Author Affiliations: Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan 430060, China (Yang F, Wang LK, Li X, Wang LW, Han XQ and Gong ZJ); Department of Infectious Diseases, Linyi People's Hospital, Linyi 276000, China (Wang LW)

Corresponding Author: Prof. Zuo-Jiong Gong, MD, PhD, Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan 430060, China (Tel: 86-27-88041911-88385; Fax: 86-27-88042292; Email: zjgong@ 163.com)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60044-8

Published online March 27, 2014.

 

 

Contributors: YF proposed the study. WLK wrote the first draft. YF, WLK, LX, WLW and HXQ collected and analyzed the data. YF and WLK contributed equally to this work. All authors contributed to the design and interpretation of the study and to further drafts. GZJ is the guarantor.

Funding: This study was supported by a grant from the National Natural Science Foundation of China (81071342).

Ethical approval: The study was approved by the Medical Ethics Committee, Renmin Hospital of Wuhan University.

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 serious clinical syndrome with high mortality. Sodium butyrate has been shown to alleviate organ injury in a wide variety of preclinical models of critical diseases. The aim of this study was to investigate the protective effect of sodium butyrate on ALF in rats.

 

METHODS: All rats were randomly divided into control, model and sodium butyrate treatment groups. Except the control group, the rats were induced ALF animal model by subcutaneous injection of human serum albumin+ D-galactosamine+lipopolysaccharide. After induction of ALF, the rats in the treatment group received sodium butyrate (500 mg/kg) at 12-hour or 24-hour time point. Fourty-eight hours after ALF induction, the animals were sacrificed and samples were harvested. Serum endotoxin, high mobility group box-1 (HMGB1), liver function parameters, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were measured. The expression of HMGB1 and nuclear factor-kappa B (NF-κB) p65 protein in liver tissue was detected by Western blotting. The histological changes of liver and intestine were examined. The survival duration was also observed.

 

RESULTS: Serum endotoxin, alanine aminotransferase, HMGB1, TNF-α and IFN-γ were significantly increased and the liver histology showed more severe histopathological injury in the model group compared with the control group (P<0.05). Compared to the model group, sodium butyrate treatment significantly improved the histopathological changes in the liver and intestine, reduced serum endotoxin and inflammatory cytokines, suppressed HMGB1 and NF-��B p65 proteins in liver tissue, and prolonged the survival duration regardless of treatment at 12 hours or 24 hours after induction of ALF (P<0.05).

 

CONCLUSIONS: Sodium butyrate protected the liver from toxin-induced ALF in rats. The mechanisms may be due to direct hepatoprotection and decreased intestinal permeability.

 

(Hepatobiliary Pancreat Dis Int 2014;13:309-315)

 

KEY WORDS: acute liver failure; high mobility group box-1; nuclear factor-kappa B p65; animal model; sodium butyrate

Acute liver failure (ALF) is a serious clinical entity, characterized by massive or submassive necrosis.^[1] ALF in Asia, especially in China, is the most common type of liver failure with high mortality and its pathogenesis remains to be elucidated.^[2-4]

 

Many studies have indicated that intestinal endotoxemia plays a significant role in the pathogenesis and progression of liver failure,^{[5, 6]} which is resulted from overgrowth of gram-negative bacteria in the gut, and bacteria translocation into the peritoneal cavity due to the high permeability of the intestinal wall.^{[7, 8]} Recent studies have shown that high mobility group box-1 (HMGB1) protein, a monocyte-derived late-acting inflammatory mediator^{[9, 10]} is involved in the progression of endotoxemia^{[11, 12]} and contributes to the pathogenesis of various inflammatory disorders.^[13-16] Since HMGB1 is secreted by immunostimulated macrophages,^{[9, 17-19]} enterocytes^[20] and necrotic cells,^[21] massive hepatocyte necrosis in ALF releases a great deal of HMGB1, later in turn enhances liver damage.

 

Sodium butyrate, a low-molecular weight four-carbon chain volatile fatty acid, is a main end-product of intestinal microbial fermentation of dietary fiber.^[22] As an important energy source for intestinal epithelial cells, sodium butyrate inhibits intestinal pathogenic bacteria and maintains gastrointestinal homeostasis.^[23] Recent studies^{[24, 25]} indicated that sodium butyrate could alleviate inflammatory reaction by inhibiting the expression of inflammatory mediators such as HMGB1, nuclear factor-kappa B (NF-��B), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). These findings suggest that sodium butyrate could be a candidate for liver failure treatment.

 

The present study is to investigate the protective effects of sodium butyrate on ALF in rats. Liver histology, intestinal permeability and proinflammatory cytokines were evaluated.

 

 

Specific pathogen free Wistar rats, weighing 120 to 160 g, were purchased from the Experimental Animal Center of Hubei Province. All rats had been acclimated to the research laboratory for 5 days before the experiments, and maintained in a light-controlled room (12 hours light and dark cycle) at an ambient temperature of 25 �� with free access to water and standard chow. The rats were given humanistic care in accordance with the animal laboratory guidelines. The study was approved by the Medical Ethics Committee, Renmin Hospital of Wuhan University.

 

An ALF rat model was induced by injection of human serum albumin (HSA, Octapharma m.b.H., Austria), D-galactosamine (D-Gal, Sigma-Aldrich Co., USA) and lipopolysaccharide (LPS, Sigma-Aldrich Co., USA) as previously described.^[26] In brief, HSA 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; 14-day intervals between the second and third time; 10-day intervals between the third and fourth time). After sensitization by HSA, the rats were injected 4 mg HSA into the tail vein twice a week for 6 weeks. The chronic liver injury in rats was firstly induced by HSA. Secondly, the rats were administrated by intraperitoneal injection with D-Gal at a dose of 400 mg/kg combined with LPS at a dose of 100 µg/kg, inducing ALF on the basis of chronic liver injury.

 

Fifty-five animals were randomly divided into control group (n=10), model group (n=15), group treated with sodium butyrate at 12 hours (n=15), and group treated with sodium butyrate at 24 hours (n=15). The time of administration of D-Gal combined with LPS was as baseline (time point 0). Rats in the control group were free from D-Gal and LPS challenge. The rats in the sodium butyrate treated groups were injected intravenously with sodium butyrate solution (Sigma-Aldrich Co., USA) at a dose of 500 mg/kg at 12 hours or 24 hours, respectively after ALF induction. The rats in the control and model groups were injected intravenously with the same volume of normal saline. Then five rats in each group were randomly sacrificed for sampling of the blood, liver and intestine at 48 hours. The remaining rats in each group were observed for survival duration after induction of ALF for 7 days.

 

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were measured using a Hitachi Automatic Analyzer (Hitachi Inc., Japan). The serum level of TNF-α, IFN-γ and HMGB1 were measured by using commercial ELISA kits (TNF-α and IFN-γ from Ebioscience Co., UK, HMGB1 from Westang Co., China). Serum endotoxin was assayed using a commercial kit (Xiamen houshiji, China) with Limulus amebocyte lysate method according to the manufacturer's instruction.

 

Liver and intestine samples were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin, sectioned at 5 µm, stained with hematoxylin and eosin (HE) staining. Liver tissue was also used for Masson's trichrome staining to evaluate the pathological changes under a light microscope. The other portions were fixed in 2.5% glutaraldehyde, embedded in epoxy resin, sliced into ultrathin sections (60 nm in thickness) and stained with uranyl acetate and lead citrate, to observe the ultrastructural changes of hepatocytes^[27] under a transmission electron microscope (TEM).^[28]

 

Intestinal permeability was determined by using an everted gut sac method as previously described.^[29] In brief, everted gut sacs were prepared in ice-cold modified normal saline solution (NSS, pH=7.4). One end of the gut segment was ligated with a 3.5 silk. The segment then was everted onto a thin wood rod, and the resulting everted gut sac was reserved in a 500 mL beaker containing NSS. The everted gut sac was distended gently by injecting 1.5 mL of NSS, and then was suspended in a 100 mL beaker containing 80 mL of warm (37 ��) NSS with FITC-labeled dextran 4000 (FD4) at a concentration of 20 mg/mL for 30 minutes. The beakers were bubbled continuously with a gas mixture containing 95% O₂ and 5% CO₂ to avoid disruption of the enterocytes. A 1.0 mL sample was taken from the sac to determine FD4 concentration. The samples were centrifuged for 5 minutes at 1000 g and 4 ��. The supernatants were measured by using a Perkin-Elmer LS-50 fluorescence spectrophotometer (Perkin Elmer Inc., USA) at an excitation wavelength of 492 nm (slit width 510 nm) and an emission wavelength of 515 nm (slit width 510 nm). The permeability of the intestine was expressed as FD4 concentration/the area of gut sac.

 

Fifty microgram protein extracts from 100 mg liver tissue samples were subjected to 12% SDS-PAGE and then transferred to a PVDF membrane (Millipore, Germany). The membrane was incubated with different primary antibodies (anti-HMGB1 antibody from Epitomics Co., USA and anti-p65 from Cell Signaling Technology Co., USA), and then with a secondary antibody (LICOR Co., USA), and finally detected by an ODESSEY infrared imaging system (LICOR Co., USA). Membranes were also probed for β-actin (Santa Cruz Co., USA) as loading controls.

 

All data were expressed as mean±SD. Differences between the two groups were assessed using unpaired Student's t test and more than 2 groups using one-way ANOVA. Animal survival data were evaluated using the Kaplan-Meier method and compared with the log-rank test. SPSS 13.0 software was used for statistical analysis. A P value less than 0.05 was considered statistically significant.

 

 

The example images of liver tissue stained with HE for each group were presented in Fig. 1. In the control group (Fig. 1A1), there was no cellular necrosis, which showed the hepatocytes arrayed radially around the central vein. Massive or submassive necrosis could be observed in the model group (Fig. 1B1). In sodium butyrate treatment group (Fig. 1C1, D1), less cellular necrosis occurred as compared with the model group.

 

As shown in Fig. 1, the representative images of liver fibrosis stained with Masson's trichrome for each group were also presented. In the control group (Fig. 1A2), there was no liver fibrosis. In the model group (Fig. 1B2), there was evidence of massive or submassive necrosis. In the sodium butyrate treatment group (Fig. 1C2, D2), there was evidence of liver fibrosis, and cirrhotic changes with formation of pseudolobule, but there is less hepatocyte necrosis.

 

Ultrastructural changes of hepatocytes were observed under TEM (Fig. 1). In the control group (Fig. 1A3), symmetrical chromatin and legible nucleoli were noted. In the model group (Fig. 1B3), there was evidence of vacuolization of hepatocyte cytoplasm, disappearance of glycogen, and formation of apoptotic body. In the sodium butyrate treated groups (Fig. 1C3, D3), there was no evidence of vacuolization of hepatocyte cytoplasm.

 

The injury of the intestine was more severe in the model group than in other groups (Fig. 1). In the model group (Fig. 1B4), intestinal epithelial cells were swollen, the central thoracic duct was expanded, villi were fallen off, and the structure of intestinal mucosa was in disorder with edema and necrosis. Compared with the model group, the damage of the intestine was ameliorated in the sodium butyrate treated groups (Fig. 1C4, D4).

 

The intestinal permeability increased significantly in the model group as compared with the control group (P<0.05), and it was greatly improved by sodium butyrate at 12 hours or 24 hours (P<0.05) (Fig. 2A).

 

The serum endotoxin was measured in the control, model, and sodium butyrate treated groups at 12 hours and 24 hours (0.19±0.03, 1.56±0.21, 0.23±0.04 and 0.45±0.06 EU/mL, respectively) (Fig. 2B). Compared with the model group, the serum endotoxin was suppressed significantly in the sodium butyrate treated groups at 12 hours and 24 hours respectively (P<0.05).

 

The levels of ALT, AST, TBIL, TNF-α, IFN-γ and HMGB1 were increased significantly in the model group (7916.07±624.50 U/L, 3201.75±360.24 U/L, 102.37±11.20 µmol/L, 216.53±31.20 ng/L, 672.53±57.20 ng/L and 40.56±3.94 µg/L, respectively) as compared with the control group (71.25±9.38 U/L, 82.63±9.7 U/L, 2.41±0.76 µmol/L, 49.47±3.76 ng/L, 92.65±7.63 ng/L and 5.16±0.53 µg/L, respectively) (Fig. 2). However, the levels of ALT, AST, TBIL, TNF-α, IFN-γ and HMGB1 were significantly decreased in the groups treated with sodium butyrate at 12 hours (836.70±95.43 U/L, 218.60±29.41 U/L, 4.33±0.95 µmol/L, 64.38±4.95 ng/L, 108.34±12.95 ng/L and 6.26±0.54 µg/L, respectively) and at 24 hours (1590.81±270.50 U/L, 579.08±86.59 U/L, 13.52±1.93 µmol/L, 93.52±12.96 ng/L, 184.52±19.08 ng/L and 11.47±1.18 µg/L, respectively) as compared with the model group (P<0.05).

 

The expressions of HMGB1 protein (Fig. 3A) and NF-��B p65 (Fig. 3B) were increased significantly in the model group as compared with the control group and decreased significantly in the groups treated with sodium butyrate at 12 hours and 24 hours as compared with the model group (P<0.05).

 

The mean survival duration of rats was 168.0 hours, 55.2 hours, 110.4 hours and 98.4 hours in the control, model and sodium butyrate treated groups at 12 hours and 24 hours, respectively (Fig. 4). Most ALF rats died within 3 days without treatment. Sodium butyrate treatment significantly prolonged the mean survival duration of the ALF rats. The log-rank test showed that there was a significant difference among the curves (χ²= 14.857, P=0.005).

 

 

It is believed that the pathogenesis of liver failure is related to "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 intestinal endotoxemia and the release of proinflammatory factors such as TNF-α.^[6] Intestinal endotoxemia, resulted from overgrowth and translocation of bacteria from the gut into peritoneal cavity due to the increased permeability of the intestinal wall, plays a significant role in the progresses of liver failure.^{[6, 7]}

 

Sodium butyrate, an important energy source for intestinal epithelial cells, has been previously shown to inhibit intestinal pathogenic bacteria and maintain gastrointestinal homeostasis.^[30] A study^[31] indicated that sodium butyrate could alleviate inflammatory reaction by suppressing of NF-κB activation and multiple proinflammatory cytokines. NF-κB is a transcription factor that controls the expression of genes encoding proinflammatory cytokines, chemokines, inducible inflammatory enzymes such as inducible NO synthase and cyclooxygenase-2, adhesion molecules, growth factors, some acute phase proteins and immune receptors.^[11] Apart from inhibition of NF-κB activation, sodium butyrate may exert an anti-inflammatory activity through inhibition of the IFN-γ production.^[32] It has been reported recently that HMGB1 protein, as a late inflammatory mediator, is involved in the processes of endotoxemia and ALF.^{[12, 33-35]} This study was designed primarily to explore the effects of sodium butyrate on liver tissue, intestinal permeability, endotoxin, HMGB1, NF-��B and multiple proinflammatory factors in ALF in rats.

 

In this study, an ALF rat model induced by injection with HSA, LPS and D-Gal was employed to evaluate the protective effect of sodium butyrate. Histological examinations showed that liver and intestinal tissue of rats in the model group showed severe pathological changes at 48-hour time point after induction of ALF. However, treatment with sodium butyrate can significantly improve the histopathology of liver and intestinal tissue, and prolong the survival duration after induction of ALF in rats. Moreover, the intestinal permeability was decreased greatly by sodium butyrate, which accompanied with the reduction of serum levels of ALT, AST, TBIL and endotoxin. These findings suggest that sodium butyrate could effectively protect liver tissue, improve intestinal permeability and reduce the mortality of ALF in rats.

 

Some studies^{[11, 31, 32]} indicated that sodium butyrate may alleviate inflammatory reaction by suppressing NF-κB activation. In our study, the expression of NF-κB p65 protein was suppressed in sodium butyrate treatment groups regardless of treating time point after induction of ALF as compared with the model group. Our data suggest that NF-κB signaling may be one of the primary pathways in the pathogenesis of ALF and could be inhibited by sodium butyrate.

 

Previous studies^{[36, 37]} have demonstrated that the inflammatory cytokines including TNF-α and IFN-γ are secreted actively by monocytes/macrophages in the patients and in animal models with liver failure. Moreover, HMGB1 is able to stimulate the release of multiple proinflammatory cytokines, including TNF-α, IFN-γ and IL-6 in many cell lines.^{[10, 38, 39]} Our results showed that sodium butyrate attenuated the injury of hepatic tissue, improved the permeability of the intestine and reduced the cytokines such as HMGB1, TNF-α and IFN-γ. It is suggested that sodium butyrate improved the intestinal permeability, alleviated intestinal endotoxemia, maintained gastrointestinal homeostasis, and consequently attenuated the cascade releases of multiple proinflammatory factors induced by endotoxemia, which may be a crucial mechanism for the protective effects of sodium butyrate on ALF in rats.

 

In conclusion, sodium butyrate is pleiotropically effective in inhibiting the expression of HMGB1 and NF-κB p65, decreasing the level of endotoxin, reducing the inflammatory cytokines, ameliorating the injury of the liver and intestine, improving intestinal permeability and survival, and resultantly protecting the rats with ALF. The mechanisms of drug action are not only related to direct hepatoprotection, but also to indirect approach through decreasing intestinal permeability and consequent intestinal endotoxemia. All of these data suggest that sodium butyrate is a potential novel therapeutic agent for ALF.

 

 

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