Acute pancreatitis (AP) is an emergent and severe disease of the peptic system, and the plasma amylase and lipase values are elevated in most patients (1). There are different factors leading to AP. However, if dangerous factors are immediately cleared off and do not produce progressive injury, the pancreatic structure and function can recover to normal. On the contrary, the pancreas would suffer injury if the life-threatening factors could not be controlled. For example, severe AP can change the structure of the pancreatic duct and finally lead to chronic obstructive pancreatitis.
The etiologic basis of acute pancreatitis (AP) is multifactorial. However, as in other inflammatory diseases, a final common pathway mediated by reactive oxygen species (ROS) appears to play a critical role in the associated tissue destruction in both the initiation and progression of AP (2-5). Augmented production of ROS in a self-perpetuating manner, in excess of antioxidant defenses, occurs predominantly in activated neutrophils. Once produced, ROS could trigger various inflammatory processes. They can directly attack the lipoid matrix of biological membranes and 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 (4). Thus, ROS could impair the microcirculation and disturb the microvascular integrity, resulting in decreased perfusion and increased capillary permeability and fluid transudation. Neutrophils infiltrating the pancreas have also been very recently demonstrated to contribute, via ROS, to the pathologic activation of digestive enzymes in acinar cells (2). Therapeutic effects of antioxidants and radical scavengers have been shown in experimental models of acute pancreatitis (6-8).
Hypericum perforatum L. (Hypericaceae), popularly called St. John's wort, has been used in popular medicine since ancient times for several disorders such as skin wounds, eczema, burns, diseases of the alimentary tract, insomnia, and mental illness, among others (9). Hypericum perforatum extract contains flavonoids such as rutin, quercetin, and quercitrin, which have demonstrated a free radical scavenging activity in a model of autooxidation of rat cerebral membranes (10). 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 (11). Therefore, Hypericum extract, with a potential antioxidant activity, may be of value in dementia among other disorders of senility in which free radical generation is implicated. In addition, besides its antidepressant activities, Hypericum perforatum, in line with popular belief, also possesses anxiolytic, antiviral, wound-healing, antimicrobial, analgesic, and antiinflammatory effects (12). Studies with other plants of the same genus have been carried out under the stimulus of great scientific interest and economic value acquired by Hypericum perforatum. Thus, antidepressant, analgesic, antiinflammatory, antioxidant, antimicrobial, and wound-healing effects have also been found for other species of the genus Hypericum (13). More recently, Hypericum perforatum extract has been reported to efficiently attenuate interferon-γ (IFN-γ)-elicited activation of STAT-1 in alveolar A549/8 and colon DLD-1 cells (14). Hypericum extract has always been considered to have a benign side-effect profile compared with tricyclic antidepressants and serotonin-specific reuptake inhibitors (15). There has not been a single fatal intoxication of the extract as a monotherapy reported in the literature (16, 17). Hypericum extract, as an efficacious antidepressant medication with a potential antioxidant activity, was therefore hypothesized to be useful in the treatment of pathological situations in which ROS play an important role such us acute inflammation (e.g., acute pancreatitis).
Thus, the aim of the present studies was to evaluate the effects of Hypericum perforatum extract in animal models of acute inflammation (cerulein-induced acute pancreatitis).In particular, we investigated the effects of Hypericum perforatum extract on the serum levels of lipase and amylase,the pancreas injury, adhesion molecule (ICAM-1) expression, the nitration of cellular proteins by peroxynitrite, and the activation of the nuclear enzyme PAR synthetase (PAR).
MATERIALS AND METHODS
Male CD mice (weight 30-40 g; Harlan Nossan, Udine, Italy) were used in these studies. Animals were kept in a controlled environment and allowed access to rodent chow and water ad libitum. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with current European Union regulations (O.J. of E.C. L 358/1 12/18/1986).
Hypericum perforatum extract
Hypericum perforatum methanolic extract was a kind gift of Indena (Milano, Italy), and it was defined by the producer as containing 0.34% of hypericin, 4.1% of hyperforin, 5% of flavonoids (rutin, kaempferol, luteolin, myricetin, quercetin, quercitrin, isoquercitrin), 10% tannins, and the remaining part is composed of polysaccharides represented by maltodextrins.
Induction of pancreatitis
Animals were randomly divided into four groups (n = 10 for each group). The first group was treated with saline solution (0.1 mL 0.9% NaCl) intraperitoneally (i.p.) and served as a sham group. The second group was treated hourly (×5) with cerulein (50 μg/kg, suspended in 0.1 mL saline solution, i.p.). In the third group, animals received Hypericum perforatum extract (30 mg/kg, suspended in 0.2 mL of saline solution, o.s.), only 1 h before the induction of pancreatitis and thereafter received hourly cerulein i.p. (×5). The fourth group, like the sham group, was treated with Hypericum perforatum extract. Mice were killed by exsanguination at 6 h after the induction of pancreatitis. Blood samples were obtained by direct intracardiac puncture. Pancreas were removed immediately, frozen in liquid nitrogen, and stored at −80°C until assayed. Portions of these organs were also fixed in formaldehyde for histological and immunohistochemical examination. In another set of experiments, mice were randomized to receive treatment regimens that were identical to the ones listed above (n = 20 for each group) but were monitored for 5 days to monitor their survival rate. The dose of 30 mg/kg was based on previous study (17).
Paraffin-embedded pancreas samples were sectioned (5 μm), stained with hematoxylin/eosin, and examined by an experienced morphologist, who was not aware of the sample identity. Acinar-cell injury/necrosis was quantified by morphometry as previously described (5). For these studies, 10 randomly chosen microscopic fields (×125) were examined for each tissue sample, and the extent of acinar-cell injury/necrosis was expressed as the percentage of the total acinar tissue. The criteria for injury/necrosis were the following: (a) the presence of acinar-cell ghosts or (b) vacuolization and swelling of acinar cells and the destruction of the histoarchitecture of whole or parts of the acini, both of which had to be associated with an inflammatory reaction.
Determination of pancreas edema
The extent of pancreatic edema was assayed by measuring tissue water content. For these latter measurements, freshly obtained blotted samples of pancreas were weighted on aluminum foil, dried for 12 h at 95°C, and reweighed. The difference between wet and dry tissue weight was calculated and expressed as a percentage of tissue wet weight.
Localization of nitrotyrosine, PAR, and ICAM-1 by immunohistochemistry
At the end of the experiment, the tissues were fixed in 10% (w/v) phosphate-buffered saline (PBS, 0.01 M, pH 7.4)-buffered formaldehyde, and 5-μm sections were prepared from paraffin-embedded tissues. After deparaffinization, 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% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin and avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin, respectively (DBA, Milan, Italy). Sections were incubated overnight with antinitrotyrosine rabbit polyclonal antibody (1:500 in PBS, v/v) or with mouse antirat antibody directed at ICAM-1 (CD54) (1:500 in PBS, v/v) (DBA, Milan, Italy) and anti-PAR antibody (1:500 in PBS, v/v).Specific labeling was detected with a biotin-conjugated goat antirabbit or goat antimouse IgG and avidin-biotin peroxidase complex (DBA, Milan, Italy). To verify the binding specificity for ICAM-1, some sections were also incubated with primary antibody only (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. 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 (10 mM) to verify the binding specificity.
Serum amylase and lipase levels were measured at 6 h after cerulein injection by a clinical laboratory. Results are expressed in international units per liter. Trypsin was measured at 37°C using 64 μM BOC-Gln-Ala-Arg-7-amino-4-methylcoumarin (Bachem, California) as a substrate (18). To increase specificity, trypsin activity was expressed as fluorescence increase sensitive to soybean trypsin inhibitor (SBTI; 2 μM). Trypsinogen content was measured as trypsin activity after preincubation with an excess amount of enteropeptidase over a period of 30 min. The trypsin activity was corrected for substrate cleavage by enteropeptidase. Tissue contents of trypsin and trypsinogen were standardized to a purified trypsin preparation (T-8003; Sigma Chemical Co., Milan, Italy) whose activity was determined by active site titration. Parallel titrations of the standard trypsin and mouse trypsin activity with SBTI showed that the specific activities of both were comparable.
The usefulness of measuring myeloperoxidase (MPO) activity to assess neutrophil infiltration has been previously reported (19). Briefly, after weighing, a segment of pancreas was suspended in 0.5% hexadecyltrimethylammonium bromide (pH 6.5, 50 mg tissue/mL) and was then homogenized. After freezing and thawing of the homogenate three times, the tissue levels of MPO were determined by using 0.0005% hydrogen peroxide as a substrate for the enzyme. One unit of MPO activity is defined as that degrading 1 μmol of peroxide per minute at 25°C and is expressed in units per gram weight (U/g) of wet tissue.
Thiobarbituric acid-reactant substances measurement
Thiobarbituric acid-reactant substances measurement is considered a good indicator of lipid peroxidation in the pancreas tissues. Tissues, collected 6 h after cerulein administration, were homogenized in 1.15% KCl solution. An aliquot (100 μL) of the homogenate was added to a reaction mixture containing 200 μL of 8.1% SDS, 1500 μL of 20% acetic acid (pH 3.5), 1500 μL of 0.8% thiobarbituric acid, and 700 μL distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3000 × g for 10 min. The optical density at 650 nm (OD650) as measured using an ELISA microplate reader (SLT-Labinstruments, Salzburg, Austria). Thiobarbituric acid-reactant substances were calculated by comparison with OD650 of standard solutions of 1,1,3,3-tetramethoxypropane 99% malondialdehyde bis (dimethyl acetal) 99% (Sigma, Milan). The absorbance of the supernatant was measured by spectrophotometry at 650 nm.
Biotin-blocking kit, biotin-conjugated goat antirabbit IgG, primary antinitrotyrosine antibody, and avidin-biotin peroxidase complexes were obtained from DBA, Milan, Italy. All other reagents and compounds used were obtained from Sigma Chemical Company (Sigma, Milan, Italy).
All values in the figures and text are expressed as mean ± SE (sem) of the mean of n observations. In 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 three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A P value of less than 0.05 was considered significant. Statistical analysis for survival data was calculated by Fisher's exact probability test. For such analyses, P < 0.05 was considered significant.
Acute pancreatitis is reduced in Hypericum perforatum extract treatment
Cerulein-induced pancreatitis in mice was associated with significant rises in the serum levels of lipase and amylase (Fig. 1). The increase in lipase and amylase was markedly reduced in cerulein-treated mice after Hypericum perforatum extract administration (Fig. 1).
In sham-saline (Fig. 2A, Table 1) and sham-Hypericum perforatum extract-treated mice (Fig. 2B, Table 1), the histologic features of the pancreas were typical of a normal architecture. Histologic examination (at 6 h after the injection of cerulein) of pancreas sections from cerulein-treated mice revealed tissue damage characterized by edema, inflammatory cell infiltrates, and acinar cell necrosis (Fig. 2C), Table 1. Hypericum perforatum extract treatment resulted in a significant reduction of pancreatic injury (Fig. 2D, Tab 1). Particularly, a significant pancreas edema was observed at 6 h after cerulein administration (Fig. 3). In contrast, no significant pancreas edema was found at 6 h after cerulein injection in the tissue from Hypericum perforatum extract-treated mice (Fig. 3).
ICAM-1 expression and neutrophil infiltration are reduced by Hypericum perforatum extract treatment
A hallmark of acute pancreatitis is the accumulation of neutrophils in the pancreas, which augments the tissue damage. Therefore, we have evaluated the extent of the ICAM-1 adhesion molecules, which play a pivotal role in the firm attachment of neutrophils to the endothelium. Assessment of neutrophil infiltration into the pancreas was also performed by measuring the activity of MPO, an enzyme that is contained in (and specific for) PMN lysosomes. Thus, tissue levels of MPO directly correlate with the number of neutrophils in any given tissue. MPO activity was significantly increased after cerulein administration in mice (Fig. 4A). The increase in MPO activity was associated and correlated with the increase of imunohistochemical staining for ICAM-1 (Fig. 5C) in the vessels wall of the inflamed pancreas (see particle C1). The tissue MPO activity (Fig. 4A) and ICAM-1 (Fig. 5D) immunostaining were markedly reduced by Hypericum perforatum extract treatment. Staining of pancreas tissue sections obtained from sham-saline (Fig. 5A) and sham-Hypericum perforatum extract-treated mice (Fig. 5B) with anti-ICAM-1 antibody showed a specific staining along vessels, demonstrating that ICAM-1 is constitutively expressed.
Hypericum perforatum extract treatment reduced nitrotyrosine formation and PARS activation
The generation of reactive oxygen and nitrogen-derived radicals contributes significantly to the tissue necrosis and dysfunction associated with acute pancreatitis (20).
At 6 h after cerulein administration, pancreas tissues were investigated for thiobarbituric acid-reactant substances, indicative of lipid peroxidation. As shown in Figure 4B, thiobarbituric acid-reactant substance levels were significantly increased in the pancreas of carrageenan-treated mice. In mice treated with Hypericum perforatum extract, pancreas thiobarbituric acid-reactant substance levels were significantly reduced in comparison to those of vehicle-treated mice (Fig. 4B). Furthermore, a positive staining for nitrotyrosine, a marker of nitrosative stress and injury, was found in the injured pancreas of cerulein-treated mice (Fig. 6A). Immunohistochemical analysis for poly(ADP-ribose) from pancreas sections obtained from mice treated with cerulein also revealed a positive staining in injured pancreas (Fig. 7A). Hypericum perforatum extract treatment of mice subjected to acutepancreatitis reduced the formation of nitrotyrosine (Fig. 6C), C1) and PAR activation (Fig. 7C), indicating the reduction of oxidant-induced tissue damage.
There was no staining for either nitrotyrosine or PAR in pancreas obtained from the sham-saline (Fig. 6A, 7A) and from sham-Hypericum perforatum extract treated mice (Fig.6B, 7B).
Hypericum perforatum extract treatment reduced reduces the mortality caused by cerulein
To imitate the clinical scenario of acute pancreatitis, mice were treated hourly (×5) with cerulein (50 μg/kg, suspended in saline solution, i.p.). Approximately 50% of the animals died at 5 days after cerulein administration. The treatment with Hypericum perforatum extract significantly reduced the cerulein-induced mortality (Fig. 8).
This study provides the first evidence that Hypericum perforatum extract attenuates (a) the development of cerulein-induced pancreatitis, (b) morphological injury, (c) nitrotyrosine formation and PARS activation, (d) neutrophil infiltration, and (e) the ICAM-I expression. All of these findings support the view that Hypericum perforatum extract attenuates the degree of AP.
What, then, is the mechanism by which Hypericum perforatum extract protects against inflammatory injury? 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). Hypericum perforatum has been used traditionally for the treatment of mild to moderate depression, and clinical studies have suggested that extract of Hypericum perforatum is as effective as traditional antidepressants and has superior efficacy to placebo. However, the antidepressive mechanism of Hypericum perforatum is unclear and controversial; more than 10 components have been found in Hypericum perforatum, including flavonoids, phloroglucinols, and naphthodianthrones. Flavonol derivatives such as quercitrin, rutin, and astilbine, naphthodiathrones such as hypericin and pseudohypericin, and phloroglucinols such as hyperforin and adhyperforin showed antidepressant activity in different antidepressive model systems (22). Moreover, two recent clinical studies have shown that Hypericum perforatum was ineffective in the treatment of moderately severe major depression (23). Flavonoids are rich in Hypericum perforatum, in which the content is 11.71% in flowers and 7.4% in leaves and stems. Recently, an antioxidant activity for Hypericum perforatum extract has been reported. In particular it has been shown, in amouse model exhibiting chronic fatigue syndrome, that Hypericum perforatum extract significantly reduced elevated lipid peroxidation and restored GSH levels decreased as a result of chronic swimming (24). In addition, it has been reported that Hypericum perforatum extract has antioxidant properties, evaluated in both human placental vein tissues and a cell-free system (25). Furthermore, different standardized extracts of Hypericum perforatum demonstrated a free radical scavenging activity, because they prevented a color reaction produced by the horseradish peroxidase-catalyzed formation of hydroxyl free radicals from hydrogen peroxide (26). Such free radical scavenging capacity was found to correlate with the content of several flavonoids including quercetin and hyperoside.
It has been suggested that oxygen free radicals are responsible for a wide variety of diseases or conditions (27). Oxygen free radicals have been known to play an important role in the pathogenesis of pancreatitis of some experimental models (28). Oxygen free radicals are involved in initiation of pancreatitis (6). Also, it was reported that oxygen free radicals act as important mediators of tissue damage in experimental acute pancreatitis (28). These species are cytotoxic agents, inducing lipid peroxidation and other cellular oxidative stress by cross-linking proteins, lipids, and nucleic acids, which then cause cellular dysfunction, damage, and eventually death. In the present study, we found that the pancreas damage induced by cerulein administration was associated with high concentrations of thiobarbituric acid-reactant substances, which are considered a good indicator of lipid peroxidation. 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 (29). Nitrotyrosine can be formed from the reaction of nitrite with hypochlorous acid or the reaction of nitrite with MPO and hydrogen peroxide (30). In our experiments, we found increased immunohistochemical expression of nitrotyrosine mostly localized in the injured area, 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 findings that immunohistochemical staining for nitrotyrosine was localized in the injured pancreas during cerulein-induced acute pancreatitis (18,31). In the present study we observed that pancreas injury was significantly less in mice treated with Hypericum perforatum. Indeed, Hypericum perforatum treatment prevented the formation of tissue thiobarbituric acid-reactant substances and nitrotyrosine staining in cerulein-treated mice. Three distinct mechanisms of protection were found and included increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca2+ despite high levels of ROS. Thus, the antioxidant property of Hypericum perforatum may contribute to the attenuation by this agent of tissue thiobarbituric acid-reactant substances and nitrotyrosine staining.
Moreover, ROS produce strand breaks in DNA, which trigger energy-consuming DNA repair mechanisms and activates the nuclear enzyme PARS resulting in the depletion of its substrate NAD+ and a reduction in the rate of glycolysis. As 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 "the PARS Suicide Hypothesis." We demonstrate here that Hypericum perforatum extract attenuates the increase in PARS activity in the pancreas from cerulein-treated mice.
In addition to attenuating ONOO− production and PARS activation, Hypericum perforatum extract also reduced the development of edema and neutrophil accumulation and had an overall protective effect on the degree of pancreas injury as assessed by histologic examination. In previous studies ROS has been found to increase both neutrophil infiltration and adhesion (32). Endothelial adhesion molecules are major regulators of neutrophil traffic, regulating the process of neutrophil chemoattraction, adhesion, and emigration from the vasculature to the tissue. ICAM-1, constitutively expressed on the surface of endothelial cells, is then involved in the neutrophil adhesion (33). We observed that cerulein induced the expression of ICAM-1 on endothelial cells in mice. In contrast, we demonstrated significant reduction in the expression of ICAM-1 in Hypericum perforatum extract-treated mice at 6 h after cerulein administration.
In conclusion, this study provides evidence that Hypericum perforatum extract cause a substantial reduction of intestinal cerulein-induced pancreatitis. We speculate that Hypericum perforatum extract and related compounds may be useful in the therapy of conditions associated with acute pancreatitis.
This study was supported by grant from PRIN 2003 to S.C. and H.S. and Cariverona Project 2002 to H.S. The authors would like to thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study and Mrs. Caterina Cutrona and Miss Valentina Malvagni for editorial assistance with the manuscript.
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St. John's wort; neutrophil infiltration; reactive oxygen species (ROS)