Protective effect of Hypericum perforatum in zymosan-induced multiple organ dysfunction syndrome: Relationship to its inhibitory effect on nitric oxide production and its peroxynitrite scavenging activity

Nitric Oxide 16 (2007) 118–130 www.elsevier.com/locate/yniox

Protective eVect of Hypericum perforatum in zymosan-induced multiple organ dysfunction syndrome: Relationship to its inhibitory eVect on nitric oxide production and its peroxynitrite scavenging activity Rosanna Di Paola a,1, Emanuela Mazzon a,b,1, Carmelo Muià a, Concetta Crisafulli a, Tiziana Genovese a,b, Paolo Di Bella b, Emanuela Esposito c, Marta Menegazzi d, Rosaria Meli c, Hisanori Suzuki d, Salvatore Cuzzocrea a,b,¤ a

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 b IRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, Italy c Dipartimento di Farmacologia Sperimentale, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy d Biochemistry Division, Department of Neuroscience and Vision, University of Verona, Verona, Italy Received 24 March 2006; revised 23 May 2006 Available online 15 June 2006

Abstract Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely Xavonoids and phenolic acids. Since polyphenolic compounds have high antioxidant potential, we have investigated the eVects of H. perforatum extract on the development of multiple organ dysfunction syndrome caused by zymosan (500 mg/kg, administered i.p. as a suspension in saline) in mice. Organ failure and systemic inXammation in rats was assessed 18 h after administration of zymosan and/or H. perforatum extract and monitored for 12 days (for loss of body weight and mortality). Treatment of mice with H. perforatum extract (30 mg/kg i.p., 1 and 6 h after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan, pulmonary, intestinal and pancreatic injury, and renal dysfunction as well as the increase in myeloperoxidase in the lung and intestine. Immunohistochemical analysis for inducible nitric oxide synthase (iNOS), nitrotyrosine, and poly(ADP-ribose) (PAR) revealed positive staining in lung and intestine tissues obtained from zymosan-injected mice. The degree of staining for nitrotyrosine, iNOS, and PAR was markedly reduced in tissue sections obtained from zymosan-treated mice, which received H. perforatum extract. In conclusion, this study provides evidence, for the Wrst time, that H. perforatum extract attenuates the degree of zymosan-induced multiple organ dysfunction syndrome in mice. © 2006 Elsevier Inc. All rights reserved. Keywords: Hypericum perforatum; Nitric oxide; Oxidative stress; Antioxidant; Zymosan induced multiple organ failure

Multiple organ dysfunction syndrome (MODS), also known as multiple organ system failure (MOF), remains a principal cause of death after severe shock or trauma, with or without evidence of sepsis [1,2]. Current theories suggest that MODS results from an overwhelming systemic inXammatory reaction, resulting in sequential deterioration of *

1

Corresponding author. Fax: +39 090 221 3300. E-mail address: [email protected] (S. Cuzzocrea). These authors contributed equally to this work.

1089-8603/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2006.05.006

organ function, usually starting with respiratory failure and progressing to other organs not directly associated with the primary insult [3–5].. Though MODS was Wrst recognized in the 1970s, no major advances in treatment exist today [6]. Therapy is limited to supportive measures and to attempts to halt the progression of organ failure and correct underlying insults. Several animal models have been described in order to understand the pathophysiological mechanism. Among them, administration of zymosan, a non-bacterial,

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non-endotoxic agent, produces acute peritonitis and multiple organ failure in experimental animals characterized by functional and structural changes in liver, intestine, lung, and kidneys [7–11]. After zymosan is phagocytosed, macrophages release lysosomal enzymes [12], reactive oxygen metabolites, arachidonic acid [13], and tumor necrosis factor- (TNF-) [14]. Because zymosan is not degradable, phagocytosis by macrophages results in a prolonged inXammatory response. The zymosan-induced generalized inXammation model is the only long-term experimental animal model for MODS. The onset of the inXammatory response caused by zymosan in the peritoneal cavity was associated with systemic hypotension, high peritoneal and plasma levels of NO, maximal cellular inWltration, exudate formation, cyclooxygenase activity, and pro-inXammatory cytokines production [15]. We have recently discovered that administration of zymosan results in excessive reactive oxygen species (ROS) formation by activated polymorphonuclear leukocytes (PMNs) including superoxide anion and lipid peroxidation in plasma, intestine, and lung [16–18]. Once produced, ROS could trigger various inXammatory processes. They can directly attack the lipoid matrix of biological membranes, stimulate arachidonic acid metabolism with increased production of prostaglandins, thromboxane, and leukotrienes, thereby enhancing the accumulation and adherence of neutrophils and platelets to the capillary wall [19]. Thus, ROS could impair the microcirculation and disturb the microvascular integrity, resulting in decreased perfusion, increased capillary permeability and Xuid transudation. Hypericum perforatum L. (Hypericaceae), popularly called St. John’s wort, is an herbaceous perennial plant belonging to the family Clusiaceae, which is used in popular medicine and phytotherapy for its well documented antiseptic and antidepressant eVects [20,21]. Moreover, it has been proposed to have antibacterial and antiviral eVects and to exert antiinXammatory and analgesic activity [22,23]. Hypericum perforatum extract contains Xavonoids such as rutin, quercetin, and quercitrin, which demonstrated a free radical scavenging activity in a model of autooxidation of rat cerebral membranes [24]. An antioxidant activity of quercetin was also demonstrated by inhibition of brain lipid peroxidation, as manifested by lowering MDA while elevating phospholipid contents in a rat model of endotoxemia [25]. Previous studies demonstrated that the eVect of Hypericum in vivo on carrageenan-induced paw oedema and in vitro on down-regulation of COX-2 and iNOS enzyme expression strongly supports the anti-inXammatory activity [26]. Recent study reported the eVects of H. perforatum extract on the lung injury associated with carrageenaninduced pleurisy demonstrating that it has strong antiinXammatory properties resulting in a reduced TNF- production, PMN inWltration, up-regulation of ICAM-1 expression, NF-B, STAT-3 and nuclear enzyme PARS activation, and the degree of peroxynitrite formation and tissue injury [27].

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Thus, the aim of the present studies was to evaluate the eVects of H. perforatum extract in a model of non-septic shock induced by zymosan in mice determining the following endpoints of the inXammatory response (a) plasma NOx levels and inducible nitric oxide synthase (iNOS), (b) neutrophils accumulation in the peritoneal exudate and inWltration in the intestinal and lung tissue by measuring myeloperoxidase (MPO) activity, (c) parameters of renal, hepatic, and liver function, (d) nitration of tyrosine residues, as an indicator of the formation of migration in the peroxynitrite and the activation of the nuclear enzyme PAR synthetase (PARS) by immunohistochemistry, and (e) intestinal and lung damage by histology, and (f) clinical scoring of systemic toxicity. Materials and methods Animals Male CD mice (weight 20–25 g; Harlan Nossan, Milan, Italy) were used in these studies. The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientiWc purposes (D.M. 116192) as well as with EEC regulations (O.J. of E.C. L358/1 12/18/1986). Hypericum perforatum extract Hypericum perforatum methanolic extract was a kind gift of Indena (Milano, Italy) and it was deWned by the producer as containing 0.34% of hypericin, 4.1% of hyperforin, 5% of Xavonoids (rutin, kaempferol, luteolin, myricetin, quercetin, quercitrin, and isoquercitrin), 10% tannins, and the remaining part is composed of polysaccharides represented by maltodextrins. Experimental groups Mice were randomly allocated into the following groups: (i) Zymosan group: mice were injected intraperitoneally (i.p.) with zymosan (500 mg/kg, suspended in saline solution, i.p.) (N D 10); (ii) Hypericum group (ZYM + Hypericum): identical to the Zymosan group but were administered H. perforatum (30 mg/kg, i.p.) at 1 and 6 h after zymosan (N D 10); (iii) Sham + vehicle. Sham group were administered saline solution i.p. (N D 10), (iv) Sham + Hypericum group. Identical to Sham group, except for the administration of Hypericum (30 mg/kg) 1 and 6 h after saline administration (N D 10). Eighteen hours after administration of zymosan, animals were assessed for multiple organ dysfunction syndrome as described below. Blood samples were obtained by direct intracardiac puncture. Lungs and ileum were removed immediately, frozen in liquid nitrogen, and stored at ¡80 °C until assayed. Portions of these organs were also Wxed in formaldehyde for histological and immunohistochemical examination. In another set of experiments,

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mice were randomized to receive treatment regimens, which were identical to the ones listed above (n D 20 for each group), but were monitored for 12 days after zymosan or saline administration in order to monitor their survival rate, loss of body weight, and mortality. The doses of H. perforatum used here to reduce acute lung injury were chosen in agreement with a dose–response study (26). Clinical scoring of systemic toxicity Clinical severity of systemic toxicity (conjunctivitis, ruZed fur, diarrhea, and lethargy) in mice was scored 18 h after zymosan or saline administration on a subjective scale ranging from 0 to 3 (0 D absence, 1 D mild, 2 D moderate, 3 D serious). All clinical scores were determined by an independent investigator who had no knowledge of the treatment regimen received by the respective animals. Assessment of acute peritonitis At 18 h after zymosan administration or saline injection, animals were killed under ether anesthesia to evaluate the development of acute inXammation in the peritoneum. The abdominal cavity was carefully opened and the peritoneal cavity washed with 7 ml phosphate-buVered saline (PBS: NaCl 137 mM, KCl 2.7 mM, NaH2PO4 1.4 mM, and Na2HPO4 4.3 mM, pH 7.4). Washing buVer was removed with a plastic pipette and was transferred into a 10 ml centrifuge tube. The amount of exudate was calculated by subtracting the volume injected (10 ml) from the total volume recovered. Exudates contaminated with blood were discarded. Peritoneal exudate was centrifuged at 7000g for 10 min at 4 °C. Cells were suspended in PBS and counted with optical microscope by Burker’s chamber after vital staining with trypan blue.

and 8 m sections were prepared from paraYn embedded tissues. After deparaYnization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min. Non-speciWc adsorption was minimized by incubating the section in 2% normal goat serum in phosphate-buVered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with primary antiiNOS antibody (1:500), anti-nitrotyrosine antibody (1:500, Upstate), and anti-poly (ADP-ribose) (PAR) antibody (1:500) or with control solutions. Controls included buVer alone or non-speciWc puriWed rabbit IgG. SpeciWc labelling was detected with a biotin-conjugated speciWc secondary anti-igG and avidin–biotin peroxidise complex (Vector, DBA, Italy). In order to conWrm that the immunoreactions for the nitrotyrosine were speciWc some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding speciWcity. To verify the binding speciWcity for iNOS and PAR, some sections were also incubated with the primary antibody (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections indicating that the immunoreactions were positive in all the experiments carried out. Determination of myeloperoxidase activity MPO activity, an indicator of PMN inWltration into lung and intestinal tissue, was determined as previously described [29]. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was deWned as the quantity of enzyme degrading 1 mol of peroxide min at 37 °C and was expressed in milliunits per gram weight of wet tissue.

Measurement of nitrite/nitrate concentrations in plasma QuantiWcation of organ function and injury Nitrite/nitrate (NO2¡/NO3¡s) production, an indicator of NO synthesis, was measured in plasma samples collected 18 h after zymosan or saline administration, as previously described [15]. The optical density at 550 nm (OD550) was measured using an enzyme-linked immunosorbent assay (ELISA) 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. Immunohistochemical localization of iNOS, nitrotyrosine and PAR Tyrosine nitration and PARP activation was detected as previously described [28] in ileum and lung sections using immunohistochemistry. At 18 h after zymosan or saline injection, tissues were Wxed in 10% (w/v) PBS-buVered formalin

Blood samples were taken at 18 h after zymosan or saline injection. The blood sample was centrifuged (1610g for 3 min at room temperature) to separate 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 alanine aminotransferase (ALT, a speciWc marker for hepatic parenchymal injury) and aspartate aminotransferase (AST, a non-speciWc marker for hepatic injury). (2) Renal dysfunction was assessed by measuring the rise in plasma levels of creatinine (an indicator of reduced glomerular Wltration rate, and hence, renal failure). (3) In addition, serum levels of lipase and amylase were determined as an indicator of pancreatic injury.

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Light microscopy Lung and small intestine samples were taken 18 h after zymosan or saline injection. The tissue slices were Wxed in dietric solution [14.25% (v/v) ethanol, 1.85% (w/v) formaldehyde, and 1% (v/v) acetic acid] for 1 week at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA). Sections (thickness 7 m) were deparaYnized with xylene, stained with hematoxylin and eosin, and observed in Dialux 22 Leitz microscope. Materials Unless otherwise stated, all compounds were obtained from Sigma–Aldrich Company (Milan, Italy). Reagents and secondary and non-speciWc IgG antibody for immunohistochemical analysis were from Vector Laboratories Inc. A biotin-blocking kit, biotin-conjugated goat anti-rabbit IgG, primary anti-nitrotyrosine, anti-poly(ADP-ribose) synthetase (PARS) antibodies, primary anti-inducible nitric oxide synthase (iNOS), and avidin–biotin–peroxidase complex (ABC) were obtained from DBA (Milan, Italy). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter Healthcare, Thetford, UK). Data analysis All values in the Wgures and text are expressed as mean § standard error of the mean (SEM) of n observations. For the in vivo studies n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the Wgures shown are representative of at least three experiments performed on diVerent experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni’s post hoc test for multiple comparisons. A p-value of less than 0.05 was considered as signiWcant. Statistical analysis for survival data was calculated by

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Fisher’s exact probability test. For such analyses, p < 0.05 was considered as signiWcant. The Mann–Whitney test was used to examine the diVerences between the body weight and organ weights of control and experimental groups. When this test was used, p < 0.05 was considered as signiWcant. Results EVect of H. perforatum on acute peritonitis At 18 h after zymosan administration, increased formation of turbid exudate was observed (Fig. 1a; p < 0.01 vs. Sham). Trypan blue staining revealed a signiWcant leukocyte inWltration in the peritoneal cavity of zymosan-injected mice vs. Sham (Fig. 1b; p < 0.01). The degree of peritoneal exudation and PMN migration was signiWcantly reduced in H. perforatum-treated-mice (Fig. 1; p < 0.01 vs Zymosan group). No abnormalities in the peritoneal cavity or Xuid were observed in H. perforatum-treated Sham mice. EVect of H. perforatum treatment on NO formation and iNOS expression The biochemical and inXammatory changes observed in the peritoneal cavity of zymosan-injected mice were associated with a signiWcant elevation of plasma nitrite/nitrate levels (Fig. 2; p < 0.01 vs Sham). The degree of plasma nitrate/nitrite levels was signiWcantly reduced in mice treated with H. perforatum (p < 0.01 vs Zymosan group), while H. perforatum treatment did not cause signiWcant changes of NO2 levels in Sham mice (Fig. 2). At 18 h following i.p. administration of zymosan sections of lung and ileum was also analyzed for the evidence of iNOS expression. Immunohistochemical analysis of lung (Fig. 3a) and ileum (Fig. 3c) sections obtained from zymosan-injected mice revealed positive staining for iNOS. A marked reduction was found in the lung (Fig. 3b) and intestine (Fig. 3d) of the zymosan-treated mice that had been received H. perforatum. In contrast, no staining for iNOS

Fig. 1. EVect of H. perforatum on zymosan induced peritonitis. The increase in volume exudate (a) and accumulation of polymorphonuclear cells (PMNs, b) in pleural cavity at 18 h after zymosan administration was inhibited by H. perforatum (30 mg/kg, orally). Data are means § SEM of 10 mice for each group. *p < 0.01 vs. Sham; °p < 0.01 vs. zymosan.

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Fig. 2. EVect of Hypericum treatment on zymosan-induced nitrite/nitrate plasma production. The increase in plasma nitrite/nitrate production at 18 h after zymosan was inhibited by H. perforatum treatment. Data are means § SEM of 10 mice for each group. *p < 0.01 vs. Sham; °p < 0.01 vs. zymosan.

was found into lung and ileum from Sham-treated mice (data not shown). EVect of H. perforatum administration on nitrosative stress and PAR accumulation At 18 h following i.p. administration of zymosan, lung and intestine sections was also analyzed for the evidence of nitrotyrosine formation. Immunohistochemical analysis

using a speciWc anti-nitrotyrosine antibody revealed positive staining in lung (Fig. 4a) and intestine (Fig. 4c) from zymosan-injected mice. A marked reduction in nitrotyrosine staining was found in lung (Fig. 4b) and intestine (Fig. 4d) of the zymosan-injected mice that had been received H. perforatum (30 mg/kg i.p.). Immunohistochemical analysis of lung and intestine sections obtained from mice injected with zymosan also revealed a positive staining for PAR in lung (Fig. 5a) and intestine (Fig. 5c), indicating PARP activation. In contrast, no positive staining for PAR was found in lung (Fig. 5a) and intestine (Fig. 5c) in the lung and intestine of zymosan-injected mice which had been treated with H. perforatum. There was no staining for either nitrotyrosine or PAR in lung and intestine obtained from Sham-treated mice (data not shown). EVect of Hypericum perforatum on neutrophil inWltration Therefore, we evaluated neutrophil inWltration into lung and ileum tissue by measuring the activity of MPO, an enzyme that is contained in, and is speciWc for, PMN lysosomes. Thus tissue levels of MPO were directly correlated with the number of neutrophils in each given tissue. MPO activity was signiWcantly increased at 18 h after zymosan administration in lung and intestine from zymosan-injected mice (Fig. 6). H. perforatum treatment signiWcantly reduced the activity of MPO in lung and intestine from zymosaninjected mice (Fig. 6; p < 0.01 vs. Sham).

Fig. 3. Immunohistochemical localization of iNOS in mouse lung and intestine. Eighteen hours following zymosan injection, positive iNOS staining was found in the lung (a) and intestine (c). The intensity of the positive staining for iNOS was markedly reduced in the lung (b) and intestine (d) section obtained from zymosan-treated mice that received H. perforatum. Figure is representative of at least three experiments performed on diVerent experimental days.

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Fig. 4. Immunohistochemical localization for nitrotyrosine in the lung and intestine. Eighteen hours following zymosan injection, positive nitrotyrosine staining was found in the lung (a) and intestine (c). The intensity of the positive staining for nitrotyrosine was markedly reduced in the lung (b) and intestine (d) section obtained from zymosan-treated mice that received H. perforatum. Figure is representative of at least three experiments performed on diVerent experimental days.

Fig. 5. Immunohistochemical localization for PAR in the lung and intestine. Eighteen hours following zymosan injection, positive PAR staining was found in the lung (a) and intestine (c). The intensity of the positive staining for PAR was markedly reduced in the lung (b) and intestine (d) section obtained from zymosan-treated mice that received H. perforatum. Figure is representative of at least three experiments performed on diVerent experimental days.

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Fig. 6. Myeloperoxidase (MPO) activity in the lung (a) and small intestine (b) of zymosan-treated rats. MPO activity was signiWcantly increased in the lung and small intestine of the zymosan-treated rats in comparison to Sham-treated mice. H. perforatum treatment signiWcantly reduces the zymosan-induced increase in MPO activity Data are means § SEM of 10 mice for each group. *p < 0.01 vs. Sham; °p < 0.01 vs. zymosan.

EVects of Hypericum perforatum on the multiple organ dysfunction syndrome caused by zymosan EVects on the liver injury In Sham mice the administration of saline or Hypericum did not result in any signiWcant alterations in the plasma levels of AST (Fig. 7a), ALT (Fig. 7b), bilirubin (Fig. 7c) or alkaline phosphatase (Fig. 7d). Compared with Sham mice, zymosan administration resulted in signiWcant rises in the plasma levels of AST, ALT, bilirubin, and alkaline phosphatase (Fig. 7, p < 0.01 vs. Sham group), demonstrating the development of hepatocellular

injury. Pre-treatment of zymosan-injection mice with H. perforatum abolished the liver injury caused by zymosan (Fig. 7, p < 0.01 vs. Zymosan group). EVects on pancreatic injury In Sham mice the administration of saline or H. perforatum did not result in any signiWcant alterations in the plasma levels of amylase and lipase (Fig. 8a and b, respectively). Compared with Sham mice, zymosan administration resulted in signiWcant rises in the plasma levels of lipase and amylase, demonstrating the development of pancreatic injury (Fig. 8a and b, respectively). Pre-treatment of zymo-

Fig. 7. EVect of H. perforatum extract on liver injury. AST (a), ALT (b), bilirubin (c), and alkaline phosphatase (d) plasma levels were signiWcantly increased in zymosan-treated mice. H. perforatum (30 mg/kg orally) treatment signiWcantly reduced all these parameters in plasma-treated mice. Data are means § SEM of 10 mice for each group. *p < 0.01 vs. Sham; °p < 0.01 vs. zymosan.

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Fig. 8. EVect of H. perforatum extract on amylase, lipase, and creatinine serum levels. EVect of H. perforatum extract on amylase (a), lipase (b) and creatinine (c) serum levels (U/L) in zymosan-injected mice. Data are means § SEM of 10 mice for each group. *p < 0.01 vs. Sham; °p < 0.01 vs. zymosan.

san-injected mice with H. perforatum abolished the pancreatic injury caused by zymosan (Fig. 8).

EVect of Hypericum treatment on zymosan-induced body weight loss and animal mortality

EVects on the renal dysfunction In Sham mice the administration of saline or H. perforatum did not result in any signiWcant alterations in the plasma levels of creatinine (Fig. 8c). Compared with Sham mice, zymosan administration resulted in signiWcant rises in the plasma levels of creatinine, demonstrating the development of renal dysfunction. Pre-treatment of zymosan-injected mice with H. perforatum abolished the renal dysfunction caused by zymosan (Fig. 8c).

Zymosan caused a severe illness in the mice characterized by a systemic toxicity and signiWcant loss of body weight (Fig. 10a and b, respectively). At the end of observation period (12 days), 90% of zymosan-injected mice were dead (Fig. 10c). H. perforatum treatment (30 mg/kg) prevented the development of systemic toxicity (Fig. 10a), body weight loss (Fig. 10b), and mortality (Fig. 10c) caused by zymosan. H. perforatum did not cause signiWcant changes in these parameters in Shamtreated mice.

Hypericum perforatum reduces lung and intestine injury (histological evaluation) caused by zymosan

Discussion

At 18 h after zymosan administration, the tissue injury in lung and small intestine was evaluated by histology. At histological examination, the lung and small intestine (see representative sections at Fig. 9) revealed pathological changes. The examination of the lung biopsies revealed extravasion of red cells and neutrophils and macrophage accumulation (Fig. 9a). Sections from the distal ileum revealed a signiWcant edema formation (Fig. 9c) and inXammatory cells inWltration (Fig. 9c1). H. perforatum treatment resulted in a signiWcant reduction of pulmonary (Fig. 9b) and intestinal injury (Fig. 9d). No histological alteration was observed in Sham-treated mice (data not shown).

Human MODS is invariably precipitated by one or more events which cause marked disruption of homeostasis, e.g., trauma, severe hemorrhage or sepsis. After the patient is resuscitated, a well-deWned sequence of organ failure occurs; starting with lung dysfunction, progressing to the coagulation system and subsequently involving the kidneys, and ultimately resulting in liver failure [1,30]. In humans, this is associated with sustained hypermetabolism and negative nitrogen balance [31]. The progression to additional organ failure increases the mortality risk to 40–60%. The mechanisms of this prevalent and costly disease state remain uncertain and thus treatment continues to be supportive in nature. Animal

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Fig. 9. Morphologic changes of lung and intestine. Representative lung sections from zymosan-injected mice demonstrate inXammatory cells inWltration (a). Lung sections from a zymosan-treated mice treated with H. perforatum demonstrate reduced inXammatory cells inWltration (b). Representative ileum sections from zymosan-injected mice demonstrate edema (c) and inXammatory cells inWltration (see particles c1). Ileum sections from zymosan-injected mice treated with H. perforatum demonstrate reduced ileum injury (d). Figure is representative of at least three experiments performed on diVerent experimental days.

Fig. 10. EVect of H. perforatum on (a) toxicity score, (b) body weight change and (c) mortality induced by zymosan administration. A signiWcant systemic toxicity, loss of body weight and mortality were observed 12 days after zymosan administration. H. perforatum (30 mg/kg orally) signiWcantly reduced all of these parameters. Data are means § SEM of 20 mice for each group. *p < 0.01 vs. vehicle; °p < 0.01 vs. zymosan.

models of MODS allow a systematic investigation of the processes and mechanisms of this disease, and are likely to contribute to a greater understanding of the relevant pathophysiology, and ultimately will help to improve

treatment. In the experimental model of multiple organ dysfunction syndrome used here, a severe systemic inXammatory response is induced by local i.p. injection of zymosan.

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This study provides the Wrst evidence that pre-treatment of mice with H. perforatum extract attenuates: (i) the development of zymosan-induced peritonitis, (ii) the inWltration of the lung and intestine with PMNs (histology and MPO activity), (iii) the degree of the renal dysfunction (biochemical analysis), and (iv) the liver, lung, pancreatic, and intestinal injury (biochemical and histological analysis) caused by injection of zymosan administration. All of these Wndings support the view that H. perforatum extract attenuates the degree of MODS induced by zymosan in the mice. What, then, is the mechanism by which H. perforatum extract reduces non multiple organ dysfunction syndrome? Hypericum perforatum, known as St. John’s Wort, is a perennial herbaceous plant of the Hypericaceae family and is distributed in Europe, Northern Africa, Northern America, and the Shandong, Hebei, and Guizhou provinces in China [21]. Moreover, more than 10 components have been found in H. perforatum, including Xavonoids, phloroglucinols, and naphthodiathrones. Flavonol derivatives such as quercitin, rutin, and astilbin, naphthodiathrones such as hypericin and pseudohypericin, and phloroglucinols such as hyperforin and adhyperforin [32]. In addition, it has been reported that H. perforatum extract has antioxidant properties when such activity was evaluated in vitro on both human placental vein tissues and a cell-free system [33]. Furthermore, diVerent standardized extracts of H. perforatum demonstrated a free radical scavenging activity since they prevented a colored reaction produced by the horseradish peroxidase catalyzed formation of hydroxyl free radicals from hydrogen peroxide [34]. Such free radical scavenging capacity was found to correlate with the content of several Xavonoids including quercetin and hyperoside. One consequence of increased oxidative stress is the activation and inactivation of redox-sensitive proteins [35]. Recent evidence suggests the fact that the activation of NFB may also be under the control of oxidant/antioxidant balance [36,37]. Recently, we have clearly demonstrated that H. perforatum extract in vivo prevents the NF-B activation in a well established model of acute inXammation [38]. NF-B plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inXammation (e.g., iNOS) [39–42]. In mice, zymosan also causes an overproduction of NO due to induction of iNOS, which contributes to the inXammatory process [43,44]. We demonstrate here that H. perforatum extract attenuates the expression of iNOS in the lung and intestine as well as the nitrite/nitrate plasma levels from zymosan-injected mice. Therefore, the inhibition of iNOS expression and NO production by H. perforatum described in the present study is most likely attributed to the inhibitory eVect the activation of NF-B. Simultaneous production of both oxygen and nitrogencentered free radicals favors the production of a toxic reaction product, the oxidant peroxynitrite [45], as it has been recently demonstrated in various forms of inXammation [46].

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Although peroxynitrite may play a role in normal cellular processes [47], excessive amounts of peroxynitrite will cause injury/death in a variety of cell types including leukocytes, neutrophils, endothelial cells, and nerve cells [48,49]. Both ROS [7,50–52] and NO [51–53] have also played a role in the pathophysiology of zymosan-induced shock and inXammation. Interventions which reduce the generation or the eVects of ROS exert beneWcial eVects in a variety of models of inXammation including the zymosan-induced non-septic shock model used here. These therapeutic interventions include melatonin [54], a superoxide dismutasemimetic [16,55], and PDTC [56]. Recent evidence indicates that nitration of tyrosine can result from a number of chemical actions, and can be considered as a global marker of nitrosative stress [45]. Nitrotyrosine can be formed from the reaction of nitrite with hypochlorous acid or the reaction of nitrite with MPO and hydrogen peroxide [57]. In our experiments, we found increased immunohistochemical expression of nitrotyrosine in the intestine and lung mostly localized in the area of inWltrated inXammatory cells, suggesting that peroxynitrite or other nitrogen derivatives and oxidants are formed in vivo and may contribute to tissue injury. These data are consistent with previous Wndings that immunohistochemical staining for nitrotyrosine was localized on inXammatory cells during zymosan-induced multiple organ dysfunction syndrome [58]. In the present study, we observed that lung and intestine positive staining for nitrotyrosine was signiWcantly reduced in zymosan-injected mice which received H. perforatum. Three distinct mechanisms of protection were found and included increasing intracellular GSH, directly lowering levels of ROS, and preventing the inXux of Ca2+ despite high levels of ROS. Thus, the anti-oxidant property of H. perforatum may contribute to the attenuation by this agent of nitrotyrosine staining. In addition, the reduction of the expression of iNOS by H. perforatum may also contribute to the attenuation by this agent of nitrotyrosine in the lung and intestine from zymosan-injected mice. Both ROS and peroxynitrite produce cellular injury and necrosis via several mechanisms including peroxidation of membrane lipids, protein denaturation, and DNA damage. ROS produce strand breaks in DNA which triggers energyconsuming DNA repair mechanisms and activates the nuclear enzyme PARS, resulting in the depletion of its substrate NAD+. Since NAD+ functions as a cofactor in glycolysis and the tricarboxylic acid cycle, NAD+ depletion leads to a rapid fall in intracellular ATP. This process has been termed as ‘the PARS Suicide Hypothesis’. There is recent evidence that the activation of PARS may also play an important role in zymosan-induced multiple organ dysfunction syndrome [59–62]. We demonstrate here that H. perforatum attenuates the increase in PARS activity in the lung and intestine from zymosan-treated mice. Thus, we propose that the antiinXammatory eVects of H. perforatum reported here are–at least in part–due to the prevention of the activation of PARS.

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Several studies have pointed out that ROS and peroxynitrite directly or via PARP activation causes contraction of endothelial cells, resulting in increased permeability of postcapillary venues [63,64] and in increased adhesion of leukocytes to endothelial cells [65]. Activated PMNs are known to play an important role in tissue and organ damage [66–68]. It is now known that PMN migration and activation, occurring at the time of invasion, are controlled locally by cytokines [69,70]. Thus, recently it has been suggested that both the SIRS and the MODS, associated with severe acute peritonitis, may be secondary to the excessive activation of leukocytes [66–68], which in turn, would result in the release of secondary pro-inXammatory mediators [15,71], all of which play an important role in the pathogenesis of zymosan-induced MOF. In accordance with these Wndings, we observed that H. perforatum extract reduces the zymosan-induced neutrophils inWltration in the lung and intestine as well as prevent the organ dysfunction/ injury (as evaluated by histological examination). Thus, we propose the following positive feedback cycle in zymosan-induced MODS: early ROS production À PARP activationÀNF-B activationÀ endothelial injury À PMN inWltration À more ROS production. H. perforatum extract would intercept this cycle at the level of early ROS formation. Therefore, to its eVect on preserving the cellular energetic status and protecting against oxidant-induced cell necrosis, regulation of neutrophil recruitment may represent a novel important additional anti-inXammatory mode of action of H. perforatum extract. However, the exact mode of action of H. perforatum still remains to be determined. Since H. perforatum is a well tolerated substance in vivo at the dose used in this study (30 mg/kg), further studies investigating other possible mechanism are strongly warranted. References [1] E.A. Faist, E. Baue, Dittmer, Multiple organ failure in poly-trauma patients, J. Trauma 23 (1983) 775–787. [2] J.R. Shayevitz, C. Miller, K.J. Johnson, J.L. Rodriguez, Multiple organ dysfunction syndrome: end organ and systemic inXammatory response in a mouse model of nonseptic origin, Shock 4 (1995) 389–396. [3] M.J.J.M. Jansen, T. Hendriks, M.T.E. Vogels, J.W.M. van der Meer, R.J.A. Goris, InXammatory cytokines in an experimental model for the multiple organ dysfunction syndrome, Crit. Care Med. 24 (1996) 1196–1202. [4] G.H. El-Sokkary, R.J. Reiter, S. Cuzzocrea, A.P. Caputi, A-FMM Hassanein, D.-X. Tan, Role of melatonin in reduction of lipid peroxidation and peroxynitrite formation in nonseptic shock induced by zymosan, Shock 12 (1999) 402–408. [5] M.J.J.M. Jansen, T. Hendriks, M.F.C.M. Knapen, L.C.L.T. van Kempen, J.W.M. van der Meer, R.J.A. Goris, Chlorpromazine down-regulates tumor necrosis factor-alpha and attenuates experimental multiple organ dysfunction syndrome in mice, Crit. Care Med. 26 (1998) 1244–1250. [6] O.C. Kirton, J.M. Civetta, Ischemia-reperfusion injury in the critically ill: a progenitor of multiple organ failure, New Horizons 7 (1999) 87–95. [7] R. Demling, U. Nayak, K. Ikegami, C. LaLonde, Comparison between lung and liver peroxidation and mortality after zymosan peritonitis in the rats, Shock 2 (1994) 222–227.

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