Sepsis and septic shock as a result of an invasive infection are challenging problems in critically ill patients and frequently end in acute organ dysfunction. Despite significant advances in intensive care technologies and tremendous enthusiasm in the development of new antibiotics, severe sepsis still claims 40% mortality in Western countries. Systemic activation of the inflammatory system is believed to be of pivotal importance in producing this symptom complex. Membrane components from bacteria such as LPS from gram-negative bacteria and exotoxins from gram-positive bacteria, as well as bacterial DNA, are known to induce inflammatory responses. These toxic components of invading microorganisms are able to produce rapid activation of the innate immune system, leading to a release of excessive amounts of proinflammatory cytokines such as TNF-α and IL-1β. However, antagonists of these cytokines have not been proven efficacious in the treatment of sepsis in the clinical settings (1). One of the main reasons could be that these cytokines are released early in the development of sepsis, thus leaving a narrow therapeutic window for administration of antagonists, because the septic process has already begun, and the therapeutic agents fail to suppress it due to the time lag.
High-mobility group box protein 1 (HMGB1) has recently been extensively studied. It is a nuclear protein that binds DNA, stabilizes nucleosomes, and facilitates gene transcription. It is found to be released by activated monocytes and macrophages in response to various stimuli such as LPS, TNF-α, or IL-1β with a delay of 9 to 16 h (2) because its serum level increases significantly during endotoxemia and sepsis with a significant retardation in comparison with TNF-α and IL-1β (2). Secretion of HMGB1 in turn triggers the release of several cytokines from monocytes and neutrophils (3), and at the same time, it stimulates the release of HMGB1 itself from the same cells it activates (2) as an autocrine to maintain an inflammatory cascade. Thus, HMGB1 acts as a late mediator of inflammation. However, when it is passively released by necrotic cells, HMGB1 shows a greatly reduced ability to promote inflammation, which proves that the release of HMGB1 can signal the demise of a cell in its neighborhood (4). It has also been demonstrated that HMGB1 level in dead patients is markedly higher than in survivors (2). The serum content of HMGB1 was found to be increased after hemorrhagic shock (5), and its expression in the lungs increased after hemorrhage (6), suggesting that HMGB1 participated in hemorrhage-induced acute lung injury. One prospective study in a population of patients with sepsis, severe sepsis, or septic shock showed that HMGB1 levels remained high in most patients up to 1 week after admission (7). Also, HMGB1 may enhance and prolong the inflammatory processes and, thus, illness, in malaria (8). Stearoyl lysophosphatidylcholine significantly attenuates circulating HMGB1 levels in endotoxemia and sepsis by suppressing endotoxin-induced HMGB1 release from macrophages/monocytes (9). Treatment with anti-HMGB1 antibody and other antagonists has been found to protect animals against sepsis and other potentially fatal systemic inflammatory disorders even when administered significantly later than any other cytokine-targeted interventions (10). Anti-HMGB1 antibodies lower the lethality of sepsis even when given 24 h after the onset of infection, proposing a clinically relevant time frame that offers a significantly wider treatment option for reversal of lethal sepsis (10).
Butyrate is a 4-carbon fatty acid normally produced as a result of bacterial fermentation of fiber in mammalian intestines. The short-chain fatty acid butyrate is capable of maintaining epithelial cell differentiation (11) and the healthy differentiated state of normal colonic epithelial cells and inhibiting cell growth and promoting differentiation of neoplastic cells (12). Moreover, butyrate is known to induce apoptosis of cells in a number of cancers, including colon cancer cell lines (13). Several studies suggest that butyrate induces apoptosis through a histone hyperacetylation-mediated pathway, which results in the conversion of caspase 3 from its proenzyme form to the catalytically active protease, a process that is dependent on new protein synthesis (14).
Recently, butyrate has received a lot of attention because it possesses anti-inflammatory activities. Although its precise mechanisms of action are not well understood, the anti-inflammatory effect of butyrate is probably mediated by the suppression of nuclear factor kappa B (NF-κB) activation, which is a well-known inflammatory mediator (15, 16). The antiproliferative and apoptotic effects of butyrate and other short-chain fatty acids may be linked to histone hyperacetylation (11). Butyrate inhibits histone deacetylase, resulting in a relative hyperacetylation of core histone proteins (H3 and H4) (17). Hyperacetylation of histones disrupts ionic interactions with the adjacent DNA backbone, creating less densely packed chromatin or euchromatin, and allowing transcription factors to activate specific genes (18). Up to now, it has not been fully elucidated how HMGB1 is produced in sepsis, whether it plays a key role in sepsis-induced multiple organ damage, and whether sodium butyrate possesses the effect of inhibition of late proinflammatory mediator HMGB1 in sepsis. In this study, a cecal ligation and puncture (CLP) model was used to reproduce sepsis in rats, and sodium butyrate was given to find out whether it was beneficial in inhibiting HMGB1 expression to block septic process.
MATERIALS AND METHODS
To reproduce intra-abdominal infection and sepsis, CLP was performed as described by Wichmann et al. (19). This procedure reproducibly results in polymicrobial peritonitis, bacteremia, and sepsis. Briefly, male Wistar rats weighing 220 to 250 g were anesthetized with sodium pentobarbital (80 mg/kg, i.m.). The abdomen was opened under aseptic condition. The cecum was ligated and punctured to produce 3 holes with an 18-gauge needle. A strip of rubber was inserted into the cecum to prevent inadvertent sealing of the puncture. The cecum was then replaced, and the abdomen was closed. All animals received subcutaneous injection of Ringer solution (40 mL/kg of body weight) on the back for resuscitation after the procedure.
One hundred Wistar rats were randomly divided into 4 groups as follows: (1) control group (10 rats); (2) sham operation group (10 rats), with laparotomy only; (3) CLP group (60 rats), which were further divided into 6 subgroups with 10 rats in each subgroup, with animals being killed at 2, 6, 12, 24, 48, and 72 h, after CLP; (4) sodium butyrate (Sigma Chemical Co, St Louis, Mo) treatment group (20 rats), which received 2 injections of sodium butyrate (500 mg/kg, i.v.) at 0.5 and 4 h post-CLP, and they were further divided into 2 subgroups of 10 rats each, which were killed at 12 and 24 hs after CLP, respectively. We took CLP group of corresponding time points as contrast group. Blood samples and tissues were harvested from liver, lungs, kidneys, and small intestine for examinations.
To testify the therapeutic efficacy of sodium butyrate in septic rats, in another group with 22 rats, in addition to the administration of sodium butyrate (500 mg/kg, i.v.) at 0.5 and 4 h after CLP, antibiotics (gentamicin, 8 mg/kg; metronidazole, 25 mg/kg; and aminobenzylpenicillin, 150 mg/kg, i.m.) were given at 0.5, 12, 24, 36, 48, and 60 h after CLP. A CLP group consisting of 35 rats as contrast received i.v. Ringer solution and antibiotics after CLP. Mortality of rats in each group was recorded up to 7 days after the procedure.
The dosage of sodium butyrate for the rats was decided according to the results of toxicology experiment and pharmacokinetics study of the drug (20).
All experimental manipulations were undertaken in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals with the approval of the Scientific Investigation Board of Postgraduate Medical College of PLA, Beijing.
Total RNA extraction, HMGB1, and TNF-α mRNA assay
Tissues (50 mg) of the liver, lungs, kidneys, and small intestine were collected under aseptic condition. Total cell RNA was isolated by a single-step technique of acid guanidinium thiocyanate-chloroform extraction (21) (Promega Co, Madison, Wis). Tissue HMGB1 and TNF-α mRNA expression was amplified by the reverse-transcription-polymerase chain reaction, and semiquantitated glyceraldehyde 3-phosphase dehydrogenase (GAPDH) was taken as an internal standard. Photos of amplified genes were taken after agarose gel (2%) electrophoresis. The integral optic density of electrophoresis zone was measured by image analysis system (Leica, Wetzlar, Germany). The relative value of HMGB1 or TNF-α was the ratio of its mRNA value to GAPDH mRNA. The following gene-specific primers were used for rat HMGB1 (680 bp): 5′-ATG GGC AAA GGA GAT CCT A-3′ (sense strand); 5′-ATT CAT CAT CAT CAT CTT CT-3′ (antisense strand) (2); rat TNF-α (415 bp): 5′-GTA GCC CAC GTC GTA GCA AA-3′ (sense strand); 5′-CCC TTC TCC AGC TGG AAG AC-3′ (antisense strand) (22); and rat GAPDH (309 bp): 5′-TCC CTC AAG ATT GTC AGC AA-3′ (sense strand); 5′-AGA TCC ACA ACG GAT ACA TT-3′ (antisense strand) (23).
Blood samples were obtained by cardiac puncture. Serum levels of blood alanine aminotransferase (ALT) and creatinine (Cr) were measured by using commercially available clinical assay kits.
Lung tissue myeloperoxidase determination
Myeloperoxidase (MPO) is a major constituent of neutrophil cytoplastic granules, and its activity is usually taken as an indirect indicator of neutrophil infiltration and acute lung injury (24). Myeloperoxidase activity was determined by the method of Kuebler et al. (24). Briefly, lung tissue was homogenized in a 9-fold volume potassium phosphate buffer (20 mol/L; pH 7.4) and was centrifuged for 30 min at 35,000 rpm at 4°C. Myeloperoxidase activity per gram of wet lung (gwl) was calculated by the following formula: Myeloperoxidase activity (U/gwl) = (ΔA460) × (13.5) per lung weight (g), where ΔA460 is the change in absorbance of 460 nm from 30 to 90 s after the initiation of the reaction. The coefficient 13.5 was determined empirically such that 1 U MPO activity is the amount of enzyme that will reduce 1 μmol peroxide/min.
Determination of diamine oxidase in intestinal tissue
The activity of the mucosal enzyme diamine oxidase (DAO) serves as a marker of intestinal mucosal maturation and integrity. Diamine oxidase is an intracellular enzyme found in high activity in the intestinal mucosa of all mammalian species, including humans. Its activity is localized primarily within the cytoplasm of the mature differentiated upper villous cells (25). Tissue DAO activity was assayed by the modified method of Koike as described in our previous studies (26).
Analysis of data
Data were expressed as mean ± SE. To analyze the data statistically, independent-sample t test and 1-way ANOVA were used to determine which means were significantly different from the mean of the control group periods. Chi-square was used to determine the differences in mortality rate between groups, and correlation analysis was used to confirm the relationship between tissue HMGB1 mRNA expressions and organ damage indices. A P value of 0.05 or less was considered to indicate statistical significance.
HMGB1 gene expression
As shown in Figure 1, A-D, HMGB1 mRNA expression was found with low levels in the liver, kidneys, lungs, and small intestine in both control and sham operation groups. Two hours after CLP, no obvious changes in HMGB1 mRNA expression were observed in various tissues. HMGB1 mRNA levels began to be markedly up-regulated in the liver, lungs, and small intestine at 6 h but only slightly in kidneys at 12 h after CLP (0.516, 0.523, 0.429, and 0.466, respectively). The values maintained high levels up to 72 h compared with baseline values (P < 0.05-0.01). High-mobility group box 1 protein mRNA levels in the various tissues peaked at 24 h after CLP (P < 0.01). Among 4 tissues, the highest level was found in the liver, and its peak value was 3.13 times of that of the control group.
Effect of sodium butyrate on HMGB1 gene expression
As shown in Figure 2, A and B, early therapy with sodium butyrate can significantly reduce HMGB1 mRNA expressions in the liver, lungs, kidneys, and small intestine compared with that of CLP group at 12 (P < 0.05) and 24 h (P < 0.05-0.01) after CLP.
TNF-α gene expression
As shown in Figure 3, A-D, TNF-α mRNA expression was found with low levels in various tissues in both control and sham operation groups. Rapid increases in TNF-α mRNA expression were observed in the liver, lungs, kidneys, and small intestine at 2 h after CLP (P < 0.01). TNF-α mRNA levels gradually decreased to normal range at 24 h in liver and lungs, at 48 h in kidneys, and at 6 h in small intestine. At 48 h after CLP, TNF-α mRNA expression in liver and lungs increased again, and 2 peaks were observed at 2 and 48 h.
Effect of sodium butyrate on TNF-α gene expression
As shown in Figure 4, A and B, treatment with sodium butyrate could not inhibit TNF-α mRNA expressions in various tissues compared with that of CLP group at both 12 and 24 h after CLP except renal expression at 24 h after CLP (P = 0.047).
Protective effect of sodium butyrate on organ injury in septic rats
As shown in Figure 5, A-D, sodium butyrate can protect the injury of the liver, kidneys, and lungs but not small intestine in septic animals. Treatment with sodium butyrate decreased serum ALT contents of septic rats at 12 h after CLP compared with CLP group (81 ± 11 vs. 127 ± 26, P = 0.008), but showed no such effect at 24 h. Meanwhile, it decreased serum Cr at both 12 and 24 h after CLP in sodium butyrate treatment group compared with CLP group (12 h: 49 ± 2 vs. 57 ± 2, P = 0.008; 24 h: 52 ± 2 vs. 58 ± 2, P = 0.050). Pulmonary MPO activity was decreased after sodium butyrate treatment only at 24 h after CLP (2.5 ± 0.6 vs. 7.4 ± 1.1, P = 0.014). However, there was no change in DAO activity of intestinal mucosa after treatment with butyrate.
Effect of sodium butyrate on mortality of post-CLP septic rats
As shown in Figure 6, survival rate of the septic rats treated with sodium butyrate was significantly increased compared with the rats treated with Ringer solution only from the first day to the sixth day (P < 0.05-0.01). However, at 12 h after the onset of experimental peritonitis, butyrate treatment showed no marked therapeutic effect on mortality rate compared with CLP group [95.5% (21/22) vs. 77.1% (27/35)]. It was notable that the difference in survival rate between these 2 groups became significant (P = 0.034) at 24 h after CLP; the difference remained significant up to the sixth day (P = 0.001-0.003).
In the CLP group, a positive correlation was found between hepatic HMGB1 mRNA expression and serum ALT content (r = 0.2901, P = 0.0210) and also between pulmonary HMGB1 mRNA expression and MPO activity (r = 0.2988, P = 0.0331). No significant correlation was noted between renal HMGB1 mRNA expression and serum Cr or intestinal HMGB1 mRNA expression and DAO activity. No correlations were found between hepatic TNF-α mRNA expression and serum ALT content, between pulmonary TNF-α mRNA expression and MPO activity, between renal TNF-α mRNA expression and serum Cr, and between intestinal HMGB1 mRNA expression and DAO activity (all P > 0.05).
In the present experiment, we observed increased HMGB1 mRNA expression in tissues from 6 to 72 h after CLP peaking at 24 h. Not only are liver and kidneys damaged as shown by significantly elevated serum levels of ALT and Cr, but lungs and small intestine mucous membrane are also damaged severely in view of a significant increase in MPO activity and marked lowering of DAO activity secondary to sepsis. Rat serum ALT content and lung MPO activity manifested significant positive correlation with HMGB1 mRNA in the liver and lungs, respectively. It was reported that serum HMGB1 level was found to be remarkably elevated in septic patients, and HMGB1 level in nonsurvivors was significantly higher than in survivors, denoting that HMGB1 level is closely related to degree of sepsis and its prognosis (2). Furthermore, giving HMGB1 intratracheally can produce acute inflammatory injury in the lung, with dense neutrophil infiltration, development of lung edema, and an increase in production of IL-1β, TNF-α, and macrophage inflammatory protein 2 in the lung (27). In endotoxin-induced acute lung inflammation, administration of anti-HMGB1 antibody either before or after endotoxin exposure decreased the aggregation of neutrophils in the lungs and edematous change (27).
It is clear that endotoxin causes tissue damage and lethal shock through producing various inflammatory mediators. Among these mediators, TNF-α and IL-1β are the most important ones. Up to the present, however, almost all antagonists of these mediators have not been found to be efficacious in clinical settings (1). Animal experiments have confirmed that mice challenged by a lethal dosage of endotoxin all died in a few days, whereas to our bewilderment, TNF-α and IL-1β levels were usually found to have declined within normal range already. Rapid increases in TNF-α mRNA expression were observed in the liver, lungs, kidneys, and small intestine at 2 h after CLP, but TNF-α mRNA levels gradually decreased to normal range at 24 h in liver and lungs, at 48 h in kidneys, and at 6 h in small intestine. Interestingly, there was a second elevation in TNF-α mRNA expression in liver and lungs at 48 h after CLP, whereas no correlation was found between tissue TNF-α mRNA expression and organ damage indices. Recently, it was found that all mice given anti-HMGB1 antibody 0.5 h before the challenge of a lethal dose of endotoxin survived, whereas all control animals died. This finding indicates that HMGB1 is closely related to the pathogenetic effect of endotoxin (2). It has also been demonstrated that when an anti-HMGB1 antibody is given 2 h after a challenge of a lethal dose of endotoxin, 60% of the mice survived (2).
The present experiments showed that sodium butyrate significantly inhibited HMGB1 expression in various tissues, demonstrating that it can down-regulate excessive HMGB1 gene expression secondary to septic challenge. Butyrate was also found to give a significant protection against LPS-induced inflammatory response in rat primary microglial cells pretreated with butyrate in vitro (28). Several studies with colonocytes (15, 29), cancer cells (30), and murine macrophages (16) have shown that butyrate inhibits the activation of NF-κB signaling and the DNA binding of NF-κB complex induced by cytokines or LPS but does not inhibit IFN-γ-stimulated Janus kinase/signal transducer and activator of transcription (STAT) activation in microglia (29). Our previous study has shown that HMGB1 stimulation can activate STAT1 and STAT3 in peritoneal macrophages at 2 h (31). It makes sense that Janus kinase/STAT pathway might be involved in regulation of HMGB1-induced inflammatory responses. The possible mechanism is that butyrate may activate STAT-dephosphorylating tyrosine phosphatases, thus attenuating STAT activity. Thus, inhibition of NF-κB signaling by butyrate effectively prevents expression and release of late inflammatory mediators (32).
Our results showed that butyrate treatment can decrease the mortality rate and attenuate multiple organ damage resulting from severe sepsis. It was assumed that early treatment of sodium butyrate in septic animals can prevent damage to the liver, kidneys, and lungs to various degrees. Among them, the kidneys seemed to be best protected. Furthermore, butyrate treatment can significantly improve the outcome of septic animals from the first day to the sixth day. Therefore, it seems that sodium butyrate treatment possesses the ability of prolonging the life of septic rats probably through its inhibitory effect on HMGB1 expression.
In summary, the current experiment suggests that HMGB1 is expressed and produced in a severe polymicrobial infection, and sodium butyrate can markedly inhibit HMGB1 mRNA expression and might have a protective effect on multiple organ damage associated with severe sepsis.
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