 : 133 . 31. Beutler B , Milsark IW , Cerami AC ( 2008 ) Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin . Science , 1985 , 229 ( 4716 ): 869 - 871 . Classical article . J Immunol 181 ( 1 ): 7 - 9 . 32. Eskandari MK , Bolgos G , Miller C , Nguyen DT , DeForge LE , et al. ( 1999 ) Antitumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia . J Immunol 148 ( 9 ): 2724 - 2730 . 33. Oh YJ , Youn JH , Ji Y , Lee SE , Lim KJ , et al. ( 2009 ) HMGB1 is phosphorylated by classical protein kinase C and is secreted by a calcium-dependent mechanism . J Immunol 182 ( 9 ): 5800 - 5809 . 34. Zhang X , Wheeler D , Tang Y , Guo L , Shapiro RA , et al. ( 2008 ) Calcium/ calmodulin-dependent protein kinase (CaMK) IV mediates nucleocytoplasmic shuttling and release of HMGB1 during lipopolysaccharide stimulation of macrophages . J Immunol 181 ( 7 ): 5015 - 5023 . 35. Jiang W , Pisetsky DS ( 2006 ) The role of IFN-alpha and nitric oxide in the release of HMGB1 by RAW 264.7 cells stimulated with polyinosinic-polycytidylic acid or lipopolysaccharide . J Immunol 177 ( 5 ): 3337 - 3343 . 36. Kim JH , Kim SJ , Lee IS , Lee MS , Uematsu S , et al. ( 2009 ) Bacterial endotoxin induces the release of high mobility group box 1 via the IFN-beta signaling pathway . J Immunol 182 ( 4 ): 2458 - 2466 .


This is a preview of a remote PDF: http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371/journal.pone.0076293&representation=PDF

Eun Jung Park, Hwa Jin Jang, Konstantin Tsoyi, Young Min Kim, Sang Won Park, Hye Jung Kim, Jae Heun Lee, Ki Churl Chang. The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice, PLOS ONE, 2013, DOI: 10.1371/journal.pone.0076293