Acute liver failure (ALF) is a relatively rare liver disorder that leads to the massive death of hepatocytes. Our previous study reported that a novel small-molecule agent, (E)-5-(2,4-di-tert-butyl-6-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)-5′-methyl-7,7′-dimethoxy-4,4′-bibenzo[d][1,3]dioxole-5,5′-dicarboxylate (7k), possessed potent anti-inflammatory activity. In the present study, we further evaluated the therapeutic effects of 7k on lipopolysaccharide (LPS)-induced ALF and investigated the mechanisms of action. Our results demonstrated that 7k inhibited the migration of RAW264.7 macrophages, blocked the activity of nuclear factor-κB protein, and dose-dependently down-regulated the production of interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 as well as their corresponding mRNAs in RAW264.7 cells. Oral administration of 7k at a dose of 50 mg/kg significantly suppressed the serum level of enzyme activity and prevented the damage of liver tissue in d-galactosamine/LPS-induced ALF. Treatment with 7k also remarkably blocked the increase in the number of CD11b+- and CD68+-positive cells in the liver, and in vivo nuclear factor-κB activity, known to regulate inflammatory responses in many cell types, was effectively inhibited. The serum concentrations and hepatic mRNA expression of proinflammatory cytokines tumor necrosis factor-α, IL-1β, and IL-6 were markedly down-regulated in mice by the treatment of 7k. In summary, 7k alleviated the development and progression of d-galactosamine/LPS-induced ALF by inhibiting macrophage infiltration and regulating the expression of cytokines.
Acute liver failure (ALF) is a clinical manifestation of sudden and severe hepatic injury and arises from many causes such as excessive alcohol intake, viral hepatitis, idiosyncratic reaction to medication, and overdose of a specific drug. After the abrupt loss of hepatic metabolic and immunological functions, it further leads to hepatic coagulopathy, and, in many cases, progressive dysfunctions of multiple organ systems (Bernal et al., 2010). ALF is associated with high mortality in patients because there are no effective therapeutic strategies, except for liver transplantation, which is limited by a chronic shortage of donor livers. ALF in humans has been demonstrated to be initiated by the activation of T cells and macrophages, which either directly attack liver parenchymal cells or induce tissue damage by the release of a variety of proinflammatory cytokines (Vergani et al., 2002; Wolf et al., 2005).
d-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver failure in mice is a well established experimental model of ALF. In this model, GalN, a transcriptional inhibitor, potentiates the toxic effects of LPS, producing typical hepatic necrosis and apoptosis followed by fulminant hepatitis (Kim et al., 2008). The stimulation of monocytes and macrophages by LPS could induce many genes to express the proinflammatory cytokines and mediators, such as tumor necrosis factor (TNF)-α, IL-1β, and IL-6. A major feature of GalN/LPS-induced ALF is clearly recognized to be an increased sensitivity to TNF-mediated effects. Considerable studies have reported that TNF-α and IL-1β play pivotal roles in the pathogenesis of an endotoxin-induced experimental liver injury model (Dinarello, 1991; Thijs and Hack, 1995; Fukuda et al., 2006). IL-6 is a multifunctional cytokine and plays a crucial role in the acute inflammatory-phase response, which results in an enhanced stimulatory effect of monokines (Heinrich et al., 1990). It is generally accepted that these primary mediators are responsible for endotoxin-induced mononuclear cells migration into organs (Malik and Lo, 1996).
Nuclear factor-κB (NF-κB) is widely considered to be important in the transcriptional activation of a variety of genes involved in mammalian immune, inflammatory, and proliferative responses. These genes include cytokines, cell adhesion molecules, complement factors, antiapoptotic factors, and immunoreceptors in macrophages (Shakhov et al., 1990; Barnes and Karin, 1997; Chen et al., 1999). In unstimulated macrophages, NF-κB localized in the cytoplasm as a heterodimer consisting of p50/p65 and Iκ-Bα protein (Müller et al., 1993; Martin et al., 2006). The activation of NF-κB mediates the transcription of inducible nitric-oxide synthase, TNF-α, IL-1β, IL-6, and IL-8 (Chen et al., 1999; Yoshimura, 2006). Moreover, TNF-α and IL-1β could directly activate NF-κB to magnify the initial inflammatory response (Jimi and Ghosh, 2005). The overproduction of these mediators and overexpression of their corresponding mRNAs are closely associated with the functions of macrophages in several inflammatory diseases, including ALF, rheumatoid arthritis, and atherosclerosis (Simpson et al., 1997; Schroder and Tschopp, 2010).
(E)-5-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenyl)-5′-methyl-7,7′-dimethoxy-4,4′-bibenzo[d][1,3]dioxole-5,5′-dicarboxylate (7k) is a bifendate derivative containing (Z)-5-benzylidene-thiazolidine-2,4-dione, which is linked by ester moiety as shown in Fig. 1A. In a previous study, we reported that 7k exhibited potential hepatoprotective effects in ConA-induced hepatitis rats (Wang et al., 2011). The aim of the present study was to explore the effects and action of mechanisms of 7k on proinflammatory cytokines in LPS-induced macrophages and GalN/LPS-induced ALF rat model.
Materials and Methods
Chemicals and Regents.
Compound 7k (Fig. 1A) with a purity of 99.5% has been synthesized in our laboratory and identified by high-performance liquid chromatography, mass spectrometry, and NMR (Wang et al., 2011). 7k was dissolved in a vehicle (0.25% carboxymethylcellulose; Sigma, St. Louis, MO). d-(+)-GalN and LPS (Escherichia coli 0111:B4 and Salmonella enteric) were purchased from Sigma.
Female BALB/c mice were purchased from Hua Fukang (Beijing, China) and bred at a temperature of 22°C and relative humidity of 55.5% with 12-h dark-light cycles for at least 2 weeks before the experiment. All experimental procedures were performed according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
Cell Viability Assay.
Raw 264.7 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium with 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2. The cells at a density of 105 per well were plated in a 96-well plate. Cells were serum-starved for 12 h, and then cultured with various concentration of 7k with or without LPS (1 μg/ml, E. coli) for 24 h. Each well was added to 20 μl of 3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/ml in normal sodium), and then cells were incubated for 3 h. The medium was carefully removed, and 150 μl of dimethyl sulfoxide was added. The plate was shaken mechanically for 10 min, and then absorbance readings at a wavelength of 570 nm were performed on a spectrophotometer (Molecular Devices, Sunnyvale, CA).
RAW264.7 cells were cultured at a seeding density of 105 cells/well in a 96-well plate and incubated for 12 h. Then cells were stimulated in the presence of different concentrations of 7k with or without 1 μg/ml of LPS (E. coli) for up to 24 h. Culture medium was removed to measure the nitrite concentration by using Griess reagent at 540 nm.
Cell Migration Assay.
The migration of RAW264.7 cells was determined by using a modified Boyden chamber. A total of 2 × 105 cells in 300 μl of RPMI 1640 medium with 1% fetal calf serum were seeded in the upper chamber with 8-μm pore size membrane. Right hundred microliters of RPMI 1640 medium with 10% fetal calf serum was added to the lower chamber. Different concentrations of 7k (10, 15, and 20 μM) or indometacin (20 μM) were added to both the lower and upper chambers, and the cells were incubated for 7 h. Cells trapped in the membrane pores or adherent under the surface were fixed with 4% paraformaldehyde, stained with viola crystalline, and counted.
Measurement of TNF-α, IL-1β, and IL-6 in Culture Supernatants and Serum.
RAW264.7 cells were cultured in RPMI 1640 medium with 10% fetal calf serum for 24 h. Then the medium was replaced by serum-free 1640 medium. Cells were incubated with different concentrations of 7k and LPS (1 μg/ml, E. coli) for up to 24 h. As negative control, isovolumetric LPS-free RPMI 1640 medium was added in other cultures. Mice were injected with GalN/LPS (S. enteric). After 8 h the animals were killed, and blood was collected. The cytokine levels (TNF-α, IL-1β, and IL-6) were measured in the medium of RAW264.7 cells and in mice serum by using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Reverse Transcription-Polymerase Chain Reaction Analysis.
The expression of TNF-α, IL-1β, and IL-6 mRNA in RAW264.7 and liver tissue was measured by reverse transcription-polymerase chain reaction (RT-PCR). A total of 1 × 105 cells were cultured in 10-cm tissue culture plates with 10% fetal calf serum and RPMI 1640 medium for 24 h, and the medium was changed to serum-free 1640 medium. LPS (1 μg/ml, E. coli) combined with various concentrations of 7k (10, 15, and 20 μM) was added to the medium for 4 h. Total content of RNA was isolated from cells or liver by using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA (2 μg/each) was reverse-transcribed to obtain cDNA by using the Prime Script RT reagent Kit (Takara, Kyoto, Japan). The sequences of primers for the cytokines genes were as follows: TNF-α (418 bp), forward 5′-GAG TGA CAA GCC TGT AGC C-3′ and reverse 5′-AAC ACC CAT TCC CTT CAC-3′; IL-1β (503 bp), forward 5′-AGG CTC CGA TGA ACA A-3′ and reverse 5′-AAG GCA TTA GAA ACA GTC C-3′; IL-6 (497 bp), forward 5′-GGA AAT CGT GGA AAT GAG-3′ and reverse 5′-GCT TAG GCA TAA CGC ACT-3′; and GAPDH (516 bp), forward 5′-GTG CTG TAT GTC GTG GAG TCT-3′ and reverse 5′-GTG GAA GAA TGG GAG TTG CTG T-3′. PCR amplification was performed for 25 cycles as follows: denaturation at 95°C for 30 s, primer annealing for 30 s at the annealing temperatures of 57°C, and extension at 72°C for 10 min.
Preparation of Nuclear Extracts from Cells or Liver.
A total of 1 × 105 cells were cultured in 10-cm tissue culture plates with 10% fetal calf serum and RPMI 1640 medium for 24 h, and the culture medium was changed to serum-free 1640 medium. After incubation with 10, 15, and 20 μM 7k for 30 min, LPS (E. coli) was added to the culture media at a final concentration of 1 μg/ml. After LPS was challenged for 30 min, nuclear protein was extracted from cells. Liver tissue was removed from mice, which was homogenized with an homogenizer. In brief, cells or liver were added to 100 μl of sucrose buffer (0.32 M sucrose, 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet-P40, 0.5 mM DTT, and 0.5 mM PMSF). After centrifugation of the samples, precipitates containing crude nuclei were resuspended in 30 μl of low-salt buffer (20 mM HEPES, pH 8.0, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, and 0.5 mM PMSF) and the same volume of high-salt buffer (20 mM HEPES, pH 8.0, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25% glycerol, 1% Nonidet-P40, 0.5 mM DTT, and 0.5 mM PMSF). Samples were softly shaken for 30 min on ice and then centrifuged at 14,000g at 4°C for 15 min to obtain the supernatant-containing nuclear extracts.
Electrophoretic Mobility-Shift Assay.
Nuclear extracts (6 μg) isolated from liver tissue or cells were mixed with double-stranded NF-κB oligonucleotide. EMSA was performed by using instructions for the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific, Waltham, PA). The following oligonucleotides were used: NF-κB sense, 5-AGCTTCAGAGGGGACTTTCCGAGAGG-3 and NF-κB antisense, 5- TCGACCTCTCGGAAAGTCCCCTCTGA-3.
In Vivo GalN/LPS Shock Model.
BALB/c mice (n = 6) were injected intraperitoneally with 700 mg/kg d-galactosamine and 4 μg/kg LPS (S. enteric). After 30 min, one group was orally administrated with 7k (50 mg/kg). The remaining group was the control. After 8 h, the animals were sacrificed. Sera were obtained by centrifugation (3000 rpm for 15 min). Liver was removed, fixed with 10% formalin, and stored in liquid nitrogen. The plasma content of ALT and AST was determined by an automated enzyme assay, commercial kits, and automated analyzers (Roche Diagnostics, Mannheim, Germany). The serum levels of TNF-α, IL-1β, and IL-6 were measured by using an ELISA kit as above. Hepatic TNF-α, IL-1β, and IL-6 expression was determined by RT-PCR.
Histological and Immunohistochemical Examination.
After mice were sacrificed, the liver was fixed in 10% buffered formalin and embedded in paraffin for histological analysis. Sections (4 μm thick) were prepared and subjected to staining with hematoxylin and eosin. In an immunohistochemistry assay, anti-CD11b and anti-CD68 (Cell Signaling Technology, Danvers, MA) were used as the primary antibodies for paraffin-embedded material after antigen retrieval. Immunoreactivity was visualized by using peroxidase-diaminobenzidine. The numbers of positive areas were counted at 200× magnification.
DNA fragmentation was detected in liver sections by using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacturer's instructions and examined by confocal laser-scanning microscopy (Olympus Bx60; Olympus, Tokyo, Japan).
All results were expressed as means ± S.E. Differences between groups were evaluated by using an independent-samples t test with SPSS 16.0 (SPSS Inc., Chicago, IL). The results were considered significantly different at *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
Effects of 7k on LPS-Induced NO Production.
Initially, we examined any potential toxicity of 7k in RAW264.7 cells. The assay of 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide indicated that cell viability was not affected by the treatment of 7k with concentrations up to 40 μM (Fig. 1C). In subsequent experiments, low concentrations of 7k (1–20 μM) were used to determine their inhibitory effects on LPS-induced production of NO in RAW264.7 cells. After stimulation by LPS (1 μg/ml) for 24 h, NO production was 10-fold increased compared with control, which was significantly inhibited by the administration of 7k in a concentration-dependent manner (Fig. 1B).
7k Inhibited the Migration of RAW264.7 Cells.
To elucidate the inhibition of monocyte migration, RAW264.7 cells were examined after the administration of 7k or indometacin in an experiment of chemotaxis. RAW264.7 cells increased the migration and invasion through the pores with increasing cultured times. Simultaneous incubation of cells with 7k (10, 15, and 20 μM) for 7 h dose-dependently inhibited the migration of macrophages in a transwell assay (Fig. 1D). Another 20 μM 7k in the culture medium resulted in a 68% inhibition of migration compared with control, which was considered as resulting in 100% migration. The inhibition of indometacin was 57% compared with control (Fig. 1E). 7k showed prominent effects on the migration of RAW264.7 cells in vitro, prompting us to assess whether 7k could inhibit the accumulation of hepatic macrophages in an animal model.
7k Inhibited the Production of TNF-α, IL-1β, and IL-6 In Vitro.
In vitro, TNF-α, IL-1β, and IL-6, the key inflammatory cytokines, were highly expressed in LPS-induced macrophages. To explore the effects of 7k on inflammation, the inflammatory cytokines were measured from different test groups. RAW264.7 cells were treated with LPS (1 μg/ml) alone or in combination with different concentrations of 7k. After 24 h, cytokine assays in the media were determined by using commercial ELISA kits. As depicted in Fig. 2, the incubation of RAW264.7 cells with LPS caused 63-, 26-, and 24-fold increases of TNF-α, IL-1β, and IL-6 levels, respectively, in culture medium. 7k (20 μM) markedly inhibited the concentration of TNF-α, IL-1β, and IL-6 levels induced by LPS. 7k dose-dependently suppressed the production of the cytokines, indicating that 7k blocked the expression of these genes involved in the inflammatory process. Furthermore, total RNA was separated from 7k-treated cells for 4 h. Reverse transcription-PCR analysis implied that 7k effectively down-regulated the mRNA levels of TNF-α, IL-1β, and IL-6 in a concentration-dependent manner (Fig. 2D). These results thus suggested that 7k prevented LPS-induced production of inflammatory cytokines in RAW264.7 macrophages.
7k Down-Regulated NF-κB Activity.
Previous study had demonstrated that 7k effectively decreased the levels of TNF-α, IL-1β, and IL-6. Activation of NF-κB transcribing these genes is necessary for the production of cytokines. Therefore, the inhibition of 7k on LPS-induced NF-κB activation in RAW264.7 cells was examined. LPS increased the binding activity of NF-κB to its consensus DNA sequence, whereas 7k suppressed the increase in the binding intensity of NF-κB in a dose-dependent manner (Fig. 3). The value of band intensity of NF-κB was decreased by 15% compared with control after administration with 15 μM 7k and was further decreased by 33% after the treatment with 20 μM 7k.
7k Prevented GalN/LPS-Induced Acute Liver Injury.
Serum levels of ALT and AST in a time-gradient manner were measured. Peak levels of transaminase release were reached at approximately 8 h after challenge (Supplemental Fig. 1). Mice were primarily injected with GalN/LPS, and 7k was orally administrated 30 min after challenge. All mice were sacrificed at 8 h after GalN/LPS shock. The levels of ALT and AST were significantly elevated at 8 h after GalN/LPS challenge, and signs of hepatic injury were observed in all of the GalN/LPS-induced mice. Treatment with 7k prevented GalN/LPS-induced liver failure as demonstrated by the reduction of serum ALT and AST release (Fig. 4A; n = 6; **, P < 0.01). In the 7k-administered group, the levels of ALT and AST resulted in 89.12% (p < 0.01) and 76.12% (p < 0.01) decreases, respectively, compared with GalN/LPS-induced mice. Livers from GalN/LPS-hepatitis mice exhibited the monocyte's infiltration, congestion and dilatation of blood vessel, and widespread hepatocellular necrosis in liver lobules. In contrast, necrotic regions and the number of monocytes invading into the liver were remarkably reduced by 7k treatment (Fig. 4B). Apoptotic hepatocytes were detected by TUNEL staining. A large number of TUNEL-positive hepatocytes were observed in the mice liver tissues obtained 8 h after GalN/LPS treatment. However, a few TUNEL-positive hepatocytes were observed in the livers by 7k treatment (Fig. 4C).
7k Reduces the Expression of CD11b and CD68 of Immunological Cells In Vivo.
On a cellular level, the activation of liver-resident macrophages, traditionally called Kupffer cells, and the vast infiltration of monocytes into the injured liver have been identified as major pathogenic factors (Baeck et al., 2011). Hence, we determined the effect of 7k on invading macrophages in vivo. CD11b, a C3b receptor, is present on the surface of monocytes/macrophages, granulocytes, and natural killer cells. CD68 is also used as a marker of macrophages, including Kupffer cells (Kinoshita et al., 2010). Liver tissue sections were first stained with same protocol without primary antibody, which showed negative control (Supplemental Fig. 2). Then sections stained with the primary antibody of CD11b+ and CD68+ showed a large number of positive cells. Sections from the 7k-treated group revealed fewer CD11b+- and CD68+-positive cells (Fig. 5A); compared with the GalN/LPS group, positive cells in the 7k-treated group decreased by 87.51% (n = 3; *, P < 0.05) and 71.61% (n = 3; *, P < 0.05), respectively.
7k Reduced the Serum Levels of Proinflammatory Cytokines.
TNF-α, IL-1β, and IL-6 play crucial roles in the progression of hepatitis. To explore the effects of 7k on the modulation of these cytokines in vivo, serum levels of TNF-α, IL-1β, and IL-6 were measured by ELISA kits. GalN/LPS injected in animals for 8 h caused 106-, 234- and 276- fold increases in TNF-α, IL-1β, and IL-6 levels, respectively, in serum compared with untreated mice. The concentrations of these inflammatory cytokines from 7k-administrated mice were significantly lower than those in the GalN/LPS group (Fig. 6, A–C). The serum level of TNF-α in the 7k-treated group was down-regulated by 68% (n = 6; ***, P < 0.001) compared with that in the GalN/LPS group. Levels of IL-1β and IL-6 were decreased by 33% (n = 6; **, P < 0.01) and 71% (n = 6; ***, P < 0.001), respectively, compared with the GalN/LPS group. To further confirm the above-mentioned results, the mRNA expression of TNF-α, IL-1β, and IL-6 was evaluated by RT-PCR. The levels of mRNA in the liver expressed significant 45-, 53-, and 29-fold increases of TNF-α, IL-1β, and IL-6, respectively, at 8 h after injection with GalN/LPS. Treatment of 7k (50 mg/kg) remarkably suppressed the TNF-α, IL-1β, and IL-6 mRNA levels by 35, 62, and 71%, respectively (Fig. 6D).
7k Suppressed NF-κB Activity In Vivo.
In vivo, previous studies showed that 7k inhibited the production of cytokines in plasma and down-regulated expression of these cytokines. Therefore, NF-κB activity in the liver of the GalN/LPS model was measured. NF-κB activity in the liver was increased to 2.5-fold 8 h after injection with GalN/LPS. When orally administrated with 7k (50 mg/kg), the activation of NF-κB decreased to 53% (Fig. 6E).
In previous study, a series of novel small-molecule bifendate derivatives were synthesized and evaluated for anti-inflammation. Compound 7k was found to possess potent anti-inflammatory activity and effectively protected the liver injury in mice. However, to date, no study has focused on the involved mechanisms of action of 7k on ALF. In the present study, the therapeutic effects and the underlying mechanisms in macrophages of 7k were investigated in detail.
LPS, an endotoxin in the outer membrane of Gram-negative bacteria, is a major trigger of septic shock. The innate immune cells recognize LPS through Toll-like receptors (TLR) (Aderem and Ulevitch, 2000), which could directly activate macrophages and trigger the production of proinflammatory mediators, such as NO, TNF-α, ILs, and leukotrienes (Watson et al., 1999; Kubes and McCafferty, 2000). Bacterial LPS acts on macrophages to release TNF-α, and then the secreted TNF induces the cells to produce IL-1β and IL-6 (West et al., 1995; Xaus et al., 2000; Jean-Baptiste, 2007). Thus, the down-regulation of LPS-induced proinflammatory cytokines was regarded as one of the essential conditions for alleviating a variety of inflammatory disorders caused by the activation of macrophages.
In vitro, compound 7k effectively decreased LPS-induced NO production in RAW264.7 cells and significantly inhibited the cell migration in a dose-dependent manner, indicating that 7k has a favorable effect on the activation of macrophages. Further work is required to determine the anti-inflammatory effects of 7k in RAW264.7 cells. Our results demonstrated that 7k reduced the production of TNF-α, IL-1β, and IL-6 (Fig. 2, A–C), which supported the hypothesis that 7k could affect the expression of these corresponding cytokine genes. The induction of these cytokines also depends on NF-κB activation (Chen et al., 1995). 7k suppressed the mRNA expression of TNF-α, IL-1β, and IL-6 (Fig. 2D). The results were consistent with the above-mentioned findings. In particular, the inhibitory effects on TNF-α and IL-1β production and their corresponding mRNA expression by 7k were more potent than those of IL-6 and its mRNA. NF-κB is a mammalian transcriptional factor that controls a number of genes, such as inducible nitric-oxide synthase, cyclooxygenase-2, TNF-α, IL-1β, and IL-6, which are responsible for immune and inflammatory diseases, and NF-κB could be activated by LPS (Barnes and Karin, 1997). In unstimulated cells, NF-κB is present in the cytosol as a heterodimer and is linked to IκB protein. The activation of NF-κB results from the phosphorylation, ubiquitination, and proteasome-mediated degradation of IκB protein, which is followed by the nuclear translocation and DNA binding of NF-κB (Brown et al., 1993; Rodriguez, et al., 1999). Our study indicated that 7k suppressed LPS-induced NF-κB activity (Fig. 3).
Macrophages in the liver tissue exert various and crucial functions, e.g., the perpetuation or resolution of hepatic inflammation, which depend on their activation from local hepatic microenvironments (Duffield et al., 2005; Karlmark et al., 2010). Therefore, one classic experimental model of AFL is established to evaluate for anti-inflammatory and therapeutic effects. GalN/LPS-induced AFL is an animal model of fulminant hepatic failure (Goto et al., 2006), which is mediated by macrophages via the induction of proinflammatory cytokines (Freudenberg et al., 1986; Silverstein, 2004). Moreover, the infiltration of macrophages could release essential proinflammatory cytokines, which thereby stimulated hepatocellular stress responses (Baeck et al., 2011). Previous studies reported that hepatocyte necrosis was followed by the up-regulation of serum transaminases and the infiltration of inflammatory cells (Simpson et al., 1997). The results of time-dependent experiments showed an increase in the serum levels of ALT and AST, starting at 6 h and reaching a maximum at 8 h. At 8 h after the injection of GalN/LPS, severe hepatic necrosis was observed as a result of GalN/LPS-induced fulminant hepatic failure (Kim et al., 2008), in which the serum ALT levels were increased at 6 h and reached a maximum 8 h after the GalN/LPS injection (Nakama et al., 2001). Through pathological examination, massive death of hepatocytes and the number of monocytes were found in hepatic vessels from the GalN/LPS-induced model. Compound 7k showed a hepatoprotective effect against GalN/LPS-induced liver injury and decreased the levels of ALT and AST in serum. Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure, which has been observed in animal models induced by GalN/LPS (Kim et al., 2008). Therefore, TUNEL staining was performed to detect the effect of 7k on hepatocyte apoptosis. The results from the model of GalN/LPS-induced ALF demonstrated striking hepatocyte apoptosis. Moreover, treatment with 7k inhibited the apoptotic process occurring in AFL (Fig. 4C), which translates to a protective effect. Hepatic macrophages were identified by using CD11b and CD68, two commonly used murine macrophage markers (Leenen et al., 1994). Furthermore, immunohistochemistry of CD11b+- and CD68+-positive cells indicated that the activation of macrophages was inhibited by 7k. This was in accordance with the previous study showing that 7k inhibited the migration of macrophages in vitro. Another group reported that bowel disease and rheumatoid arthritis were satisfactorily treated by suppressing monocyte/macrophage infiltration (Ma et al., 2010; Peng et al., 2010). A range of cytokines, such as TNF-α, TGF-β, IL-1β, IL-6, IL-10, IL-12, and IL-18, and chemokines, such as macrophage inflammatory protein, could contribute to the migration of macrophages/monocytes to the inflammatory site. Activated intrahepatic macrophages may take on a cytotoxic profile and destroy infected hepatocytes directly (Tomita et al., 1994). Hence, we determined the serum levels of TNF-α, IL-1β, and IL-6 and hepatic TNF-α, IL-1β, and IL-6 mRNA expression. The results showed that 7k suppressed the production of these cytokines and the expression of their corresponding mRNAs. In vivo, 7k protected mice from hepatic failure induced by GalN/LPS, which was paralleled by a significant decrease of cytokines and invaded macrophages. Furthermore, 7k could inhibit the transcriptional activity of NF-κB in hepatocytes. One proposed hepatoprotective mechanism of 7k was that it exerted its effect on the NF-κB pathway, which was mediated by the levels of cytokines, TNF-α, IL-1β, and IL-6, and targeted macrophages.
In conclusion, our study confirmed the in vitro anti-inflammatory effect and therapeutic potency of 7k on ALF. Hepatic protection of 7k contributed to its inhibitory effect on the production of proinflammatory mediators and further suppressed the infiltration of macrophages. Compound 7k might offer an effective therapeutic strategy in the treatment of liver disease.
Participated in research design: Deng, Liu, G. Wang, and Chen.
Conducted experiments: Deng, Liu, G. Wang, Ma, Xie, X. Wang, Li, and Chen.
Contributed new reagents or analytic tools: Chen.
Performed data analysis: Deng, Liu, and Chen.
Wrote or contributed to the writing of the manuscript: Deng, Liu, and Chen.
We thank Xian-huo Wang, Hao Zheng, Yuan Liu, and Lin Deng of the State Key Laboratory of Biotherapy at Sichuan University for technical assistance.
This work was supported by the National Key Technologies R&D Program of China [Grant 2009ZX09501-015].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- acute liver failure
- alanine aminotransferase
- aspartate transaminase
- tumor necrosis factor
- nuclear factor-κB
- enzyme-linked immunosorbent assay
- electrophoretic mobility-shift assay
- reverse transcription-polymerase chain reaction
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- phenylmethylsulfonyl fluoride
- base pairs
- nitric oxide
- glyceraldehyde-3-phosphate dehydrogenase
- Received October 22, 2011.
- Accepted January 9, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics