Author Affiliations: Department of Infectious Diseases (Yan BZ, Yang BS and Wang XR), Department of Gastroenterology and Hepatology (Li H, Pei FH and Liu BR) and Department of Laboratory Diagnosis (Zhang YF), The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China; Department of Histology, The First Hospital of Harbin City, Harbin 150001, China (Zhu AC)

Corresponding Author: Bing-Rong Liu, MD, Department of Gastroenterology and Hepatology, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China (Tel/Fax: +86-451-86605980; Email: bingrongliu@qq.com)

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60044-3

Published online December 31, 2015.

 

 

Acknowledgements: Authors are grateful to the assistance provided by Dr. Ma Ning and other colleagues at Translational Medicine Center of Northern China (Harbin Medical University).

Contributors: YBZ, YBS and LBR designed the study. YBZ wrote the manuscript. YBZ, LH, ZYF, PFH, ZAC and WXR performed most of the experiments. YBZ and YBS collected and analyzed the data. All authors contributed to the interpretation of the study and future drafts. LBR is the guarantor.

Funding: This study was supported by a grant from the Science and Technology Research Foundation of Educational Committee, Heilongjiang Province, China (12531294).

Ethical approval: This study was approved by the Ethics Committee of Harbin Medical University (HMUIRB20150003).

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Acute liver failure (ALF) is a severe and life-threatening clinical syndrome resulting in a high mortality and extremely poor prognosis. Recently, a water-soluble CO-releasing molecule (CORM-3) has been shown to have anti-inflammatory effect. The present study was to investigate the effect of CORM-3 on ALF and elucidate its underlying mechanism.

 

METHODS: ALF was induced by a combination of LPS/D-GalN in mice which were treated with CORM-3 or inactive CORM-3 (iCORM-3). The efficacy of CORM-3 was evaluated based on survival, liver histopathology, serum aminotransferase activities (ALT and AST) and total bilirubin (TBiL). Serum levels of inflammatory cytokines (TNF-α, IL-6, IL-1β and IL-10) and liver immunohistochemistry of NF-κB-p65 were determined; the expression of inflammatory mediators such as iNOS, COX-2 and TLR4 was measured using Western blotting.

 

RESULTS: The pretreatment with CORM-3 significantly improved the liver histology and the survival rate of mice compared with the controls; CORM-3 also decreased the levels of ALT, AST and TBiL. Furthermore, CORM-3 significantly inhibited the increased concentration of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and increased the anti-inflammatory cytokine (IL-10) productions in ALF mice. Moreover, CORM-3 significantly reduced the increased expression of iNOS and TLR4 in liver tissues and inhibited the nuclear expression of NF-κB-p65. CORM-3 had no effect on the increased expression of COX-2 in the ALF mice. An iCORM-3 failed to prevent acute liver damage induced by LPS/D-GalN.

 

CONCLUSION: These findings provided evidence that CORM-3 may offer a novel alternative approach for the management of ALF through anti-inflammatory functions.

 

KEY WORDS: acute liver failure; CO-releasing molecule-3; cytokines; inflammation

Acute liver failure (ALF) is defined as severe liver injury characterized by increased levels of liver enzymes, hepatic encephalopathy, severe coagulopathy and jaundice.^{[1, 2]} ALF results in extremely high mortality and poor prognosis, despite significant advances in liver transplantation and liver support systems.^[3,4] Although the treatment strategies for ALF have been extensively studied in recent years, there are still no validated therapeutic approaches.^{[5, 6]} Therefore, the study of effective therapies for ALF is of great importance.

 

Lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced acute hepatic damage has been extensively used as an experimental animal model that imitates the pathological processes of human ALF, which is characterized by widespread and massive necrosis of the liver.^{[7, 8]} Increasing evidences indicate that inflammatory responses play a vital role in the pathogenesis of ALF.^[9] The release of hepatic and circulating inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10), is associated with the development and prognosis of ALF.^{[10, 11]} Additionally, LPS can stimulate the production of inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which participate in the inflammatory response through the NF-κB signaling pathway.^[12] Among the Toll-like receptors (TLRs) family, TLR4 is involved in the pathogenesis of ALF.^[13] The inhibition of TLR4 over-expression is also regarded as a potential therapeutic target in LPS/D-GalN-induced ALF.^[14] Theoretically, the regulation of inflammatory mediators is considered to be a feasible strategy for the management of LPS/D-GalN-induced ALF.^[15]

 

As an important signaling gas molecule, carbon monoxide (CO) was found to have potent cytoprotective effects including anti-inflammatory, anti-apoptotic, anti-proliferative and vasodilator effects.^[16-18] However, the therapeutic use of gaseous CO has been limited due to its toxic properties, which result in an increased concentration of carboxyhemoglobin (COHb). CO-releasing molecules (CORMs) have recently been synthesized, and they facilitate the delivery of CO to biological systems without increase of COHb levels.^[19] Therefore, CORMs are regarded as very valuable tools to assess CO bioactivity and give rise to new drug candidates for the experimental use of gaseous CO. Among the different classes of CORMs, a water-soluble CORM-3 has been characterized and successfully tested in various animal models of inflammation.^{[20, 21]}

 

However, it is unclear whether CORM-3 is effective in the treatment of ALF. The present study was to investigate the therapeutic effect of CORM-3 on ALF and its possible mechanisms.

 

 

Pathogen free C57BL/6 male mice (8 to 10 weeks old) were obtained from the Experimental Animal Center of Harbin Medical University. The mice were caged in a room with standard conditions including light (12-hour light/dark cycle), temperature (22±2 ��) and humidity (55%±5%). All animals had free access to a standard laboratory diet and water. The mice received humane care in accordance with the guidelines of the Institutional Research Board of Harbin Medical University for the use of experimental animals (HMUIRB20150003).

 

LPS (Escherichia coli 011:B4), D-GalN and CORM-3 were purchased from Sigma-Aldrich Chemical Co. LLC (St. Louis, MO, USA); mouse ELISA kits (TNF-α, IL-6, IL-1β and IL-10) were from Blue Gene (Shanghai, China); alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBiL) detection kits were from Siemens Healthcare Diagnostic, Inc. (Newark, USA); rabbit anti-mouse polyclonal antibodies against COX-2, iNOS, TLR4, NF-κB-p65 and β-actin were from Abcam, Inc. (Piscataway, USA); MaxVision™ HRP-Polymer anti-mouse IHC kit (KIT-5030) and MAX007™ (DAB) were from Maixin Biotech, Co., Ltd. (Fuzhou, China).

 

Mice were simultaneously injected intraperitoneally with LPS (100 µg/kg) and D-GalN (800 mg/kg) dissolved in phosphate-buffered saline (PBS). The animals were randomly divided into four groups with ten mice in each group: (1) Control group: mice were injected with the same volume of sterile saline alone; (2) LPS/D-GalN group: mice were given only LPS/D-GalN; (3) iCORM-3+LPS/D-GalN group: mice were intraperitoneally administered iCORM-3 (10 mg/kg) dissolved in sterile PBS 30 minutes prior to LPS/D-GalN injection; (4) CORM-3+LPS/D-GalN group: mice were intraperitoneally administered CORM-3 (10 mg/kg) as described above. CORM-3 was dissolved fresh in distilled water on the day of experiment, and was stored at -20 �� prior to use. iCORM-3 (inactivated CORM-3) was made by incubating fresh CORM-3 dissolved in PBS and leaving it for 24 hours at room temperature in bubbling with nitrogen to displace the CO.^[22] The mice were sacrificed by decapitation at 6 hours after LPS/D-GalN administration. Blood and liver samples were then quickly collected and frozen at -80 �� for biochemical and histological analyses. The survival of the mice was assessed using an additional sixty mice grouped as described above 48 hours after LPS/D-GalN administration.

 

Collected blood samples were centrifuged at 1000×g for 15 minutes to obtain serum samples, which were hemolysis-free and stored at -80 �� before use. The serum levels of ALT, AST and TBiL were measured using an automatic chemistry analyser (Dimension®EXL™ with LM, SIEMENS).

 

The collected liver tissues were rinsed gently with PBS and preserved in 10% paraformaldehyde. The samples were then dehydrated and embedded in paraffin. The samples were sectioned (5 µm thick) and stained with hematoxylin and eosin (HE) and analysed under a Bio Imaging Navigator microscope (Olympus FSX100, Tokyo, Japan). Histological changes including necrosis, hemorrhage and inflammation were graded by five grades on a severity scale of “–” to “+++” (–: no change; ±: slight change; +: mild change; ++: moderate change; +++: strong change) according to the literature.^[5]

 

Collected blood samples were allowed to clot for 2 hours at room temperature and centrifuged at approximately 1000×g for 15 minutes. The concentrations of inflammatory cytokines (TNF-α, IL-6, IL-1β and IL-10) were quantified using a mouse ELISA kit according to the manufacturer’s instruction. The sensitivity of the kit was 1.0 pg/mL.

 

For immunohistochemical analysis, the following steps were performed to examine NF-κB-p65 in the liver tissue according to the instructions of MaxVision™ kit. Briefly, liver specimens were incubated with rabbit anti-mouse NF-κB-p65 antibody diluted 1:250 in PBS. After washing, the sections were incubated with goat anti-rabbit IgG antibody labeled with horse radish peroxidase (HRP) for 15 minutes. After the coloration with DAB substrate, all samples were visualized using a Bio Imaging Navigator microscope. For quantification of NF-κB-p65, NF-κB-p65-positive hepatocellular nuclei were counted in ten consecutive fields at magnification ×400 and given as cells per mm².

 

The total protein was extracted from the mouse liver tissues using a protein extract kit according to the manufacturer’s instructions. Tissues were lysed with RIPA lysis buffer (Solarbio, Beijing, China), and the protein concentrations were measured using a BCA protein assay kit. Briefly, 40 µg protein extracts were fractionated on a 10% or 12% SDS-PAGE gel. After electrophoretic separation, proteins were transferred to polyvinylidene fluoride membranes that were blocked with 5% fat-free milk dissolved in Tris-buffered saline containing 0.05% Tween (TBST) at room temperature for one hour. The membranes were incubated with the primary antibodies overnight at 4 �� followed by washing with TBST three times for five minutes and were treated with fluorescent secondary antibodies (1:10 000) for one hour. Primary antibodies against COX-2, iNOS and TLR4 (rabbit polyclonal antibodies) were used, and signals were standardized to β-actin. The image density of specific bands was quantified and was scanned using the Odyssey Infrared Imaging System (Gene Company Limited, Hong Kong, China).

 

Statistical analysis was performed using SPSS 13.0. Data were expressed as mean±standard deviation, and the differences between groups were assessed using one-way ANOVA followed by Tukey’s test. Survival rates were compared using the Kaplan-Meier method and the log-rank test. A P<0.05 was considered statistically significant.

 

 

Mice with ALF had a high death rate; therefore, we investigated CORM-3 in lowering LPS/D-GalN-induced mortality. The survival rate of mice was monitored for 48 hours after injection of LPS/D-GalN (Fig. 1). All mice survived in the control group. Mice without CORM-3 died at 8 hours, and the mortality rate was 100% (15/15) within 48 hours after injection of LPS/D-GalN. However, pretreatment with CORM-3 significantly decreased the mortality rate of mice induced by LPS/D-GalN, and the survival rate was 73.3% (11/15) at 48 hours (P<0.05). The survival rate in the iCORM-3-treated group was not different from that in the LPS/D-GalN-treated group (P>0.05).

 

To evaluate the severity of acute liver injury, we assessed the activities of serum aminotransferase and the level of serum TBiL (Fig. 2). The marked elevation of serum levels of ALT, AST and TBiL observed in the LPS/D-GalN group, which reached 2987.70±529.25 U/L, 3432.90±629.14 U/L and 48.62±4.85 µmol/L, respectively, indicated the success of the model creation. Compared to the LPS/D-GalN-treated mice, the increased levels of ALT, AST and TBiL were significantly inhibited after pretreatment with CORM-3 (P<0.05) to 1231.30±94.98 U/L, 1288.00±116.22 U/L and 13.39±1.79 µmol/L, respectively. iCORM-3 had no effect on the levels of ALT, AST and TBiL in the LPS/D-GalN-treated mice (P>0.05).

 

In the control mice, histological observation showed normal liver lobular architecture and cell structure (Fig. 3A). The histological appearance of liver tissue in mice challenged with LPS/D-GalN revealed marked morphological changes, including destruction of hepatic architecture, massive necrosis and inflammatory cells infiltration (Fig. 3B). The severity of liver injuries induced by LPS/D-GalN exposure was significantly ameliorated in the mice treated with CORM-3 but not in those treated with iCORM-3 (Fig. 3D). There were no significant differences between the LPS/D-GalN group and LPS/D-GalN+iCORM-3 group (Fig. 3C). The histological grading of LPS/D-GalN-induced ALF was significantly reduced in the CORM-3-treated mice (P<0.05) (Table).

 

To assess the underlying mechanisms, the regulatory effects of CORM-3 on the levels of multiple inflammatory cytokines were investigated by ELISA (Fig. 4). After injection of LPS/D-GalN, the serum levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were significantly elevated compared with those of the controls (P<0.05). Injection of CORM-3 significantly decreased the elevation of TNF-α, IL-1β and IL-6 levels in the LPS/D-GalN-treated mice (P<0.05). Whereas the expression of IL-10 in the serum was significantly increased in the LPS/D-GalN group, pretreatment of CORM-3 significantly increased the level of serum IL-10 (P<0.05). However, there were no significant differences in the levels of serum cytokines between the LPS/D-GalN-treated group and the LPS/D-GalN+iCORM-3-treated group (P>0.05).

 

To further investigate the potential anti-inflammatory mechanism of CORM-3, the expression of NF-κB-p65 in the liver was evaluated by immunohistochemistry. In liver tissues from the control group, the expression of NF-κB-p65 was negative in the nuclei of hepatocytes (Fig. 5A). LPS/D-GalN significantly activated NF-κB which was translocated from the cytosol into the nuclei, where the expression of NF-κB-p65 was strongly positive (Fig. 5B). In contrast, the expression of NF-κB-p65 was weakly positive in the nuclei of hepatocytes in the CORM-3-treated group (Fig. 5D). However, the expression of NF-κB-p65 in the nuclei of hepatocytes in the iCORM-3-treated group was as strongly positive as in the LPS/D-GalN group (Fig. 5C). As shown in Fig.5E, the number of NF-κB-p65-positive cells was significantly reduced after the pretreatment of CORM-3 (P<0.05).

 

To further assess the regulatory effects of CORM-3 on inflammation mediation, the expression of COX-2, iNOS and TLR4 proteins in the liver was measured using Western blotting (Fig. 6). After injection of LPS/D-GalN, the expression of iNOS, TLR4 and COX-2 proteins in the liver was significantly increased compared with the controls (P<0.05). Compared to the LPS/D-GalN group, the injection of CORM-3 significantly reduced iNOS and TLR4 protein expressions (P<0.05). CORM-3 had no effect on COX-2. iCORM-3 did not affect the expression of iNOS, TLR4 and COX-2 in the LPS/D-GalN-treated mice (P>0.05).

 

 

Recent studies^{[23, 24]} have shown that exogenous CO gas has a protective effect against acute liver damage in mice, However, CO gas as a therapeutic tool has potential risk in clinical application due to its toxic properties.^[25] Thus, it is necessary to develop a method suitable for therapeutic application of CO. Compared with gaseous CO, CORMs are considered to have the capability of delivering controlled quantities of CO gas, which is safer at its therapeutic dose without elevating the serum levels of COHb.^[26] Studies^[27-29] have shown that CORM-3, a water-soluble CORM, has therapeutic effects in experimental models depending on its striking anti-inflammatory properties. Although the anti-inflammatory effect of CORM-3 has been described, no research has been carried out to directly compare the effects of CORM-3 on ALF. Thus we suggest that controlled CO delivery through CORM-3 may offer a novel alternative approach to the treatment of ALF in the future. This study hence was designed to evaluate the effect of CORM-3 on acute liver failure and to elucidate its possible mechanisms.

 

LPS/D-GalN-induced ALF is generally used as an experimental model for screening of protective agents for the liver and studying relevant mechanisms.^{[8, 30]} Furthermore, exposure to LPS and D-GalN results in histological and biochemical changes closely resembling human ALF.^{[7, 8]} In the present study, we adopted an animal model of ALF induced by LPS/D-GalN to observe the potential protective effects of CORM-3. In this experiment, severe liver injuries caused by LPS/D-GalN were observed, including classical histological changes and a rapid loss of liver function, which are consistent with a previous report.^[6] In addition, some of the mice died quickly in 8 hours, but most of them died within 48 hours after injection of LPS/D-GalN. Our results showed that the survival rate of the mice was significantly improved in the presence of CORM-3 but not the iCORM-3. Histological analysis of the liver exposed to LPS/D-GalN demonstrated severe injuries with massive hepatic necrosis and inflammatory cell infiltration compared with the control group. The severity of LPS/D-GalN-induced liver injury was alleviated in the mice treated with CORM-3 but was not in those treated with iCORM-3. In parallel, histological grading was significantly reduced by CORM-3 after injection of LPS/D-GalN. The levels of serum aminotransferase and total bilirubin were important indicators of the severity of ALF. CORM-3, not iCORM-3, significantly controlled the elevation of serum ALT, AST and TBiL in ALF, indicating that CORM-3 is effective in preventing liver injury through CO release. This is also evidenced by the improvement of survival rate, liver function and histology.

 

It is well documented that the activation of the inflammatory cytokines cascade plays a crucial role in the development of ALF, which is concomitant with a massive release of inflammatory cytokines including TNF-α, IL-6, IL-1β and IL-10.^[31-33] In contrast to other pro-inflammatory cytokines, IL-10 is regarded as a major anti-inflammatory cytokine through the inhibition of TNF-α production.^{[34, 35]} Therefore, anti-inflammatory responses are thought to be a target for the treatment and prevention of ALF.^[36] To investigate the underlying mechanisms of CORM-3-mediated protective effects, we determined whether CORM-3 can provide significant anti-inflammatory action against ALF. We observed that CORM-3 not only reduces the overproduction of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) but also increases the production of anti-inflammatory cytokine (IL-10). There were no significant differences in the regulation of inflammatory cytokines in the iCORM-3-treated mice compared with the LPS/D-GalN-treated mice. To further verify the underlying anti-inflammatory mechanisms, we observed the effects of CORM-3 on the expression of inflammatory mediators including COX-2, iNOS and TLR4. Previous studies^{[12, 37]} proved that iNOS and COX-2 play a crucial role in the pathological and inflammatory process of acute liver failure. Additionally, TLR4 signaling plays a vital role in the initiation of the inflammatory response in ALF induced by LPS/D-GalN.^{[14, 38]} The present study showed that pretreatment with CORM-3 significantly reduced the increased expression of iNOS and TLR4 proteins after injection of LPS/D-GalN but did not significantly reduce the expression of COX-2 protein.

 

Increasing evidence confirmed that the induction of most pro-inflammatory cytokines and the production of inflammatory mediators mainly depend upon the activation of NF-κB in vitro and in vivo.^{[39, 40]} Once activated, NF-κB translocates from the cytosol into the nucleus, where it causes the transcription of multiple inflammatory cytokines and mediators.^[41] The activation of NF-κB is considered to play an important role in LPS/D-GalN-induced inflammatory process.^[42] Therefore, we also determined whether CORM-3 can inhibit the activation of NF-κB. In the present study, exposure to LPS/D-GalN resulted in the translocation of NF-κB and the increase of nuclear expression of NF-κB-p65. Interestingly, pretreatment of CORM-3 blocked NF-κB nuclear translocation. However, iCORM-3 had no effect on the translocation of NF-κB in ALF. In line with these observations, the anti-inflammatory actions were at least in part related to interfering with the NF-κB pathway. Our study may provide a theoretical support for CORM-3 for the treatment of ALF instead of exogenous CO gas. Indeed, the pathogenic process of ALF is rather complex, and it is involved in multiple pathogenic factors. Although these findings showed that the hepatoprotective effect of CORM-3 was likely related to the inhibition of inflammatory responses, we could not exclude other possible mechanisms of CORM-3 against ALF.

 

In conclusion, the administration of CORM-3 was associated with increased survival and alleviation of liver injury in mice induced by LPS/D-GalN. The results prove that CORM-3 is effective in the treatment of ALF through the inhibition of NF-κB activation and the regulation of expression of inflammatory cytokines and mediators in mice. The use of CORM-3 may be a novel strategy for the treatment of ALF.

 

 

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