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ABSENCE OF PEROXISOME PROLIFERATORS-ACTIVATED RECEPTORS (PPAR) α ENHANCED THE MULTIPLE ORGAN FAILURE INDUCED BY ZYMOSAN

Di Paola, Rosanna*; Esposito, Emanuela; Mazzon, Emanuela*‡; Genovese, Tiziana*‡; Muià, Carmelo*; Crisafulli, Concetta*; Malleo, Giuseppe*; Sessa, Edoardo; Meli, Rosaria; Cuzzocrea, Salvatore*‡

doi: 10.1097/01.shk.0000230299.78515.2c
Basic Science Aspects
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ABSTRACT The peroxisome proliferator-activated receptor (PPAR) α is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid, and thyroid hormone receptors. The aim of the present study is to evaluate the role of PPAR-α receptor on the development of multiple-organ dysfunction syndrome (MODS) induced by zymosan. MODS was induced by peritoneal injection of zymosan (dose, 500 mg/kg i.p. as a suspension in saline) in PPAR-α wild-type (PPAR-αWT) and PPAR-α knockout (PPAR-αKO) mice, was assessed 18 h after the administration of zymosan, and was monitored for 12 days (for loss of body weight and mortality). A severe inflammatory process, induced by zymosan administration in wild-type mice, coincided with the damage of liver, kidney, pancreas, and small intestine. Myeloperoxidase activity, indicative of neutrophil infiltration, and lipid peroxidation were significantly increased in zymosan-treated wild-type mice. Zymosan in the wild-type mice also induced a significant increase in the plasma levels of nitrite/nitrate. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine and Fas ligand in the intestine of zymosan-treated wild-type mice. In contrast, the degree of (1) peritoneal inflammation and tissue injury, (2) nitrotyrosine formation and Fas ligand expression, and (3) neutrophil infiltration were markedly enhanced in intestinal tissue obtained from zymosan-treated PPAR-αKO mice. Zymosan-treated PPAR-αKO mice also showed a significantly increased mortality. Taken together, the present study clearly demonstrates that PPAR-α pathway modulates the degree of MODS associated with zymosan-induced nonseptic shock.

*Dipartimento Clinico Sperimentale di Medicina e Farmacologia, Facoltà di Medicina e Chirurgia, Università di Messina, Italy; IRCCS Centro Neurolesi "Bonino-Pulejo," Messina, Italy; and Dipartimento di Farmacologia Sperimentale, Università di Napoli "Federico II," Via D. Montesano 49, 80131 Napoli, Italy

Received 12 Apr 2006; first review completed 28 Apr 2006; accepted in final form 18 May 2006

Address reprint requests to Salvatore Cuzzocrea, PhD, Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina, Italy. E-mail: salvator@unime.it.

Rosanna Di Paola and Emanuela Esposito contributed equally to this work.

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INTRODUCTION

Multiple-organ dysfunction syndrome (MODS) is a cumulative sequence of progressive deterioration of function occurring in several organ systems, frequently observed after septic shock, multiple trauma, severe burns, or pancreatitis (1, 2). Zymosan is a nonbacterial, nonendotoxic agent that produces acute peritonitis and multiple-organ failure characterized by functional and structural changes in liver, intestine, lung, and kidneys (3). The organ dysfunction in zymosan-treated animals may be, in part, dependent on bacterial translocation (4). We have recently reported that zymosan administration to mice causes both signs of peritonitis and of organ injury within 18 h (5). The onset of the inflammatory response caused by zymosan in the peritoneal cavity was associated with systemic hypotension, high peritoneal and plasma levels of nitric oxide (NO), maximal cellular infiltration, exudate formation, and cyclooxygenase activity (5). In addition, the generation of free fatty acids and eicosanoids has been demonstrated in the zymosan-induced peritonitis model (6).

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid, and thyroid hormone receptors (7). The PPAR subfamily comprises of 3 members: PPAR-α, PPAR-β, and PPAR-γ (8). In rats, PPAR-α is most highly expressed in brown adipose tissue, then in liver, kidney, heart, and skeletal muscle (9). Recently, it has also been demonstrated that PPAR-α is also expressed in the digestive tract, mainly localized in the intestinal mucosa in the small intestine, colon (10), and pancreas. PPAR-α binds to a diverse set of ligands, (i.e., arachidonic acid metabolites [prostaglandins and leukotrienes] and plasticizers) and synthetic fibrate drugs, including clofibrate, fenofibrate, and bezafibrate (11). Although PPAR-α has been less studied than PPAR-γ, PPAR-α ligands have also been shown to regulate inflammatory responses (12). In addition, we and other authors have clearly demonstrated that PPAR-α-deficient mice have abnormally prolonged responses to different inflammatory stimuli (13, 14). However, the role of endogenous PPAR-α ligands in conditions associated with experimental MODS has not yet been investigated.

In this study, we have investigated the role of endogenous PPAR-α ligands in a model of zymosan-induced nonseptic shock. To characterize the role of endogenous PPAR-α ligands in this model of nonseptic shock, we have determined the following endpoints of the inflammatory response: (1) neutrophils infiltration, (2) lipid peroxidation, (3) nitrotyrosine formation, (4) TNF-α production, (5) Fas ligand (FasL) activation, (6) organ injury, and (7) mortality.

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MATERIALS AND METHODS

Animals

Mice (age, 4-5 weeks; weight, 20-22 g) with a targeted disruption of the PPAR-α gene (PPAR-αKO) and littermate wild-type controls (PPAR-αWT) were purchased from Jackson Laboratories (Harlan Nossan, Italy). Mice homozygous for the PparatniJGonz targeted mutation mice are viable, fertile, and seem healthy in appearance and behavior. Exon 8, encoding the ligand-binding domain, was disrupted by the insertion of a 1.14-kb neomycin resistance gene in the opposite transcriptional direction. After electroporation of the targeting construct into J1 ES cells, the ES cells were injected into C57BL/6N blastocysts. This strain was created on B6, 129S4 background, and is maintained as a homozygote on a 129S4/SvJae background by brother-sister mating. The study was approved by the University of Messina review board for the care of animals. The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care complied with the regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986), and United States (Animal Welfare Assurance No. A5594-01, US Department of Health and Human Services).

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Zymosan-induced peritonitis in mice

The animals were randomly divided into 4 groups (n = 10 for each group). The first group (PPAR-αWT) was treated with saline solution (concentration, 0.9% NaCl i.p.) and served as sham group. The second group (PPAR-αWT) was treated with zymosan (dose, 500 mg/kg i.p. [suspended in saline solution]). In the third and fourth groups, PPAR-αKO mice received saline or zymosan administration, respectively. In another set of experiments, animals (n = 20 for each group) were randomly divided into 4 groups (as previously described) and monitored for loss of body weight and mortality for 12 days after zymosan or saline administration.

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Clinical scoring of systemic toxicity

The clinical severity of systemic toxicity (conjunctivitis, ruffled fur, diarrhea, and lethargy) in the mice was measured for 12 days after zymosan or saline injection on a subjective scale ranging from 1 to 3 (0 indicates absence of toxicity; 1, mild; 2, moderate; and 3, serious). All clinical score measurements were performed by an independent investigator, who had no knowledge of the treatment regimen received by each group of animals.

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Acute peritonitis assessment

At 18 h after zymosan injection, the animals were killed under ether anesthesia to evaluate the development of acute inflammation in the peritoneum. The abdominal cavity was carefully opened and the peritoneal cavity was washed with 3 mL of phosphate-buffered saline (PBS) solution (composition in mmol/L: NaCl, 137; KCl, 2.7; NaH2PO4, 1.4; Na2HPO4, 4.3) The PBS had a pH value of 7.4. The peritoneal exudate and washing buffer were removed by aspiration, and the total volume was measured. Exudates contaminated with blood were discarded. Peritoneal exudate was centrifuged at 7000 g for 10 min at room temperature. The cells were suspended in PBS and counted with optical microscope using Burker chamber after vital Trypan blue staining.

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Measurement of nitrite/nitrate

Nitrite/nitrate (NO2/NO3) production, an indicator of NO synthesis, was measured in plasma samples collected 18 h after zymosan or saline administration. Plasma was incubated with nitrate reductase (concentration, 0.1 U/mL), nicotinamide adenine dinucleotide phosphate (concentration, 1 mmol/L), and flavin adenine dinucleotide (concentration, 50 μmol/L) at 37°C for 15 min, followed by another incubation with lactate dehydrogenase (concentration, 100 U/mL) and sodium pyruvate (concentration, 10 mmol/L) for 5 min. The nitrite concentration in the samples was measured by the Griess reaction by adding 100 μL of Griess reagent (0.1% [wt/vol] naphthylethylenediamide dihydrochloride in H2O and 1% [wt/vol] sulphanilamide in 5% [vol/vol] concentrated H2PO4; vol. 1:1) to the 100-μL sample. The optical density at 550 nm (OD550) was measured using an enzyme-linked immunosorbent assay microplate reader (SLT-Lab Instruments, Salzburg, Austria). Nitrate concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution.

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Immunohistochemical localization of TNF-α, nitrotyrosine, MPO, and FasL

At the end of the experiment, the tissues were fixed in 10% (wt/vol) PBS-buffered formaldehyde; then, 8-μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (vol/vol) hydrogen peroxide in 60% (vol/vol) methanol for 30 min. The sections were permeabilized with 0.1% (wt/vol) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (vol/vol) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy), respectively. Sections were incubated overnight with (1) rabbit antinitrotyrosine antibody (concentration, 1:500 [wt/vol] in PBS; Upstate, DBA, Milan, Italy), (2) anti-TNF-α antibody (concentration, 1:500 [wt/vol] in PBS; Santa Cruz Biotechnology, DBA, Milan, Italy), or (3) anti-FasL antibody (concentration, 1:500 [vol/vol] in PBS; Santa Cruz), or (4) anti-MPO antibody (concentration, 1:500 [vol/vol] in PBS; Santa Cruz). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat antirabbit immunoglobulin G and avidin-biotin peroxidase complex (DBA, Milan, Italy). The counter stain was developed with 3c3-diaminobenzidine HCl (brown) and nuclear fast red (red background). To confirm that the immunoreactions for the nitrotyrosine were specific, some sections were also incubated with the primary antibody (antinitrotyrosine) in the presence of excess nitrotyrosine (concentration, 10 mmol/L) to verify the binding specificity. To verify the binding specificity for MPO, TNF-α, or FasL, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments performed.

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Measurement of cytokines

The ileum tissue, collected at 18 h after zymosan administration, was homogenized, as previously described (15), in PBS containing 2 mmol/L of phenylmethylsulfonyl fluoride (Sigma); then, the tissue levels of TNF-α were evaluated. The assay was performed by using a colorimetric, commercial kit (Calbiochem-Novabiochem Corporation) according to the manufacturer instructions. All cytokine determinations were performed in duplicate serial dilutions.

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Quantification of organ function and injury

Blood samples were taken at 18 h after zymosan or saline injection. The blood sample was centrifuged (1610 g for 3 min at room temperature) to separate the plasma. All plasma samples were analyzed within 24 h by a veterinary clinical laboratory using standard laboratory techniques. The following marker enzymes were measured in the plasma as biochemical indicators of multiple-organ injury/dysfunction: (1) liver injury was assessed by measuring the rise in plasma levels of bilirubin, alkaline phosphatase, alanine aminotransferase (ALT), a specific marker for hepatic parenchyma injury, and aspartate aminotransferase (AST), a nonspecific marker for hepatic injury (25); (2) renal dysfunction was assessed by measuring the rise in plasma levels of creatinine (an indicator of reduced glomerular filtration rate and, hence, renal failure); and (3) the serum levels of lipase and amylase were determined as an indicator of pancreatic injury.

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Myeloperoxidase activity

Myeloperoxidase (MPO) activity, an index of polymorphonuclear (PMN) leukocyte accumulation, was determined as previously described (16). Ileum tissues, collected at the specified time point, were homogenized in a solution containing 0.5% hexadecyltrimethyl ammonium bromide dissolved in 10 mmol/L potassium phosphate buffer (pH, 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (concentration, 1.6 mmol/L) and 0.1 mmol/L H2O2. The rate of change in absorbance was measured by a spectrophotometer at 650-nm wavelength. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per minute at 37°C and was expressed in units per gram weight of wet tissue.

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Lipid peroxidation measurement

The levels of malondialdehyde (MDA) in the ileum tissue samples were determined as an indicator of lipid peroxidation (17). Ileum samples were collected at the specified time and were homogenized in 1.15% KCl solution. An aliquot (volume, 100 μL) of the homogenate was added to a reaction mixture containing 200 μL of 8.1% sodium dodecyl sulfate, 1500 μL of 20% acetic acid (pH, 3.5), 1500 μL of 0.8% thiobarbituric acid, and 700-μL distilled water. The samples were then boiled for 1 h at 95°C and centrifuged at 3000 g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 650-nm wavelength.

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Light microscopy

Ileum samples were taken 18 h after zymosan injection. The tissue slices were fixed in Dietric solution (composition: ethanol, 14.25%; formaldehyde, 1.85%; acetic acid, 1%) for 1 week at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Sections (thickness, 7 mm) were deparaffinized with xylene, stained with hematoxylin/eosin and Alcian blue periodic acid-Schiff stain (used to assess the mucopolysaccharides components), and observed in an AxioVision Zeiss (Milan, Italy) microscope. All the histological studies were performed in a blinded fashion.

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Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling assay

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling assay (TUNEL) assay was conducted by using a TUNEL detection kit according to the manufacturer's instruction (Apotag horseradish peroxidase kit; DBA, Milan, Italy). Briefly, sections were incubated with 15 μg/mL proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyl transferase (TdT) buffer containing deoxynucleotidyl transferase and biotinylated deoxyuridine 5-triphosphate in TdT buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-fluorescein isothiocyanate horseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine.

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Data analysis

All values in the figures and text are expressed as mean ± SEM of the mean of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least 3 experiments performed on different experimental days. The results were analyzed by 1-way analysis of variance, followed by a Bonferroni post hoc test for multiple comparisons. A P value less than 0.05 was considered significant. Statistical analysis for survival data was calculated by Fisher exact probability test or by Hazard statistic. For such analyses, P < 0.05 was considered significant. The Mann-Whitney test was used to examine the differences between the body weight and the organ weights of control and experimental groups. When this test was used, P < 0.05 was considered significant.

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8Materials

Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company (Milan, Italy). Primary antinitrotyrosine antibody was purchased from Upstate, DBA, Milan, Italy; the primary anti-TNF-α antibody, anti-FasL antibody, and anti-MPO antibody were obtained from Santa Cruz Biotechnology, DBA, Milan, Italy. Reagents and secondary and nonspecific immunoglobulin G antibody for immunohistochemical analysis were from Vector Laboratories (DBA, Milan, Italy). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (composition, 0.9% NaCl; Baxter Healthcare, Ltd, Thetford, Norfolk, UK).

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RESULTS

Effects of functional PPAR-α gene on the course of zymosan-induced nonseptic shock model

The administration of zymosan caused a severe illness in the mice characterized by a systemic toxicity and significant loss of body weight (Fig. 1, A and B). At the end of the observation period (12 days), 70% of zymosan-treated PPAR-αWT mice were dead (Fig. 1C). The absence of functional PPAR-α receptor in PPAR-αKO mice significantly enhanced the development of systemic toxicity (Fig. 1A), the loss in body weight (Fig. 1B), and mortality (Fig. 1C) caused by zymosan. The sham animals injected only with saline seemed healthy and active throughout the entire observation period (data not shown).

Fig. 1

Fig. 1

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Effects of functional PPAR-α gene on the upregulation of TNF-α levels during zymosan-induced nonseptic shock model

Zymosan-induced nonseptic shock model results in the upregulation of proinflammatory cascades in the intestine and in other organs. The inflammatory response includes the expression of TNF-α at 18 h after zymosan administration. The TNF-α levels were significantly increased in the ileum from the zymosan-treated PPAR-αWT mice at 18 h after zymosan administration in comparison with those of the sham-treated animals (Fig. 2A). The ileum levels of TNF-α were significantly higher in the PPAR-α-deficient mice in comparison with those of the PPAR-αWT animals (Fig. 2A). In addition, ileum tissue sections obtained from the PPAR-αWT animals at 18 h after zymosan administration demonstrate positive staining for TNF-α (Fig. 2B), mainly localized in the infiltrated inflammatory cells. In the zymosan-treated PPAR-αKO mice, the staining for TNF-α (Fig. 2C) was visibly and significantly increased in comparison with that in the PPAR-αWT mice. No positive stainings for TNF-α were observed in the ileum section obtained from the sham-treated mice (data not shown).

Fig. 2

Fig. 2

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Effects of functional PPAR-α gene on the inflammatory cells infiltration

A hallmark of zymosan-induced MODS is the accumulation of neutrophils in the intestine, which augments the tissue damage. Therefore, we have evaluated, at 18 h after zymosan administration, the extent of inflammatory cell infiltration in the pleritoneal cavity and in the ileum tissues by measurement of the activity of MPO. The PMN leukocytes infiltration in the peritoneal cavity and MPO activity were significantly elevated at 18 h after zymosan administration in PPAR-αWT mice in comparison with sham-treated animals (Fig. 3, A and B). The PMN leukocytes infiltration in the peritoneal cavity and the intestinal MPO activity were significantly enhanced in PPAR-α-deficient mice in comparison with those of PPAR-αWT animals (Fig. 3, A and B). In addition, the ileum tissue sections obtained from PPAR-αWT animals at 18 h after zymosan administration demonstrate positive staining for MPO (Fig. 3C). In the zymosan-treated PPAR-αKO mice, the staining for MPO (Fig. 3D) was visibly and significantly increased in comparison with that in the PPAR-αWT mice. No positive stainings for MPO were observed in the ileum section obtained from sham-treated mice (data not shown).

Fig. 3

Fig. 3

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Effects of functional PPAR-α gene on NO formation, lipid peroxidation, and nitrosative stress

The biochemical and inflammatory changes observed in the peritoneal cavity of zymosan-treated mice were associated with a significant elevation of plasma NO2 levels. The nitrite/nitrate levels were significantly elevated in zymosan-treated PPAR-αWT mice in comparison with those in sham-treated mice (Fig. 4A). The degree of plasma nitrate/nitrite levels was significantly enhanced in PPAR-αKO mice at 18 h after zymosan administration (Fig. 4A).

Fig. 4

Fig. 4

As shown in Figure 4B, the MDA levels, indicative of lipid peroxidation, were significantly increased in the intestine of zymosan-treated PPAR-αWT mice. The degree of intestinal MDA levels was significantly enhanced in PPAR-αKO mice at 18 h after zymosan administration (Fig. 4B). At 18 h after intraperitoneal administration of zymosan, the sections of the intestine were also analyzed for the evidence of nitrotyrosine formation. Immunohistochemical analysis, using a specific antinitrotyrosine antibody, revealed a positive staining in intestine (Fig. 4C) from zymosan-treated PPAR-αWT mice. A marked enhancement of positive nitrotyrosine stainings was found in the intestine (Fig. 4D) of the zymosan-treated PPAR-αKO mice.

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Multiple-organ dysfunction syndrome caused by zymosan is enhanced in PPAR-α-deficient mice

Effects on pancreatic injury

In sham mice, the administration of saline did not result in any significant alterations in the plasma levels of lipase and amylase (Fig. 5, A and B). When compared with sham-treated mice at 18 h after zymosan administration, a significant rise in the plasma levels of lipase and amylase was observed in wild-type mice, demonstrating the development of pancreatic injury (Fig. 5, A and B). The absence of functional PPAR-α receptor 5-LO in PPAR-αKO mice increased the pancreatic injury caused by zymosan (Fig. 5, A and B).

Fig. 5

Fig. 5

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Effects on the renal dysfunction

In sham-treated mice, the administration of saline did not result in any significant alterations in the plasma levels of creatinine (Fig. 5C). When compared with sham-treated mice at 18 h after zymosan administration, a significant rise in the plasma levels of creatinine was observed in PPAR-αWT mice, demonstrating the development of renal dysfunction (Fig. 5C). The absence of functional PPAR-α receptor in PPAR-αKO mice enhanced the renal dysfunction caused by zymosan (Fig. 5C).

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Effects on the liver injury

In sham-treated mice, the administration of saline did not result in any significant alterations in the plasma levels of AST (Fig. 6A), ALT (Fig. 6B), bilirubin (Fig. 6C), and alkaline phosphatase (Fig. 6D). When compared with sham-treated mice at 18 h after zymosan administration, a significant rise in the plasma levels of AST, ALT, bilirubin, and alkaline phosphatase was observed in PPAR-αWT mice, demonstrating the development of hepatocellular injury (Fig. 6). The absence of functional PPAR-α receptor in PPAR-αKO mice increased the liver injury caused by zymosan (Fig. 6).

Fig. 6

Fig. 6

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Effects of functional PPAR-α gene on the expression of FasL during zymosan-induced nonseptic shock model

The potential effect of functional PPAR-α gene on apoptosis in zymosan-induced nonseptic shock was evaluated by immunohistochemical detection of FasL. At 18 h after zymosan administration, positive staining for FasL are readily detected in the ileum tissues from wild-type mice, mainly localized in vascular wall and in inflammatory cells (Fig. 7A). The presence of positive staining for FasL in vascular wall and in inflammatory cells was significantly increased in the absence of a functional PPAR-α gene in PPAR-αKO mice (Fig. 7B). No positive staining for FasL was observed in the intestinal tissues from sham-treated wild-type mice and from sham-treated PPAR-αKO mice (data not shown).

Fig. 7

Fig. 7

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Effects of functional PPAR-α gene on apoptosis during zymosan-induced nonseptic shock model

To investigate whether zymosan-induced nonseptic shock is associated with apoptotic cell death, we measured the TUNEL-like staining in small intestinal tissues. A few apoptotic cells were observed at the villus tips in the intestine of sham-treated wild-type mice (data not shown). The number of apoptotic cells in the intestine of the wild-type mice increased at 18 h after zymosan administration (Fig. 8A). The presence of apoptotic cells was significantly increased in the intestine of PPAR-αKO mice (Fig. 8B).

Fig. 8

Fig. 8

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Intestinal injury (histological evaluation) caused by zymosan is enhanced in PPAR-α-deficient mice

At 18 h after zymosan administration, the tissue injury in small intestine was evaluated by histology. At histological examination, the small intestine (see representative sections at Fig. 9) revealed pathological changes. Sections from the distal ileum revealed a significant villus tips damage, edema in the space bounded by the villus and in submucosa, and inflammatory cell infiltration (Fig. 9A). Moreover, zymosan administration induced a presence of acid mucine (Fig. 9C). The absence of a functional PPAR-α gene in PPAR-α-deficient mice resulted in a significant enhancement of intestinal injury (Fig. 9B) and of presence of acid mucine (Fig. 9D).

Fig. 9

Fig. 9

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DISCUSSION

This study provides the important evidence that the absence of PPAR-α (PPAR-α knockout mice or PPAR-ƒÑαKO mice) increased (1) the development of zymosan-induced peritonitis; (2) the infiltration of the intestine with PMN leukocytes (histology and MPO activity); (3) the degree of liver, kidney, and pancreas injury or organ dysfunction (biochemical markers); and (4) the degree of intestinal injury (histology) caused by the injection of zymosan. All of these findings support the view that PPAR-α receptor modulates the degree of MODS induced by zymosan in the mice.

PPAR-α expression is relatively high in the hepatocytes, the heart, the enterocytes, the muscle, and the kidney (18). PPAR-α regulates the genes involved in the β-oxidation of fatty acids and lipoprotein metabolism. Various studies have clearly demonstrated, using PPAR-α-deficient mice, that this receptor is involved in high-density lipoprotein and triglyceride metabolism and in hepatic regulation of apolipoprotein and fatty acid β-oxidation enzyme expression (19, 20).

The first evidence for a role of PPAR-α in inflammation was suggested in studies using PPAR-α knockout mice. In particular, it has been shown that the ear inflammation induced by leukotriene (LT) B4 or arachidonic acid (but not by the tetradecanoylphorbol acetate) was prolonged in PPAR-αKO mice (21). Recently, many PPAR-α ligands, including the naturally occurring ligand, LT B4, and the synthetic ligands, fenofibrate and Wy-14,643, have been used to investigate the role of PPAR-α receptor in inflammation (18).

Recently, it has been demonstrated that PPAR-α activators suppress interleukin (IL) 1-induced C-reactive protein and IL-6-induced fibrinogen expression, the major acute-phase response (APR) proteins in humans (22) whose plasma concentrations are elevated not only in acute but also in chronic inflammatory states. This anti-inflammatory action of PPAR-α is not restricted to these genes but applies more generally to other APR genes, such as serum amyloid A and fibrinogens A and B (23). The activation of PPAR-α leads to a reduction in the formation of nuclear C/EBPbBp50-NFκB complexes and thereby reduces the C-reactive protein promoter activation. Moreover, PPAR-α increases IκBa expression, thus preventing nuclear p50/p65 NF-κB translocation and arresting their nuclear transcriptional activity. Moreover, long-term treatment with fibrates decreases hepatic C/EBPb and p50-NF-κB protein expression in mice in a PPAR-α-dependent manner (23). This latter effect likely contributes to the generalized anti-inflammatory effects of fibrates on the expression of a wide range of APR genes containing response elements for these transcription factors in their promoters.

The activation of NF-κB is crucially involved in FasL expression induced by DNA-damaging agents, such as genotoxic drugs and UV radiation (24). FasL plays a central role in apoptosis induced by a variety of chemical and physical insults (25). Recently, it has been pointed out that Fas-FasL signaling plays a central role in acute inflammation (25). Furthermore, the cell death induced by reactive oxygen species (ROS) depends on FasL expression mediated by redox sensitive activation of NF-κB (26). The generation of ROS has been implicated in induction of MODS caused by zymosan (27) through NF-κB activation and expression of FasL. We confirm in this study that zymosan-induced nonseptic shock leads to a substantial activation of FasL in the intestinal tissues, which likely contribute in different capacities to the evolution of tissues injury. In the present study, we found that the genetic inhibition of PPAR-α receptors lead to a substantial increase of FasL activation. FasL activation also induced a proinflammatory response characterized by a release of IL-1β, chemokines macrophage inflammatory protein (MIP) 1α, MIP-1β, and MIP-2 (28). There is evidence that the proinflammatory cytokines TNF-α and IL-1β help propagate the extension of MODS (29). We confirm in this study that the zymosan administration leads to a substantial increase in the levels of TNF-α in the intestinal tissues, which likely contribute in different capacities to the evolution of multiple-organ failure. Interestingly, the levels of this proinflammatory cytokine are significantly higher in the absence of functional PPAR-α gene, suggesting that the PPAR-α receptor modulates the activation and the subsequent expression of proinflammatory genes. However, in an in vivo study, it has been demonstrated that the treatment of CD-1 mice with fenofibrate or Wy14,643 leads to 5-fold higher TNF-α plasma levels induced by lipopolysaccharide administration and to a significantly lower, 50% lethal dose than it does with control mice (30). These results were also confirmed in PPAR-α-deficient mice (30). On the contrary, in the same studies, treatment of wild-type mice with Wy14,643 resulted in a modest decrease of TNF expression in peritoneal macrophages (30). Neutrophils play a crucial role in the development and full manifestation of inflammation and shock, including the zymosan model of nonseptic shock (31). A number of recent studies have demonstrated the importance of specific adhesion molecules in the recruitment of inflammatory cells into an area of inflammation (32). PPAR-α is expressed in various types of human endothelial cells (33), suggesting a role for PPAR-α receptor in the downregulation of endothelial cells' inflammatory responses. Neutrophil infiltration into inflamed tissue represents a major source of free radicals in injured tissues. In this study, as previously published (34), we demonstrate that zymosan-induced nonseptic shock leads to a significant inflammatory cells infiltration in the peritoneal cavity and in the intestine. Thus, the enhanced inflammatory cells infiltration observed in the absence of endogenous PPAR-α ligand suggests that endogenous PPAR-α ligand reduced the neutrophil infiltration.

In mice, zymosan also causes an overproduction of NO because of the induction of inducible nitric oxide synthase, which contributes to the inflammatory process (5). We demonstrate in this study that the absence of functional PPAR-α gene increases the NO formation in the plasma. Thus, the increase of the NO formation by the absence of functional PPAR-α gene may contribute to the increase of lipid peroxidation and of the nitrotyrosine formation in the intestine of zymosan-treated PPAR-αKO mice. Nitrotyrosine formation, along with its detection by immunostaining, was initially proposed as a relatively specific marker for the detection of the endogenous formation "footprint" of peroxynitrite (35). However, recent evidence showed that certain other reactions can also induce tyrosine nitration (e.g., the reaction of nitrite with hypochlorous acid and the reaction of MPO with hydrogen peroxide can lead to the formation of nitrotyrosine; (36). Therefore, increased nitrotyrosine staining is considered as an indication of increased nitrosative stress rather than as a specific marker of the generation of peroxynitrite. Thus, we propose that the increase of the nitrosative stress caused by the absence of the PPAR-α receptor contributes to the enhancement and the development of nonseptic shock in mice.

In conclusion, this study demonstrates that PPAR-α pathway modulates the degree of zymosan-induced nonseptic shock in mice, reducing (1) the renal dysfunction, (2) the hepatocellular dysfunction, (3) the intestinal injury, and (4) the pancreatic dysfunction caused by zymosan administration in mice. Further experiments with multiple approaches can be expected to unlock the full potential of modulating the PPAR-α pathway for therapeutic purposes. Thus, we propose the following cycle: nonseptic shock, NF-κB activation, FasL expression, cytokines release, endothelial injury, PMN leukocytes infiltration, more proinflammatory mediator release (e.g., cytokines, ROS), and organ damage. The PPAR-α receptor activation would intercept this cycle before endothelial injury. However, the confirmation of this proposed feedback cycle requires further investigation. This study provides evidence that PPAR-α pathway modulates the degree of nonseptic shock and supports the potential use of PPAR-α ligands as therapeutic agents in the therapy for conditions associated with nonseptic shock.

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ACKNOWLEDGMENTS

The authors thank Giovanni Pergolizzi and Carmelo La Spada for excellent technical assistance during this study, Caterina Cutrona for secretarial assistance, and Valentina Malvagni for editorial assistance with the manuscript.

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Keywords:

PPAR-α; multiple-organ failure; inflammation; shock; zymosan; systemic inflammation

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