Thermal injury is one of the most severe forms of trauma with a high mortality rate (1). The pathogenesis is a complex process that may be rooted in the additive effects of inadequate tissue perfusion, hypermetabolic condition, free radical damage, and systemic alterations in cytokines (2). Based on current research findings, it is evident that local burn insult produces the release of large systemic inflammatory cytokines (3). However, The infection following burns is a very important cause of excessive systemic inflammatory response and multiple organ damage (4, 5). On one hand, tissue wounds on the skin surface provided the opportunistic infection condition of gram-positive bacteria (6). On the other hand, translocation of gram-negative microbiota and their toxic products resulting from disruption of the intestinal mucosal barrier after thermal injury is another vital cause (7, 8). The main types of infection were Pseudomonas, Escherichia coli, Staphylococcus, and Acinetobacter species (1).
Muramyl dipeptide (MDP) or N-acetyl-muramyl-l-alanine-D-isoglutamine is a minimally active subunit commonly found in gram-positive bacteria and gram-negative bacteria cell walls triggering the innate immune system (9). Experiments show that a certain amount of MDP is able to induce a wide range of defense responses against gram-positive bacteria and gram-negative bacteria and a large number of inflammatory cytokine production in vitro or in vivo (10).
Therefore, the aim of this investigation was to determine the effect of MDP on thermal injury–induced inflammatory responses, organ function injury, and mortality in rats—including the histopathology and enzymatic indicators of liver, kidney, heart, and lung tissues; the white blood cell (WBC) and platelet (PLT) counts; the plasma inflammatory cytokine concentration; arterial blood gas (ABG); and survival time and survival rate—and provide a possible experimental model choice for basic scientists and clinical workers in inflammatory diseases.
MATERIALS AND METHODS
Animal thermal injury procedure
Male Sprague-Dawley rats were maintained on sterile standard laboratory chow and water ad libitum in individual ventilated cages under specific pathogen–free conditions in the animal facility of Wuhan University. Wuhan University Institutional Animal Care and Use Committee approved all animal experiments. Animals were anesthetized by intraperitoneal injection of ketamine (75 mg·kg−1) plus midazolam (5 mg·kg−1). The dorsum of the rats was shaved (20% sodium sulfide) and exposed to a water bath at 99°C to 100°C for 12 s, which resulted in third-degree skin thermal injury involving 20% of the total body surface area (20% TBSA) (11). All animals were resuscitated with Ringer’s lactate solution via intraperitoneal route (50 ml·kg−1 body weight). The scald wound was treated with 1% silver sulfadiazine against wound infection. Analgesic treatment was performed with 0.1 mg·kg−1 buprenorphine every 12 h subcutaneously. At the end of study, the animals were killed via abdominal aorta bleeding under ketamine anesthesia.
Fifty male rats weighing 200 to 250 g were randomly assigned into three groups as follows. The normal control group (n = 10) underwent the same procedure except for thermal injury; their backs were immersed in a water bath at 25°C for the same period. The scald group (n = 10) was inflicted with 20% TBSA third-degree thermal injury. The MDP group (n = 30) was injected with MDP (5 mg·kg−1) (12) via the left femoral vein at 24 h after thermal injury. Blood samples and tissue specimens were collected at 24 h after burn injury in the scald group. The MDP group rats were equally divided into three subgroups: MDP I, MDP II, and MDP III groups, each subgroup of 10 rats, and the blood samples and tissue specimens were collected at 1, 6, and 24 h after MDP administration or 25, 30, and 48 h after burn injury, respectively.
In addition, 90 male rats were divided into three equal groups as follows: normal control group, scald group, MDP group, each group of 30 rats. All animals were observed for survival time, and survival rates for 72 h after the initial operation burn were calculated. When animals appear to show distress or suffering, severely declining condition, inability to move, inability to drink, shortness of breath, moribund, or even coma condition, death should be viewed as an end point; the animals would be put in a closed CO2 glass bottle and the animals will die. All efforts were made to minimize suffering.
The histopathology of liver, kidney, heart, and lung tissue
At 24 h after thermal injury in the scald group and at 1, 6, and 24 h after administration of MDP in the MDP group, the liver, kidney, heart, and lungs were then removed. The tissue specimens were fixed in 10% formaldehyde solution for 48 h, embedded in paraffin and cut into 4-μm pieces by microtome, and stained with hematoxylin and eosin (H&E), observing the pathological morphological changes.
Enzymatic indicators of liver, kidney, heart, and lung function
At 24 h after thermal injury in the scald group and at 1, 6, and 24 h after administration of MDP in MDP I, MDP II, and MDP III groups, whole blood was drawn via the abdominal aortic artery (2.0 mL each). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), blood urea nitrogen (BUN), creatinine (Cr), and creatine kinase isoenzyme-MB (CK-MB) levels were determined enzymatically by an automated VetScan chemistry analyzer (Abaxis, Union City, Calif).
White blood cell and platelet counts, plasma inflammatory cytokine levels, and analysis of ABGs
At 24 h after thermal injury in the scald group and at 1, 6, and 24 h after administration of MDP in the MDP I, MDP II, and MDP III groups, whole blood was drawn via the abdominal aortic artery (4.0 mL each). The WBC and PLT counts in the whole blood were analyzed. The plasma was immediately separated by centrifugation at 3,000 rpm for 15 min at 4°C, divided into aliquots, and stored at -70°C until assayed; plasma TNF-α, interferon-γ (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and high-mobility group box 1 (HMGB1) protein levels were quantified using enzyme-linked immunosorbent assay kits (American R & D and Bender). Arterial blood gases were immediately determined using a portable blood gas analyzer (i-STAT, Princeton, NJ).
The activity of lung tissue myeloperoxidase assay
One gram of snap-frozen lung tissue was homogenized in 0.05 M potassium phosphate buffer at pH 5.5 and centrifuged at 3,000 rpm for 10 min at 4°C. The pellet was redissolved in 10 mL of 0.05 M potassium phosphate buffer at pH 5.5 containing 0.5% hexadecyltrimethyl ammonium bromide. An aliquot of the supernatant was assayed by measuring the H2O2-dependent oxidation of tetramethyl benzidine in sodium phosphate buffer. Absorbance at 450 nm of visible light was measured, and the myeloperoxidase (MPO) activity was calculated in units per gram of lung tissue.
Data were presented as mean ± SD. Data analysis was performed using SPSS 18.0 software. One-way analyses of variance were used to evaluate differences associated with main sources of variation. When the F statistic was significant for analyses of variance comparisons, differences between individual means were tested for significance using the Bonferroni test. The Bonferroni test is a post hoc test that adjusts α for multiple comparisons. The survival curves were analyzed using the log-rank statistical method. A significant difference was presumed at a value of P < 0.05.
The histopathology of liver, kidney, heart, and lung tissues
Under light microscope observation, H&E staining revealed the normal structure of the liver, kidney, heart, and lung parenchyma in the normal control group. Thermal injury produced inflammatory cell infiltration in the hepatic gate-duct area and hepatic cell necrosis; glomerular basement membrane thickening, mesangial matrix increasing, and interstitial inflammatory cell infiltration; cardiac interstitial edema in myocardial tissues; and neutrophil cells infiltrating into alveolar interstitial and alveolar spaces in the scald group. Stimulation with MDP after thermal injury led to a wider range of organ dysfunction and inflammatory responses, including ballooning degeneration and putrescence of hepatic cells; hyperplasia of Kupffer cells, necrosis, vacuolation, and desquamation of epithelial cells in the renal tubules; cardiac fiber engorgement; cytoplasm destruction, more inflammatory cell infiltration into the alveolar interstitial and alveolar spaces, broadened pulmonary interval, and collapsed alveolar (Figs. 1–4).
Enzymatic indicators of liver, kidney, heart, and lung function
Compared with the normal control group, after thermal injury challenge alone, serum ALT, AST, and BUN levels and the pulmonary MPO activity were significantly increased (P < 0.05). Compared with the scald group, serum ALT, AST, TB, BUN, Cr, and CK-MB levels and pulmonary MPO activity were significantly elevated (P < 0.05) in the MDP group (Fig. 5).
Peripheral WBC and PLT counts
Compared with the normal control group, the whole WBC count after thermal injury challenge was increased (P < 0.05), and the PLT count had no difference. Compared with the scald group, the WBC and PLT counts decreased significantly (P < 0.05) in the MDP group (Fig. 6).
The plasma inflammatory cytokine levels
Compared with the normal control group, plasma IL-6, IL-10, and IFN-γ levels in the scald group were significantly increased (P < 0.05). Compared with the scald group, plasma TNF-α, IL-6, IFN-γ, and HMGB1 levels in the MDP group were significantly higher (P < 0.05, respectively). Plasma TNF-α levels reached a peak (49.4 ± 4.1 pg·mL−1) at the 6-h time point in the MDP group. Plasma IL-6, IFN-γ, and HMGB1 levels reached a peak: 191.2 (±12.4) pg·mL−1, 257.2 (±20) pg·mL−1, and 2.15 (±0.56) pg·mL−1 at the 24-h time point in the MDP group, respectively (Fig. 7).
Analysis of ABGs
The oxygen, carbon dioxide tension, arterial pH, and base excess did not differ significantly between the normal control group and the scald group during the experimental period. Compared with the scald group, decreased oxygen, increased carbon dioxide tension, and significantly lower arterial pH value and higher BE were observed in the MDP group (P < 0.05) (Fig. 8).
General situation and survival analysis of rats
During the 72-h experimental period after burn injury, MDP stimulation led to animals’ less behavior, sleepiness, piloerection, short- and fast-frequency breath, no reaction to surrounding environment, no drinking water, diarrhea, and pyuria, accompanied by a large number of animal death. In addition, we have recorded the survival time and survival rate. All animals in the normal control group survived, and 70% of the animals in the scald group survived, but the survival rate significantly decreased to 23.3% in the MDP group (Fig. 9).
We made use of thermal injury, and observation of the histopathology of liver, kidney, heart, and lung tissues shows different degrees of tissue hyperemia, edema, and inflammatory cell infiltration during thermal injury. Enzymatic or metabolic indicators of organ function indicated that scald injury induced high serum ALT, AST, TB, Cr, BUN, CK-MB, and MPO levels and high plasma inflammatory cytokine concentrations. However, thermal injury combined with a second hit of MDP increased enzymatic or metabolic indicators of organ function, deteriorated hypoxemia, metabolic acidosis, and inflammatory cytokines. The effect of MDP is an important finding of the present study.
In our study, the whole WBC counts increased significantly after thermal injury alone, whereas MDP administration after thermal injury caused a significant reduction in WBC and PLT, which might be associated with exaggerated inflammatory responses and suppressed immune function in rats. Muramyl dipeptide is the minimum effective component that exists generally in gram-negative bacteria and gram-positive bacteria cell wall triggering the innate immune system (9, 13). Muramyl dipeptide is recognized by leukocytes and other cells of humans and animals and then leads to production of inflammatory cytokines such as TNF-α, IL-6, and IL-10 (14). Our study suggested that MDP markedly elevated the plasma inflammatory cytokines IL-6, IL-10, IFN-γ, and HMGB1 levels after thermal injury.
In 2012, Grimes et al. (15) confirmed that MDP could directly bind to Nod2 and is a high-affinity ligand for Nod2. Muramyl dipeptide exposure led to a more serious systemic inflammatory response that might be associated with activated Nod2/Nod2 receptor–interacting protein 2 (receptor-interacting protein 2 [RIP2]) complex signaling pathways, resulting in activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, and then initiating the synthesis and release of a large number of inflammatory mediators (16). Certainly, as a traumatic factor of thermal injury, it is also an important reason leading to systemic inflammatory responses. In 2011, Ravat et al. (3) revealed that thermal injury is an inflammatory process, which is consistent with thermal injury producing massive inflammatory cytokines and multiple organ damage. In 2012, Huber et al. (17) discovered that lipopolysaccharide (LPS) caused an acute increase in serum IL-6, IL-10, and chemokine keratinocyte-derived chemokine levels after 25% TBSA scald injury. Lipopolysaccharide recognition is mediated primarily via the Toll-like receptor 4-MD2 receptor complex signaling pathway, resulting in activation of relative transcription factors and proinflammatory cytokine production (18). Toll-like receptor 4 is a transmembrane protein that can recognize pathogen-associated molecular pattern LPS at the extracellular level. How is MDP different from LPS response? Nod2 by MDP triggers a signaling cascade through the common adapter protein, receptor-interacting serine/threonine-protein kinase 2 (RICK), resulting in NF-κB and MAPK and an inflammatory cytokine/chemokine response (19). Nod2 is a nontransmembrane protein that can recognize pathogen-associated molecular pattern MDP at the intracellular level. It is involved in the innate immune response in mammalian animals.
In the present study, we have not detected the alteration of TNF-α because TNF-α is viewed as an early proinflammatory cytokine and plasma TNF-α is in comparatively lower levels at 24 h after thermal injury. High-mobility group box 1 was known as a mediator of delayed critical disease lethality, severe burn, and systemic inflammatory response. In 2002, Fang et al. (20) revealed that HMGB1 expression significantly increased at 24 h after burns and remained markedly elevated up to 72 h.
In addition, we also surveyed the animals’ general condition and survival rate during the combined second hit of MDP. The animals’ general condition manifests shortness of breath, urine turbidity, diarrhea, supine, piloerection, and no drinking water and dispirited until death. Compared with approximately 70% survival rate after scald injury alone in 72 h, only 23% survived after the combined second hit of MDP, suggesting that MDP enhanced thermal injury–induced high mortality rates in rats, which might be associated with overwhelming systemic inflammatory responses and multiple organ injury.
In summary, thermal injury and combined second hit of MDP could result in a systemic inflammatory response, multiple organ dysfunction syndrome, and high mortality in rats. The mechanism might be associated with Nod2 oligomerization, raised RIP2 by MDP stimulation, and activation of NF-κB signaling pathways, resulting in the synthesis and release of a large number of inflammatory mediators (21, 22). Nevertheless, further investigation is needed, including Nodlike receptors and negative regulatory protein-erbin and upriver genes (23, 24), the complex mechanisms involved in the signaling pathway.
Limitations of this study
The observation did not include MDP alone, which could be a limitation of the present study. In 2008, Murch et al. (12) investigated the effects of MDP on organ injury/dysfunction caused by systemic administration of a low dose of LPS in rats; administration of MDP alone did not result in a significant organ injury or increases in cytokine production and lung MPO activity compared with the vehicle group. However, administration of MDP 24 h before LPS resulted in a significant increase in the degree of organ injury, cytokine release, and lung injury compared with LPS alone. Therefore, Murch et al. thought that MDP enhanced the cytokine response to endotoxin and caused multiple organ injury in rats, which is in accordance to our study.
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Keywords:© 2014 by the Shock Society
Muramyl dipeptide; thermal injury; IL-6; HMGB1; ALT