Inflammation ( # 2014) DOI: 10.1007/s10753-013-9805-x

Inhibitions of NF-κB and TNF-α Result in Differential Effects in Rats with Acute on Chronic Liver Failure Induced by d-Gal and LPS Fan Yang,1 Xun Li,1 Li-kun Wang,1 Lu-wen Wang,1 Xiao-qun Han,1 Hong Zhang,2 and Zuo-jiong Gong1,3

Abstract—In this study, we induced an acute-on-chronic liver failure (ACLF) model by human serum albumin (HSA), D-galactosamine (D-Gal) and lipopolysaccharide (LPS) in rats. Anti-TNF-α polyclonal antibody (as TNF-α inhibitor) and pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) were used to treat the liver failure animals, respectively. The results showed that TNF-α inhibition was beneficial, but NF-κB inhibition failed to protect the rats in ACLF. However, HMGB1 levels, cytokine production and activation of TLR4-NF-κB signaling pathway were all suppressed by both TNF-α and NF-κB inhibition. In order to verify the effect of PDTC on inflammatory response, we further explored its effect in vitro. Anti-inflammatory activity of PDTC was proved in U937 cell line. To conclude, both inhibitions of TNF-α and NF-κB are able to suppress the activation of TLR4 and NF-κB signaling pathway. However, NF-κB inhibition with PDTC failed to protect the rats in ACLF induced by D-Gal and LPS. KEY WORDS: acute-on-chronic liver failure; inflammation; NF-κB; TNF-α; pyrrolidine dithiocarbamate.

INTRODUCTION Acute-on-chronic liver failure (ACLF) is a severe lifethreatening condition with a high fatality. Until now, ACLF still lacks of effective treatment. The main pathological feature is the necrosis of a great number of liver cells accompanied with inflammatory cell infiltration on the basis of chronic liver injury. Many factors have been implicated in the pathogenesis of the disease including endotoxin and inflammatory cytokines [1–3]. HMGB1 is an important inflammatory mediator which is associated with many inflammatory diseases including endotoximia, acute pancreatitis, acute lung injury, rheumatoid arthritis, hemorrhagic shock and ischemia-reperfusion injury [4–9]. In a previous study, we have demonstrated that HMGB1 plays a crucial role in the pathogenesis of ACLF in

1

Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, People’s Republic of China 2 Pharmaceutical Department, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, People’s Republic of China 3 To whom correspondence should be addressed at Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, People’s Republic of China. E-mail: [email protected]

rats and blockade of HMGB1 by anti-HMGB1 antibody can confer protective effect to ACLF [10]. HMGB1 is actively secreted by immunostimulated macrophages and enterocytes and it is also released by necrotic cells [11–16]. There are many receptors including RAGE, TLR2, TLR4, and TLR9 [17–19] that HMGB1 can bind to. Among these receptors, TLR4 is the critical receptor mediating the inflammatory activity of HMGB1 [13–15]. Once its signaling pathway was activated, nuclear factorkappaB (NF-κB) can be activated via MyD88-dependent pathway and/or MyD88-independent pathway [19]. NF-κB has a role in a variety of biological processes including immunity, inflammation, wound healing, proliferation, and apoptosis [20]. Many inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease, are associated with the activation of NF-κB [21]. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, can attenuate myocardial inflammatory response and ventricular dysfunction following coronary microembolization [22], alleviate paraquatinduced lung injury [23] and improve hepatic parenchymal microcirculation and tissue oxygenation [24]. TNF-α is one of the most common cytokines that have been implicated in the pathogenesis of inflammatory

0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York

Yang, Li, Wang, Wang, Han, Zhang, and Gong diseases. It can be induced to release by administrating HMGB1 via TLR4 signaling pathway in macrophages [25]. In addition, to be activated by HMGB1, NF-κB can also be activated by TNF-α. TNF-α binds two distinct receptors, TNF-R1 and TNF-R2, and the binding of TNF-α to TNF-R1 triggers a series of intracellular events that ultimately result in the activation of NF-κB and c-Jun [26]. NF-κB pathway plays a key role in the pathogenesis of HMGB1-mediated inflammatory diseases and TNF-α is the central mediator, because HMGB1 triggers TLR4 activation and TNF-α release which in turn mediates the activation of NF-κB. With regard to the roles of NF-κB and TNF-α in the pathway mediated by HMGB1, the aim of this study is to evaluate the therapeutic effects of NF-κB and TNF-α inhibition by PDTC and anti-TNF-α antibody in rats with ACLF.

MATERIAL AND METHODS Experimental Animals Female specific pathogen-free (SPF) Wistar rats, weighing 180 to 220 g, were purchased from the Experimental Animal Center of Wuhan University. All animals were acclimated to the laboratory for 5 days before experiments and were maintained in a light-controlled room (12h light/dark cycle) at an ambient temperature of 25 °C with free access to water and standard chow. All animals were given humane care in accordance with the institutional guidelines. Animal Model Production The ACLF rat models induced by HSA, D-Gal, and LPS were established as previously described [27]. In brief, HSA (Octapharma m.b.H., Austria) was diluted to a concentration of 8 g/L with physiological saline and emulsified with an equal amount of incomplete Freund's adjuvant. The rats were injected subcutaneously at multipoint with 0.5-ml above solutions containing 4 mg HSA at a total of four times (14-day intervals between the first and second time; 10-day intervals between the third and fourth time). After sensitization by HSA, the rats were injected 4 mg HSA into tail vein twice a week for 6 weeks. The chronic liver injury in rats was firstly induced by HSA. Secondly, the rats were attacked by intraperitoneal injection with DGal (purity of 98 %, Sigma-Aldrich Co., USA) at a dose of 400 mg/kg combined with LPS (purity of 99 %, Sigma-

Aldrich Co., USA) at a dose of 100 μg/kg, which induce acute liver failure on the basis of chronic liver injury. Experimental Design All animals were randomly divided into control group (n=15), model group (n=15), rabbit polyclonal anti-TNFα antibody (Abcam Co., UK) treatment group (n=15), isotype antibody (nonimmune rabbit IgG) treatment group (n=15) and PDTC (Sigma-Aldrich Co., USA) treatment group (n=15). Except for the rats in the control group, ACLF model was induced in all of the rats. In the antiTNF-α antibody treatment group, the rats received 100 μg/ kg anti-TNF-α antibodies 3 h after ACLF model had been induced. The rats in the isotype antibody treatment group and the PDTC treatment group received 100 μg/kg isotype antibody and 100 mg/kg PDTC, respectively 3 h after ACLF model had been induced. The rats in the control group and the model group received physiological saline as controls. Rats were killed randomly for blood and hepatic tissue collection after treatment reagents had been given 24 h in each group. The remaining 10 rats in each group were observed for survival time every 12 h for 7 days. Cell Culture and Treatment Monocyte-like cell line U-937 cells (lab preserved) were cultured and maintained at a cell density between 2× 106 and 1×107 cells/mL in RPMI 1640 (Jinuo, China) supplemented with 10 % fetal bovine serum (Sijiqing, China), 100 U/mL penicillin, and 100 μg/mL streptomycin. For macrophage-like cell differentiation, serum starved U-937 cells were plated in a 35-mm cell culture dish (2×106 cells/mL) and treated with 60 ng/mL PMA for 24 h. The non-adherent cells were removed by two PBS washes. Complete RPMI 1640 medium containing 1 μg/mL LPS or 1 μg/mL LPS combined with PDTC (different doses) was applied in subsequent experiments. Cultural supernatants were collected for cytokine detection. Determinations of Blood and Supernatant Samples Serum levels of ALT were determined by routine biochemical methods using a Hitachi Automatic Analyzer (Hitachi, Inc. Japan). Serum and supernatant levels of HMGB1, TNF-α, and IFN-γ were measured using ELISA Kits (HMGB1 from Westang Co., China; TNF-α and IFNγ from Ebioscience Co., UK) according to the manufacturer's recommendations, respectively.

Inhibitions of NF-κB and TNF-α Result in Differential Effects Determinations of TUNEL Assay Some portions of liver samples were fixed in 10 % buffered formalin for 24 h, embedded in paraffin, sliced into sections of 5-μm thickness. The sections were incubated in 0.1 % Triton X-100, for 8 min, and washed twice in PBS for 5 min. In situ labeling of apoptosis-induced DNA strand breaks (TUNEL assay) was performed using a commercial kit (In Situ Cell Death Detection kit, Roche, Switzerland). Western Blot Analysis of Hepatic Levels of HMGB1, TLR4 and NF-κB P65 In brief, 50-μg protein extracts from 100-mg liver tissue were subjected to 10 % SDS-PAGE and then transferred to a PVDF membrane (Millipore Co., USA). The membranes were incubated with different primary antibodies (anti-TLR4 antibody from Abcam Co., UK, antiHMGB1 antibody from Epitomics Co., USA, and NF-κB P65 antibodies from Cell Signaling Technology Co., USA), and then with a secondary antibody (LICOR Co., USA), and finally detected by ODESSEY infrared imaging system (LICOR Co., USA). Membranes were also probed for β-actin (Santa Cruz Co., USA) as an additional loading control. Statistical Analysis All data were expressed as means±SD. Differences among groups were assessed using Student’s t test and oneway ANOVA. SPSS 13.0 software was used for statistical analysis. P value less than 0.05 was considered to be statistically significant.

RESULTS Effects of Anti-TNF-α Antibody and PDTC on Survival Time of Rats All of the rats died within 4 days without treatments, and their median survival time was 54 h (Fig. 1). AntiTNF-α antibody treatment prolonged the median survival time of the ACLF rats to 84 h. However, the rats with PDTC treatment all died within 4 days and their median survival time was 30 h. In addition, isotype antibody treatment did not prolong the median survival time of the ACLF rats. As determined by the log-rank test, there was a significant difference among the survival curves (P<0.0001).

Fig. 1. Effects of anti-TNF-α antibody and PDTC on survival time of rats. The survival time of rats in each group (n=10). Compared to the control group, * P<0.05. Compared to the model group, # P<0.05.

Effects of Anti-TNF-α Antibody and PDTC on Serum ALT Levels and Hepatic Tissue Apoptosis As shown in Fig.2, TUNEL assay showed that apoptosis could be seldom detected in the control group, while apoptosis increased markedly in the model group. Compared with the model group, less apoptosis occurred in the anti-TNF-α antibody treatment group, while more apoptosis cells were observed in the PDTC treatment group. Serum ALT level in the model group was much higher than that in the control group. Compared with the model group, serum ALT level in the anti-TNF-α antibody treatment group was much lower (P<0.05), while its value was higher in the PDTC treatment group (P<0.05). However, there was no significant difference between the model group and the isotype antibody treatment group (P>0.05). Effects of Anti-TNF-α Antibody and PDTC on HMGB1 Serum levels of HMGB1 were measured by ELISA assay. The results showed that serum HMGB1 levels in the model group increased significantly compared with the control group (P<0.05). Treatment with anti-TNF-α antibody and PDTC reduced the HMGB1 levels in sera (P< 0.05). However, there was no statistical significance between the model group and the isotype antibody treatment group. As shown in Fig. 3, similar results were observed in hepatic HMGB1. Effects of Anti-TNF-α Antibody and PDTC on Cytokine Productions Serum levels of TNF-α and IFN-γ were also detected by ELISA assay. The results (Fig. 4) showed that serum levels of TNF-α and IFN-γ in the model group were much higher than those in the control group (P<0.05). Compared to the model group, serum TNF-α and IFN-γ levels were

Yang, Li, Wang, Wang, Han, Zhang, and Gong

Fig. 2. Effects of anti-TNF-α antibody and PDTC on serum ALT levels and hepatic tissue apoptosis. All the samples were collected 27 h after the model had been induced. a The TUNEL assay of liver tissue in each group (n=3). Apoptotic cells were identified as fluorescein positive cells under a fluorescent microscope. C: the control group, M: the model group, I: the isotype antibody treatment group, A: the anti-TNF-α antibody treatment group, P: the PDTC treatment group. b The ALT levels in each group (n=4). Compared to the control group, * P<0.05. Compared to the model group, # P<0.05.

lower in the anti-TNF-α antibody and PDTC treatment groups (P<0.05). However, their values were not decreased significantly in the isotype antibody treatment group (P>0.05). Effects of Anti-TNF-α Antibody and PDTC on TLR4 and NF-κB Pathway To explore the effect of inhibition of TNF-α and NFκB on TLR4 and NF-κB signaling pathway, hepatic levels of TLR4 and NF-κB p65 were detected by western blot analysis. The results (Fig. 5) showed that hepatic levels of TLR4 and NF-κB p65 were increased in the model group compared with the control group (P<0.05), and treatment with anti-TNF-α antibody and PDTC decreased hepatic levels of TLR4 and NF-κB p65 (P<0.05). However, there

were no significant differences between the isotype antibody treatment group and the model group (P>0.05). Effect of PDTC on Viability of U937-cell Line The viability of U937 cells was measured by cck-8 assay; the result showed that the viability of U937 cells was decreased greatly in the LPS-treated group, and PDTC improved the viability of U937 cells at the concentration of 5 and 10 μM (P<0.05) (Fig. 6). Effect of PDTC on HMGB1 in U937-cell Line HMGB1 levels in supernatant and cell lysates were detected by ELISA assay and western blot analysis, respectively. The results showed that HMGB1 levels in supernatant and cell lysate in the LPS-treated group were much

Inhibitions of NF-κB and TNF-α Result in Differential Effects

Fig. 3. Effects of anti-TNF-α antibody and PDTC on HMGB1. All the samples were collected 27 h after the model had been induced. a Hepatic HMGB1 levels were detected by western bolt analysis and β-actin was probed as a loading control. C: the control group, M: the model group, I: the isotype antibody treatment group, A: the anti-TNF-α antibody treatment group, P: the PDTC treatment group. b HMGB1 levels in sera of each group (n=4). c Semi-quantification of HMGB1 levels in each group (n=3). The relative protein expression levels from three independent experiments are represented as the density of HMGB1 bands normalized to that of β-actin bands. Compared to the control group, * P<0.05. Compared to the model group, # P<0.05.

higher than those in the control group (P<0.05). Compared to the LPS-treated group, their values in the PDTC-treated groups were much lower (P<0.05). HMGB1 protein was inhibited by PDTC in a dose-dependent manner as shown in Fig. 7.

Effect of PDTC on Cytokine Productions in U937-cell Line TNF-α and IFN-γ levels were detected by ELISA assay. The results (Fig. 8) showed that supernatant levels of TNF-α and IFN-γ in the LPS-treated group were much

Fig. 4. Effects of anti-TNF-α antibody and PDTC on cytokine productions. All the samples were collected 27 h after the model had been induced. Serum levels of TNF-α and IFN-γ in each group (n=4) were detected by ELISA assay. Compared to the control group, * P<0.05. Compared to the model group, # P<0.05.

Yang, Li, Wang, Wang, Han, Zhang, and Gong

Fig. 5. Effects of anti-TNF-α antibody and PDTC on TLR4 and NF-κB pathway. All the samples were collected 27 h after the model had been induced. a Hepatic levels of TLR4 and NF-κB P65 were detected by western bolt analysis. C: the control group, M: the model group, I: the isotype antibody treatment group, A: the anti-TNF-α antibody treatment group, P: the PDTC treatment group. b and c Semi-quantification of TLR4 and NF-κB P65 levels in each group (n=3). The relative protein expression levels from three independent experiments are represented as the density of TLR4 and NF-κB P65 bands normalized to that of β-actin bands. Compared with the control group, * P<0.05; compared with the model group, # P<0.05.

higher than those in the control group (P<0.05). Compared to the LPS-treated group, their values were lower in the PDTC-treated groups (P<0.05). PDTC inhibited TNF and

IFN-γ production in a concentration-dependent manner as shown in Fig. 8. Effect of PDTC on TLR4 and NF-κB Signaling Pathway in U937-cell Line TLR4 and NF-κB P65 were further measured by western blot analysis. Similar to the results in vivo, TLR4 and NF-κB P65 levels in cell lysates of PDTC-treated groups were much lower compared to the LPS-treated group (P<0.05) (Fig. 9). A dose-dependent manner was also observed and the differences among the PDTC-treated groups were significant (P<0.05).

DISCUSSION

Fig. 6. Effect of PDTC on viability of U937-cell line. Viability of U937 cells was measured by CCK-8 assay 24 h after LPS had been administrated (n=4 in each group). A higher densitometry value indicates higher viability of U937 cells. Compared with the control group, * P<0.05; compared with the LPS-treated group, # P<0.05.

Both NF-κB and TNF-α are important components in the HMGB1-mediated signaling pathway. In this study, we evaluated the effects of NF-κB and TNF-α inhibition on ACLF rats. We demonstrated that TNF-α inhibition improved the survival time of rats with ACLF, but PDTC administration accelerated the death of the rats. The results showed that TNF-α inhibition was beneficial, but NF-κB

Inhibitions of NF-κB and TNF-α Result in Differential Effects

Fig. 7. Effect of PDTC on HMGB1 in U937-cell line. All the samples were collected 24 h after LPS had been administrated in each group (n=3). a HMGB1 levels in cell lysates were detected by western bolt analysis and β-actin was probed as a loading control. C: the control group, LPS: the LPS-treated group, P1: cells treated with 1 μM PDTC, P5: cells treated with 5 μM PDTC, P10: cells treated with 10 μM PDTC. b HMGB1 levels in supernatant. c Semi-quantification of HMGB1 levels. The relative protein expression levels from three independent experiments are represented as the density of HMGB1 bands normalized to that of β-actin bands. Compared to the control group, * P<0.05. Compared to the LPS-treated group, # P<0.05.

inhibition was failed to protect the rats in ACLF. To verify the different effects of NF-κB and TNF-α inhibition on ACLF, we further observed the apoptosis of hepatic tissue and measured serum ALT levels of the rats. The results showed that TNF-α inhibition attenuated the apoptosis of hepatocytes and decreased the ALT levels in ACLF, however, NF-κB inhibition increased the apoptosis of hepatocytes and ALT levels in ACLF. TNF-α is an important cytokine which has been implicated in the progress of inflammatory disease. Treatment with anti-TNF-α antibody has been proven to have the ability to protect many inflammatory diseases, including Crohn's disease [28], rheumatoid arthritis [29], etc.

Although a bad side effect of treatment with anti-TNF-α antibody has been reported [30], we demonstrated that treatment with TNF-α antibody can inhibit the activation of TLR4 and NF-κB signaling pathway and confer protection to the rats with ACLF. PDTC is an inhibitor of NF-κB and it has been demonstrated to have protective effect to myocardial, lung and liver injuries [22–24]. In addition to NF-κB signaling pathway, it can also inhibit the L-arginine-nitric oxide pathway [31]. In this study, the results showed that PDTC has the ability to inhibit the activation of TLR4 and NF-κB signaling pathway. However, it failed to protect against ACLF induced by GalN/LPS.

Fig. 8. Effect of PDTC on cytokine productions in U937-cell line. The supernatant were collected 24 h after LPS had been administrated in each group (n= 3). Compared to the control group, * P<0.05. Compared to the LPS-treated group, # P<0.05.

Yang, Li, Wang, Wang, Han, Zhang, and Gong

Fig. 9. Effect of PDTC on TLR4 and NF-κB signaling pathway in U937-cell line. All the samples were collected 24 h after LPS had been administrated in each group (n=3). a TLR4 and NF-κB P65 levels in cell lysates were detected by western bolt analysis. C: the control group, LPS: the LPS-treated group, P1: cells treated with 1 μM PDTC, P5: cells treated with 5 μM PDTC, P10: Cells treated with 10 μM PDTC. b and c: Semi-quantification of TLR4 and NF-κB P65 levels. The relative protein expression levels from three independent experiments are represented as the density of TLR4 and NF-κB P65 bands normalized to that of β-actin bands. Compared with the control group, * P<0.05; compared with the LPS-treated group, # P<0.05.

To explore the possible mechanisms of the different effects, the protein levels of HMGB1, TLR4, and NF-κB P65 were analyzed. Although HMGB1, TLR4, and NF-κB P65 levels were decreased both by NF-κB and TNF-α inhibition, ALT levels, apoptosis of liver cells, and survival time were quite different in two groups. These results indicated that both inhibitions of NF-κB and TNF-α can attenuate the activation of TLR4-NF-κB signaling pathway. Inflammatory cytokines are important mediators in modulating inflammatory responses. Liver failure is also an inflammatory disease in which inflammatory cytokines play key roles. In addition to the HMGB1, TLR4, and NFκB P65, we also measured cytokines in the sera of the rats. The results showed that TNF-α inhibition greatly reduced the serum levels of TNF-α and IFN-γ and similar effect was also observed in the PDTC treatment group. These results demonstrated that both NF-κB and TNF-α inhibitions are beneficial to decrease the cytokine production during the pathogenesis of ACLF. Theoretically, inhibitions of NF-κB and TNF-α in ACLF should result in a similar protective effect. Because they are both the components of HMGB1 mediated signaling pathway, and inhibitions of NF-κB and TNF-α attenuated the activation TLR4-NF-κB signaling pathway and decreased the cytokine production, thus confer protection to ACLF in rats. In this study, inhibitions of NF-κB and

TNF-α were actually proved to have the ability to inhibit the activation of TLR4-NF-κB signaling pathway and to decrease the cytokine production in rats with ACLF. TNFα inhibition improved the survival time of rats with ACLF, however PDTC administration accelerated the death of the rats. Why were the differences observed in ACLF rats? It is documented that the pathogenesis of liver failure is related with ‘primary liver injury’ induced directly or indirectly (i.e., immunopathological damage) by hepatitis virus or other etiological factors; and ‘secondary liver injury’ induced by the release of inflammatory factors such as TNFα [32]. In this study, the productions of mediators of the ‘secondary liver injury’ were attenuated by both inhibitions of NF-κB and TNF-α. It has been reported that PDTC has differential effects on BCG/LPS-induced inflammatory liver injury and GalN/LPS-induced apoptotic liver damage [33]. GalN is a chemical drug which is metabolized mainly in liver cells, and it can lead to the apoptosis of liver cells. In this study, liver failure was induced by GalN/LPS, and the apoptosis of liver cells was also found aggravated by PDTC treatment. It is reasonable to attribute the detrimental effects of PDTC on ACLF to GalN. The monocyte-like cell line U-937 is widely used in inflammation research, after being differentiated by phorbol myristate acetate (PMA) and activated by lipopolysaccharide (LPS) [34]. In order to confirm the effect

Inhibitions of NF-κB and TNF-α Result in Differential Effects of PDTC on inflammatory response, the effects of PDTC on U937-cell line were also detected. The results showed that PDTC was able to maintain the viability of U937 cells and to decrease TNF-α and IFN-γ level supernatants. In addition, HMGB1, TLR4, and NF-κB P65 were also reduced by PDTC. These results suggest that PDTC is able to reduce the production of inflammatory mediators and to inhibit TLR4-NF-κB signaling pathway. In conclusion, both inhibitions of TNF-α and NF-κB are able to suppress the activation of TLR4 and NF-κB signaling pathway. The same as antiTNF-α antibodies, PDTC could also inhibit the inflammatory cytokines, however, because of the injury effect of PDTC to the liver cells, it failed to protect the rats in ACLF induced by GalN and LPS. In this situation, the apoptosis of liver cells was aggravated by PDTC treatment.

ACKNOWLEDGEMENT This study was supported by a grant from the National Natural Science Foundation of China (No. 81071342 and 81371789).

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