Raloxifene, a Selective Estrogen Receptor Modulator, Reduces Carrageenan-Induced Acute Inflammation in Normal and Ovariectomized Rats
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内分泌学杂志 2005年第8期
Department of Experimental Pharmacology, University of Naples Federico II, 80131 Naples, Italy
Address all correspondence and requests for reprints to: Prof. Rosaria Meli, Department of Experimental Pharmacology, via D. Montesano 49, 80131 Naples, Italy. E-mail: meli@unina.it.
Abstract
Raloxifene (RAL) is a selective estrogen receptor modulator presenting tissue-specific agonist activity. The aim of this study was to examine whether RAL has an estrogenic effect on carrageenan-induced acute inflammation. Adult female rats were ovariectomized (OVX) 7 wk before edema or pleurisy to deplete circulating estrogens. Edema formation and selected inflammatory markers in inflamed paw tissue were measured in intact (sham-operated) and OVX rats. Groups of OVX rats were treated with RAL (1, 3, or 10 mg/kg) or 17?-estradiol (E2, 25 μg/kg), and these treatments began 2 d after surgery and continued until carrageenan paw edema or pleurisy. Ovariectomy amplifies the inflammation, and we found that RAL, as well as E2, attenuates inflammation and tissue damage associated with paw edema and pleurisy. In treated rats, there is a decrease in edema development and formation, and in polymorphonuclear cell infiltration and migration, as shown by myeloperoxidase measurement and cell counting. RAL and E2 treatments decrease cyclooxygenase-2 and inducible nitric oxide synthase expression in inflamed areas and counteract the inhibition of peroxisome proliferators-activated receptor- expression caused by ovariectomy, restoring this receptor protein expression to sham-operated levels and identifying a possible peroxisome proliferators-activated receptor-dependent antiinflammatory effect of these drugs. Moreover, RAL and E2 increase cytoprotective heat shock protein 72 expression, which seems to be closely associated with the remission of the inflammatory reaction. In addition, we confirm the antiinflammatory effect of RAL in male rats, using a single administration of RAL or E2.
Introduction
HYPOESTROGENISM CAUSED BY ovariectomy, disease, or menopause is associated with a multitude of clinical symptoms. Selective estrogen receptor modulators (SERMs) are well established as synthetic estrogen substitutes and can be used to prevent or treat diseases caused by estrogen deficiency, without most of the undesirable effects of estrogen (1). Although SERMs are extensively used in postmenopausal women, not everything is known about their action mechanisms, especially on the inflammatory process. We focused on raloxifene (RAL), a benzothiophene derivative that was shown to have antiestrogenic properties in breast tissue and a neutral effect on endometrium, and it has potentially beneficial estrogen-like effects in nonreproductive tissue, such as bone (2).
Estrogens reduce bone resorption directly by inhibiting osteoclasts and indirectly by suppressing osteoblastic production of various proresorptive paracrine factors, such as IL-1?, IL-6, and TNF- (3), thus showing a relationship with inflammatory cytokines. Moreover, estrogens may also inhibit the inflammatory reaction by decreasing the expression of specific markers and attenuating the degree of inflammation and tissue damage (4, 5, 6). It has recently been demonstrated that RAL increases osteoprotegerin, a potent inhibitor of bone resorption, and suppresses the bone-resorbing cytokine, IL-6 (7).
The aim of this study was to investigate the effect of RAL in models of carrageenan-induced acute inflammation, in particular, paw edema and pleurisy in the rat.
The acute inflammatory response is characterized by edema from increased vascular permeability, leading to an extravasation of fluid, and leukocyte infiltration. Carrageenan-induced local inflammation models are commonly used to evaluate nonsteroidal antiinflammatory drugs; hence, the cellular and the molecular mechanisms of carrageenan-induced edema are well known. Histamine, serotonin, and bradykinin are involved in the initial phase of inflammation (0–1 h), whereas the late phase (1–6 h) is mainly sustained by prostaglandins (8, 9), attributed to the induction of cyclooxygenase (COX)-2 in the inflamed tissue (10). Another key mediator in acute inflammation is nitric oxide (NO), a potent vasodilator. Its involvement during the inflammatory response may be related to its ability to increase vascular permeability and edema through changes in local blood flow (11, 12). Among the three distinct isoforms of NO synthase (NOS), it has been suggested that inducible enzyme [inducible NOS (iNOS)] mainly contributes to the development and maintenance of acute inflammation. This is further supported by the observation that its protein expression is up-regulated during the late stage of inflammation in paws after injection of carrageenan (13, 14). Furthermore, NO has been shown to increase the production of proinflammatory prostaglandins in in vitro (15, 16) and in vivo studies (17, 18).
At the site of inflammation, NO is produced by leukocytes and endothelial cells (14). In the acute phase after the increase of vascular permeability, neutrophil infiltration occurs. This contributes to the inflammatory response by producing several mediators, such as oxygen-derived free radicals (19). A marker of neutrophil infiltration in tissue is myeloperoxidase (MPO), and it has been clearly shown to be related to the severity.
Recently, the peroxisome proliferator-activated receptor (PPAR)- appears to be involved in the regulation of inflammatory responses (20), and its receptor agonists inhibit inflammatory edema and hyperalgesia (21) and raise the possibility that synthetic PPAR- ligands may have therapeutic value involving macrophage activation.
In vertebrates, heat shock proteins (hsp), hsp 72 especially, may be protective against certain pathological conditions, including ischemia, infection, and inflammation; and it has been demonstrated that activation of the heat shock response is closely associated with the remission of inflammation (22).
To characterize the antiinflammatory effects of RAL in carrageenan-induced acute inflammation models, we used ovariectomized (OVX) rats and, as reference drug, 17?-estradiol (E2). We investigated the effect of RAL and E2 on paw edema and pleurisy development, polymorphonuclear leukocyte infiltration (assessing MPO levels), and COX-2, iNOS, PPAR-, and hsp72 expression. In addition, to confirm the antiinflammatory effect of the drug, we induced paw edema in male rats, which are not influenced by E2 fluctuation of estrous cycle, using a single administration of RAL or E2.
Materials and Methods
Animals
Male and female Sprague Dawley rats (Harlan Italy, San Pietro al Natisone, Udine, Italy) were housed in stainless steel cages in a room kept at 22 ± 1 C with a 12-h light, 12-h dark cycle. The animals were acclimated to their environment for 1 wk and had ad libitum access to tap water and rodent standard diet. All animal experiments complied with the Italian (D.L. no. 116 of 27 January, 1992) and associated guidelines in the European Communities Council (Directive of 24 November, 1986, 86/609/ECC).
Ovariectomy and drug treatment
At the onset of the study, female rats (mean body weight of the cohort, 176 ± 1.3 g) were bilaterally OVX under anesthesia (100 mg/kg ketamine plus 5 mg/kg xylazine ip). The sham-operated (SHAM) animals were subjected to the same general surgical procedure as OVX groups except ovarian excision.
A group of OVX rats were treated by gavage with RAL (1, 3, or 10 mg/kg once daily; Lilly Research Laboratories, Indianapolis, IN). RAL was administered in 17.5% hydroxypropyl-?-cyclodextrin (Aldrich, Milwaukee, WI) in a vol of 100 μl/100 g body weight. SHAM and OVX rats were administered the vehicle, i.e. 100 μl/100 g body weight of 17.5% hydroxypropyl-?-cyclodextrin. Moreover, a group of OVX rats received E2 (25 μg/kg, sc, twice a week; Sigma, St. Louis, MO) as reference drug. All treatments began 2 d after surgery and continued for 7 wk. Last administrations were performed 2 h before inflammation induction. Each experimental group was composed of 15 rats.
The acute experiment was done to confirm the drawn antiphlogistic effect of RAL, even in the physiological estrogenic state, by treating male rats with a single dose (acute administration). Briefly, the animals (200.6 ± 6.8 g) were divided into the following groups: control animals, rats treated with RAL (3, 10, or 30 mg/kg per os 1 h before carrageenan paw injection), or rats treated with E2 (50 μg/kg, ip, 1 h before carrageenan injection). In this experiment, each group was composed of five rats.
Carrageenan-induced paw edema
The experimental groups described in the above paragraph received a subplantar injection of 100 μl of a sterile saline containing 1% wt/vol -carrageenan or sterile saline alone (control group) in the right hind paw. Foot volumes were measured using a water plethysmometer (Ugo Basile, Milan, Italy) immediately before injection and every hour for 5 h; and edema was evaluated, in milliliters, as the difference between the paw volume at each time-point and the basal paw volume. The inflammatory effect was evaluated as area under the curve (AUC) and expressed in milliliters of edema per hour. AUC is the integrated and cumulative measure of this effect. To compare the course of the edema of all groups, we integrated all curves to obtain the cumulative areas. In this way, one value for each area under the entire curve was obtained. Statistical analysis of these values was performed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
Some female rats from selected groups were killed at 3 h or 5 h after carrageenan injection, and the subplantar edematous area from paw was excised and prepared for further determinations (see below).
MPO assay in inflamed paws
Subplantar tissue obtained from five female animals per group, killed 3 h after carrageenan or saline injection, was homogenized in 1 ml hexadecyltrimethylammonium bromide buffer (Sigma) using a Polytron homogenizer (3 cycles of 10 sec at maximum speed). After centrifugation of homogenates at 13,000 rpm for 2 min, supernatant fractions were assayed for MPO activity using the method described by Bradley et al. (23). Briefly, samples were mixed with phosphate buffer containing 1 mM O-dianisidine dihydrochloride and 0.001% hydrogen peroxide in a microtiter plate reader. Absorbance was measured at 450 nm, taking three readings at 30-sec intervals. Units of MPO were calculated considering that 1 U MPO = 1 μmol H2O2 split, and 1 μmol gives a change in absorbance of 1.13 x 10–2 nm/min. MPO activity was expressed as units of MPO per milligram of protein.
Western blot analysis
Subplantar tissue (0.1 g), obtained from female rats killed 5 h after carrageenan or saline injection, was disrupted by homogenization on ice in lysis buffer (HEPES 20 mM, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2; 1 mM EGTA; 0.2 mM EDTA; 25% (vol/vol) glycerol; 0.5% Nonidet P-40; 0.5 mM phenylmethylsulfonyl fluoride; 1.5 μg/ml trypsin inhibitor; 3 μg/ml pepstatin A; 2 μg/ml leupeptin; 0.1 mM benzamidine; and 0.5 mM dithiothreitol). After 1 h, tissue lysates were obtained by centrifugation at 100,000 x g for 15 min at 4 C. Protein concentrations were estimated by the Bio-Rad protein assay (Bio-Rad Laboratories, Segrate, Milan, Italy) using BSA as standard.
For Western blot analysis, 25 or 40 μg protein of lysates was dissolved in Laemmli’s sample buffer, boiled for 5 min, and subjected to SDS-PAGE (8% polyacrylamide). The blot was performed by transferring proteins from a slab gel to nitrocellulose membrane at 240 mA for 40 min at room temperature. The filter was then blocked with 1x PBS, 5% nonfat dried milk for 40 min at room temperature and probed with specific monoclonal antibodies against iNOS (BD Biosciences Transduction Laboratories, Lexington, KY; 1:2,000), or COX-2 (Cayman Chemical, Ann Arbor, MI; 1:1500), or PPAR- (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100), or hsp72 (Stressgen Bioreagents, Victoria, British Columbia, Canada; 1:15,000) in 1x PBS, 5% nonfat dried milk, 0.1% Tween 20 at 4 C, overnight. The secondary antibody (antimouse IgG, or antirabbit IgG, or antigoat IgG peroxidase conjugate) was incubated for 1 h at room temperature. Subsequently, the blot was extensively washed with PBS, developed using enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturer’s instructions, and exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY). -Tubulin protein (Sigma; 1:1000) Western blot was performed to ensure equal sample loading.
The protein bands of iNOS (130 kDa), COX-2 (70 kDa), PPAR- (55 kDa), or hsp72 (72 kDa) on x-ray film were scanned and densitometrically analyzed with a model GS-700 imaging densitometer (Bio-Rad Laboratories).
Carrageenan-induced pleurisy
Carrageenan pleurisy was induced in other SHAM or OVX groups of rats (n = 6 in each group). A group of OVX animals was treated by gavage with RAL at 3 mg/kg·d in 17.5% hydroxypropyl-?-cyclodextrin, as described above. Another group of OVX rats received E2 (25 μg/kg, sc, twice a week) as reference drug. SHAM and OVX untreated rats received vehicle in the same conditions. All treatments were initiated on d 2 of the surgery and continued up to 7 wk.
Animals were anesthetized with isoflurane, and pleurisy was initiated by injection, into the pleural cavity, of 200 μl of a 1% wt/vol carrageenan solution in saline. After 4 h, the rats were killed. Pleural exudates were harvested by rinsing the pleural cavity with 2 ml saline solution. Exudates with blood contamination were rejected. The exudate volumes were measured, the samples were centrifuged at 800 x g for 10 min, and the cell pellet was resuspended in saline solution for cell count. Leukocyte number was estimated after Turk solution staining with the Neubauer counting chamber.
Measurement of circulating estradiol
After the experiments, serum estradiol level was determined by an ELISA kit (Abbott Laboratories, Abbott Park, IL) to determine the hypoestrogenism induced by ovariectomy and its level in E2 or RAL-treated animals.
Statistical analysis
All data were presented as mean ± SEM. Statistical analysis was performed by ANOVA test for multiple comparisons followed by Bonferroni’s test. Statistical significance was set at P < 0.05.
Results
Effects of RAL and E2 on the course of the carrageenan-induced paw edema in OVX and male rats
Ovariectomy significantly reduced estradiol serum levels (P < 0.05), and this decrease was reversed by E2 replacement but not by RAL treatment (data not shown).
As reported in Fig. 1, hypoestrogenism amplified the acute inflammatory process induced by intraplantar injection of carrageenan. In fact, ovariectomy had a strong proinflammatory effect seen by an increase in rat paw edema, evaluated as AUC and expressed in milliliters of edema per hour (P < 0.001). This effect was inhibited by E2 or RAL treatment. Moreover, RAL acts in a dose-dependent manner, and significantly, at 3 and 10 mg/kg (P < 0.05 and P < 0.01, respectively). Figure 2 shows the time course of edema, highlighting which mediators are modified the most by drug treatment. E2 and RAL (10 mg/kg) reduced paw edema every hour, whereas RAL (3 mg/kg) treatment showed a significant effect only 3 and 5 h after carrageenan injection, the endpoints chosen for the determination of MPO activity and protein expression.
FIG. 1. Antiinflammatory effect of 7-wk RAL treatment (RAL 1, 3, or 10 mg/kg) on paw edema of OVX rats. The SHAM animals were subjected to the same general surgical procedure as OVX groups, except ovarian excision. E2 (25 μg/kg) was used as reference drug. The inflammatory effect was evaluated as AUC and expressed in milliliters of edema per hour. Values are means ± SEM from n = 10 rats for each group. ***, P < 0.001 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
FIG. 2. Time-dependent development of paw edema elicited by carrageenan in SHAM and OVX rats. Seven weeks of RAL (1, 3, or 10 mg/kg) and E2 (25 μg/kg) treatments significantly inhibited edema formation at the indicated time points. Data are means ± SEM of 10 animals for each group. **, P < 0.01; ***, P < 0.001 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
To confirm the antiphlogistic effect of RAL, we induced paw edema in male rats, where the estrogen serum levels are not influenced by the estrous cycle. The animals were treated with a single administration (60 min before carrageenan injection) of E2 (50 μg/kg) or RAL (3, 10, and 30 mg/kg). In Fig. 3, RAL showed a reduction of edema at the highest dose used (P < 0.05), and E2 (50 μg/kg) had a considerable antiinflammatory effect, confirming the protective effect of estrogen in another carrageenan-induced tissue injury (5, 6).
FIG. 3. Antiinflammatory effect of acute administration of RAL (3, 10, or 30 mg/kg) or E2 (50 μg/kg) on carrageenan paw edema of male rats. Control animals (CON) received drug vehicle. Data are means ± SEM of five animals for each group. *, P < 0.05; ***, P < 0.001 vs. CON.
The control AUC value of edema in male rats is slightly higher than in female rats. This is because male and female rats were the same age but had different weights. This difference reflects the measurement of basal paw volume before carrageenan. In fact, male vol was 0.64 ± 0.04 ml, whereas female vol was 0.39 ± 0.04 ml (P > 0.05). This difference was due to the size of the paws and not to sex.
RAL and E2 effect on some inflammatory parameters of carrageenan-induced paw edema
MPO activity, a marker of polymorphonuclear cell (PMN) accumulation in subplantar areas, was determined in control and inflamed tissues (Fig. 4). In the paws of OVX rats, a slight, but not significant, increase of MPO activity (127.3 ± 14.37 vs. 101.1 ± 10.01 SHAM values) was revealed, whereas it was undetectable in saline control paws of animals treated with vehicle or drugs (data not shown). In carrageenan-injected animals, RAL treatment significantly reduced the activity of MPO, in a dose-dependent manner, vs. OVX animals. The effect of E2 was higher and significant both vs. SHAM and OVX rats.
FIG. 4. Dose-dependent effect of RAL on MPO activity. MPO activity was measured in subplantar paw homogenates of SHAM, 7-wk OVX, and OVX rats treated with RAL (1, 3, or 10 mg/kg) or E2 (25 μg/kg). The animals were killed 3 h after carrageenan paw injection. Data are means ± SEM of five animals for each group. **, P < 0.01 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
To evaluate the effect of the drugs on inflammatory enzymes, Western blot analysis of COX-2 and iNOS was performed on lysates of subplantar tissues from animals killed 5 h after edema induction. We chose 5 h because this is the time point where these proteins are overexpressed in a very significant manner. In saline-injected paws, both these inducible enzymes were not expressed (data not shown). Injection of carrageenan induced the expression of these proteins in all groups studied, and ovariectomy clearly increased their expression (Figs. 5A and 6A). E2 (25 μg/kg) and RAL (3 mg/kg) significantly reduced COX-2 and iNOS expression to SHAM level as evidenced by densitometric analysis (Figs. 5B and 6B).
FIG. 5. Western blot analysis showing the increase of induction of COX-2 expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Seven weeks of RAL (3 mg/kg) or E2 (25 μg/kg) treatment significantly reverted the increase of COX-2 expression induced by hypoestrogenism. This effect was revealed by densitometric analysis of bands. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates obtained from two animals per group is shown, and densitometric analysis of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
FIG. 6. Western blot analysis showing the increase of induction of iNOS expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Seven weeks of RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reverted the increase of iNOS expression induced by hypoestrogenism. This effect was revealed by densitometric analysis of bands. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates from two animals per group is shown, and densitometric analysis of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
Moreover, to elucidate whether the protective effect of RAL was related to the modulation of PPAR- expression, we examined the modification of this receptor in the same subplantar tissue lysates. The basal expression of PPAR-, evaluated in saline-injected paw tissues, was enhanced in inflammatory conditions, whereas 7-wk ovariectomy induced a clear reduction of this specific receptor protein band (Fig. 7A). Both hormonal replacement and RAL treatment reversed this reduction, as revealed by densitometric analysis (Fig. 7B).
FIG. 7. Western blot analysis showing the decrease of PPAR- expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Control saline-injected paw tissue lysate is also reported (Saline). Seven weeks of RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reversed this reduction, as seen by densitometric analysis. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates from two animals per group is shown, and densitometric analysis of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
Moreover, carrageenan injection was associated with increased hsp72 compared with protein expression observed in lysates from tissues collected from saline-treated rats. In OVX animals, 5 h after carrageenan, a decreased hsp72 expression was also detected by immunoblotting analysis compared with SHAM rats (Fig. 8). In contrast, both drug treatments reverted the OVX effect, inducing an increase of protein until SHAM levels.
FIG. 8. Western blot analysis (A) showing the decrease of hsp72 expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Control saline-injected paw tissue lysate is also reported (Saline). Seven weeks of RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reversed this reduction, as revealed by densitometric analysis. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates from two animals per group is shown, and densitometric analysis (B) of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
Further antiinflammatory effect of RAL and E2: carrageenan-induced pleurisy in OVX rats
Ovariectomy amplified the acute pleurisy induced by carrageenan injection, producing an increase of turbid exudates (Fig. 9, upper panel, 2.0 ± 0.1 vs. 1.4 ± 0.1 ml/rat, OVX vs. SHAM) that contained a larger amount of polymorphonuclear leukocytes (Fig. 9, lower panel, 117.4 ± 10.4 vs. 69.7 ± 2.0 x 106cell/rat in SHAM). E2 and RAL treatment significantly reduced exudate formation (43.6% and 20.1%, 1.1 ± 0.1 and 1.6 ± 0.1, respectively) and polymorphonuclear leukocyte infiltration in the pleural cavity (47.5% and 30%, 61.5 ± 6.8 and 82.1 ± 5.3, respectively).
FIG. 9. Effect of RAL and E2 on carrageenan-induced pleurisy in OVX rats. Volume exudate (upper panel) and accumulation of PMNs (lower panel) in pleural cavity at 4 h after carrageenan injection. Both 7-wk RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reduced pleural exudation and leukocyte infiltration. Data are means ± SEM from six animals per group. ***, P < 0.001 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
Discussion
Here we demonstrated that RAL, a lipolytic SERM with tissue-specific agonist activity, plays a protective antiinflammatory role in the development of carrageenan-induced edema and pleurisy.
Rat paw edema has been well characterized and described (24), and most nonsteroidal antiinflammatory drugs have been shown to be active in this inflammatory model. Moreover, the model is useful to assess the contribution of mediators involved in vascular changes associated with acute inflammation. This study used OVX animals, and the results indicate that the estrogen state of rats is a critical issue. Indeed, the OVX animals have a more consistent inflammatory response to carrageenan (increased edema) than SHAM rats.
In several experimental models, estrogens display antiinflammatory activity: arthritis (25), uveitis (26), encephalomyelitis (27), and pleurisy (5, 6). The mechanism behind estradiol’s antiinflammatory effect may be multifactorial: the hormone-dependent blockade of cytokines such as IL-6, IL-1, and TNF-; the inhibition of macrophage infiltration in the damaged tissues; and the decrease of inflammatory mediators.
Growing evidence indicates a role for proinflammatory factors in the pathogenesis of coronary artery disease, among them: liver-derived acute-phase reactant proteins, such as C-reactive protein (CRP), fibrinogen, and albumin, as well as cytokines (28). However, in clinical studies, estrogen alone (29) or hormonal replacement therapy (HRT) (30, 31) using estrogen and progestin combination has different effects on markers of inflammation. In fact, proinflammatory effects of HRT have been demonstrated for CRP, albumin, metalloproteinase, and factor VII. It is important to consider that these effects may be modified by obesity and that the route of administration also appears to play an important role, because oral formulation of estrogens may produce a strong first-pass liver effect.
Although the impact of RAL on the venous system is generally similar to that of HRT, its effect on the arterial system and subsequent risk of atherosclerotic events appears to be either neutral or slightly favorable, based on the limited clinical data (32). RAL has a neutral effect on CRP (33) and a favorable effect on low-density lipoprotein fibrinogen and lipoprotein(a) (34).
Using OVX rats, we recently demonstrated that 7 wk of RAL treatment modified the lipid profile by reducing cholesterol and low-density lipoprotein levels. This drug also shows an estrogen agonist effect on fat mass homeostasis and weight gain (35). Seven-week ovariectomy induced in rats a mild obesity that mimics estrogen insufficiency in humans and creates a useful model to study the mechanism by which hypoestrogenism influences hormonal status and metabolic processes. On the basis of these previous data, we used the same E2 and RAL doses and route of administration, beginning the treatment on d 2 to prevent the early modifications of estrogen loss.
E2 treatment is known to reduce the carrageenan-induced inflammatory process, especially in pleurisy. Our present results confirm these findings; in fact, the pharmacological dose of E2 (25 μg/kg, a higher dose than used in replacement therapy) dramatically reduced paw edema. All of the parameters examined revealed that RAL had a significant dose-dependent, but minor, effect compared with the exogenous hormone. E2 more significantly reduced MPO activity, COX-2, and iNOS expression and increased protective parameters (hsp72 and PPAR-) but also reduced exudates and cell migration in the pleural cavity.
RAL may be antiinflammatory by reducing inducible proinflammatory enzymes (COX-2 and iNOS) and decreasing infiltration of PMNs into the inflamed paw and pleural cavity. This is revealed by the declining MPO activity after 7 wk of RAL treatment. MPO enzyme, contained in azurophilic granules of PMNs, is involved in the formation of reactive oxygen species and oxidation of biological materials responsible for tissue damage. In fact it is undetectable in noninflamed control paws, evident in SHAM and OVX inflamed tissue, and significantly reduced by E2, and dose-dependently by RAL.
The inducible isoform of COX appears to be responsible for prostaglandin production in edema 1 h after carrageenan injection (8, 36). COX-2 is usually absent, or present only in a small amount, in cells under basal conditions but is overexpressed in the immune cells or tissue after phlogogenic stimuli or cytokine induction. More recently, the substained late phase (1–6 h) of carrageenan paw edema has been attributed to the induction of COX-2 in the tissue (10). Inhibitors of NOS activity reduce the development of carrageenan-induced inflammation and support a role for NO in the pathophysiology associated with this model of inflammation (13). Although NO itself is a weak oxidant, biochemical studies have shown that NO rapidly interacts with superoxide anion to yield peroxynitrite, which then decomposes to form highly reactive oxidant species responsible for lipid peroxidation and tissue damage.
The NOS and COX pathways appear to cooperate in amplifying the inflammatory response. This is achieved by a synergistic interaction between NO and prostaglandins on blood flow and microvascular permeability (37) as well as by NO-driven COX activation, leading to the exaggerated production of prostaglandins (13, 15, 17).
We showed that ovariectomy and decreasing estrogen levels can increase both COX-2 and iNOS expression. This study provides the first evidence that RAL causes a substantial reduction of acute inflammation in the rat by reducing tissue expression of these proinflammatory enzymes.
Moreover, here we demonstrate that RAL is able to modulate PPAR- expression in inflamed paw tissue. PPARs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, which includes the classical steroid, thyroid, and retinoid hormone receptors as well as many orphan receptors. Among three related PPAR isotypes, PPAR- has been attracting considerable attention as a regulator of adipogenesis and expression of adipocyte genes involved in lipid metabolism (38). Apart from its metabolic effects, agonists of PPAR- have been demonstrated to modulate the inflammatory responses of several cells by inhibiting the expression of proinflammatory cytokines (20, 39), iNOS (40), and COX-2 (41). On the other hand, it has also been reported that cytokines, such as TNF-, antagonize the synthesis of PPAR-, block adipocyte differentiation, and contribute to insulin resistance (39). In our experimental model, RAL or E2 treatment counteracted the inhibition of PPAR- expression caused by ovariectomy, restoring this receptor protein expression to SHAM levels and identifying a possible PPAR-dependent antiinflammatory effect of these drugs. Multiple functions have been proposed for PPAR- in inflammation, but how PPAR- signaling pathway may affect the development of acute inflammation remains unclear. It is known that activated PPAR- could down-regulate activator protein-1, nuclear factor-B (NF-B), and signal transducer and activator of transcription activity (42). Among these, NF-B has been shown to activate the genes encoding chemotactic and inflammatory cytokines, cell adhesion molecules, iNOS and COX-2; and both enzymes are inhibited in inflamed paw tissues from RAL or E2-treated rats. However, it cannot be excluded that NF-B inhibition and other mechanisms participate in establishing an antiinflammatory effect of RAL and E2.
Moreover, we were the first to show a modulation of PPAR- expression after inflammation in vivo. This receptor was more expressed in inflamed paws and reduced by ovariectomy. Exogenous E2 or RAL treatment restores PPAR- expression, suggesting a contribution to their antiinflammatory effect. In addition, treatment of OVX rats with RAL or E2 caused an increase of cytoprotective hsp72 protein expression. In the case of inflammation, a protective role of hsp has been shown in a variety of experimental models (43, 44, 45), and it was recently demonstrated that carrageenan causes HSF-1-induced hsp72 expression in inflamed paw tissues and that activation of the heat shock response seems to be closely associated with the remission of the inflammatory reaction (22). The effect of these two drugs on PPAR- and hsp72 expression was evident only in inflammatory conditions, after carrageenan injection, whereas both treatments did not significantly modify their basal expression in saline-injected paws (data not shown).
We also confirmed RAL antiinflammatory activity in male rats after acute drug administration, to avoid estrogen serum level variability due to the different stages of the estrous cycle. As seen in the acute edema experiment, RAL effect was significant at the highest dose (30 mg/kg; P < 0.05) compared with effective doses in OVX rats (3 mg/kg, P < 0.05; and 10 mg/kg, P < 0.01).
Leukocyte migration at the site of inflammation is fundamental in the inflammatory process. RAL exerted a marked inhibition on leukocyte infiltration and pleural exudate in carrageenan-induced pleurisy.
All of these findings support the view that RAL attenuates the carrageenan acute inflammation in the rat in a manner similar to estrogen. E2 was more potent than RAL in inhibiting inflammation induced in OVX rats. This different potency could be due to several factors. In fact, the effect of orally dosed RAL was lower than sc injected E2, which inhibits inflammation more efficiently. This may reflect differences in the bioavailability of these agents, depending on the route of administration, the dosing time protocol, and the differences in metabolism, that is more marked in oral administration of RAL. RAL was used by oral route of administration as normally used in therapy. Overall the antiinflammatory activity profile of these two drugs is consistent with previous observations that evidence the major potency of E2 vs. RAL in lowering serum cholesterol (46) and in preventing bone loss (47). Furthermore the pharmacology of SERMs is very complex. In particular, RAL is a mixed agonist/antagonist and possesses sufficient intrinsic activity to act as an agonist in bone and liver, as well as in modulating the inflammatory process, but it is a relatively pure antagonist in uterine tissue.
Here we have focused our attention on the antiinflammatory effect of RAL, examining only some specific inflammatory or antiinflammatory parameters. However, it is well known that the inflammatory response is articulate and that other mechanisms and mediators might be differently regulated by the activity of RAL.
It is of great interest to further investigate the precise contribution of intracellular receptors, ER and ER?, in mediating these effects. Crystal structures of ER have been made with E2 and RAL in the ligand-binding site; and from the differences between them, some conclusions have been drawn: RAL deforms the binding site for coactivators on ER, thereby producing a weaker activation than E2 (48).
Acknowledgments
The authors thank Lilly Research Laboratories for providing RAL, and Dr. Joseph Sepe for editorial assistance with the manuscript.
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Address all correspondence and requests for reprints to: Prof. Rosaria Meli, Department of Experimental Pharmacology, via D. Montesano 49, 80131 Naples, Italy. E-mail: meli@unina.it.
Abstract
Raloxifene (RAL) is a selective estrogen receptor modulator presenting tissue-specific agonist activity. The aim of this study was to examine whether RAL has an estrogenic effect on carrageenan-induced acute inflammation. Adult female rats were ovariectomized (OVX) 7 wk before edema or pleurisy to deplete circulating estrogens. Edema formation and selected inflammatory markers in inflamed paw tissue were measured in intact (sham-operated) and OVX rats. Groups of OVX rats were treated with RAL (1, 3, or 10 mg/kg) or 17?-estradiol (E2, 25 μg/kg), and these treatments began 2 d after surgery and continued until carrageenan paw edema or pleurisy. Ovariectomy amplifies the inflammation, and we found that RAL, as well as E2, attenuates inflammation and tissue damage associated with paw edema and pleurisy. In treated rats, there is a decrease in edema development and formation, and in polymorphonuclear cell infiltration and migration, as shown by myeloperoxidase measurement and cell counting. RAL and E2 treatments decrease cyclooxygenase-2 and inducible nitric oxide synthase expression in inflamed areas and counteract the inhibition of peroxisome proliferators-activated receptor- expression caused by ovariectomy, restoring this receptor protein expression to sham-operated levels and identifying a possible peroxisome proliferators-activated receptor-dependent antiinflammatory effect of these drugs. Moreover, RAL and E2 increase cytoprotective heat shock protein 72 expression, which seems to be closely associated with the remission of the inflammatory reaction. In addition, we confirm the antiinflammatory effect of RAL in male rats, using a single administration of RAL or E2.
Introduction
HYPOESTROGENISM CAUSED BY ovariectomy, disease, or menopause is associated with a multitude of clinical symptoms. Selective estrogen receptor modulators (SERMs) are well established as synthetic estrogen substitutes and can be used to prevent or treat diseases caused by estrogen deficiency, without most of the undesirable effects of estrogen (1). Although SERMs are extensively used in postmenopausal women, not everything is known about their action mechanisms, especially on the inflammatory process. We focused on raloxifene (RAL), a benzothiophene derivative that was shown to have antiestrogenic properties in breast tissue and a neutral effect on endometrium, and it has potentially beneficial estrogen-like effects in nonreproductive tissue, such as bone (2).
Estrogens reduce bone resorption directly by inhibiting osteoclasts and indirectly by suppressing osteoblastic production of various proresorptive paracrine factors, such as IL-1?, IL-6, and TNF- (3), thus showing a relationship with inflammatory cytokines. Moreover, estrogens may also inhibit the inflammatory reaction by decreasing the expression of specific markers and attenuating the degree of inflammation and tissue damage (4, 5, 6). It has recently been demonstrated that RAL increases osteoprotegerin, a potent inhibitor of bone resorption, and suppresses the bone-resorbing cytokine, IL-6 (7).
The aim of this study was to investigate the effect of RAL in models of carrageenan-induced acute inflammation, in particular, paw edema and pleurisy in the rat.
The acute inflammatory response is characterized by edema from increased vascular permeability, leading to an extravasation of fluid, and leukocyte infiltration. Carrageenan-induced local inflammation models are commonly used to evaluate nonsteroidal antiinflammatory drugs; hence, the cellular and the molecular mechanisms of carrageenan-induced edema are well known. Histamine, serotonin, and bradykinin are involved in the initial phase of inflammation (0–1 h), whereas the late phase (1–6 h) is mainly sustained by prostaglandins (8, 9), attributed to the induction of cyclooxygenase (COX)-2 in the inflamed tissue (10). Another key mediator in acute inflammation is nitric oxide (NO), a potent vasodilator. Its involvement during the inflammatory response may be related to its ability to increase vascular permeability and edema through changes in local blood flow (11, 12). Among the three distinct isoforms of NO synthase (NOS), it has been suggested that inducible enzyme [inducible NOS (iNOS)] mainly contributes to the development and maintenance of acute inflammation. This is further supported by the observation that its protein expression is up-regulated during the late stage of inflammation in paws after injection of carrageenan (13, 14). Furthermore, NO has been shown to increase the production of proinflammatory prostaglandins in in vitro (15, 16) and in vivo studies (17, 18).
At the site of inflammation, NO is produced by leukocytes and endothelial cells (14). In the acute phase after the increase of vascular permeability, neutrophil infiltration occurs. This contributes to the inflammatory response by producing several mediators, such as oxygen-derived free radicals (19). A marker of neutrophil infiltration in tissue is myeloperoxidase (MPO), and it has been clearly shown to be related to the severity.
Recently, the peroxisome proliferator-activated receptor (PPAR)- appears to be involved in the regulation of inflammatory responses (20), and its receptor agonists inhibit inflammatory edema and hyperalgesia (21) and raise the possibility that synthetic PPAR- ligands may have therapeutic value involving macrophage activation.
In vertebrates, heat shock proteins (hsp), hsp 72 especially, may be protective against certain pathological conditions, including ischemia, infection, and inflammation; and it has been demonstrated that activation of the heat shock response is closely associated with the remission of inflammation (22).
To characterize the antiinflammatory effects of RAL in carrageenan-induced acute inflammation models, we used ovariectomized (OVX) rats and, as reference drug, 17?-estradiol (E2). We investigated the effect of RAL and E2 on paw edema and pleurisy development, polymorphonuclear leukocyte infiltration (assessing MPO levels), and COX-2, iNOS, PPAR-, and hsp72 expression. In addition, to confirm the antiinflammatory effect of the drug, we induced paw edema in male rats, which are not influenced by E2 fluctuation of estrous cycle, using a single administration of RAL or E2.
Materials and Methods
Animals
Male and female Sprague Dawley rats (Harlan Italy, San Pietro al Natisone, Udine, Italy) were housed in stainless steel cages in a room kept at 22 ± 1 C with a 12-h light, 12-h dark cycle. The animals were acclimated to their environment for 1 wk and had ad libitum access to tap water and rodent standard diet. All animal experiments complied with the Italian (D.L. no. 116 of 27 January, 1992) and associated guidelines in the European Communities Council (Directive of 24 November, 1986, 86/609/ECC).
Ovariectomy and drug treatment
At the onset of the study, female rats (mean body weight of the cohort, 176 ± 1.3 g) were bilaterally OVX under anesthesia (100 mg/kg ketamine plus 5 mg/kg xylazine ip). The sham-operated (SHAM) animals were subjected to the same general surgical procedure as OVX groups except ovarian excision.
A group of OVX rats were treated by gavage with RAL (1, 3, or 10 mg/kg once daily; Lilly Research Laboratories, Indianapolis, IN). RAL was administered in 17.5% hydroxypropyl-?-cyclodextrin (Aldrich, Milwaukee, WI) in a vol of 100 μl/100 g body weight. SHAM and OVX rats were administered the vehicle, i.e. 100 μl/100 g body weight of 17.5% hydroxypropyl-?-cyclodextrin. Moreover, a group of OVX rats received E2 (25 μg/kg, sc, twice a week; Sigma, St. Louis, MO) as reference drug. All treatments began 2 d after surgery and continued for 7 wk. Last administrations were performed 2 h before inflammation induction. Each experimental group was composed of 15 rats.
The acute experiment was done to confirm the drawn antiphlogistic effect of RAL, even in the physiological estrogenic state, by treating male rats with a single dose (acute administration). Briefly, the animals (200.6 ± 6.8 g) were divided into the following groups: control animals, rats treated with RAL (3, 10, or 30 mg/kg per os 1 h before carrageenan paw injection), or rats treated with E2 (50 μg/kg, ip, 1 h before carrageenan injection). In this experiment, each group was composed of five rats.
Carrageenan-induced paw edema
The experimental groups described in the above paragraph received a subplantar injection of 100 μl of a sterile saline containing 1% wt/vol -carrageenan or sterile saline alone (control group) in the right hind paw. Foot volumes were measured using a water plethysmometer (Ugo Basile, Milan, Italy) immediately before injection and every hour for 5 h; and edema was evaluated, in milliliters, as the difference between the paw volume at each time-point and the basal paw volume. The inflammatory effect was evaluated as area under the curve (AUC) and expressed in milliliters of edema per hour. AUC is the integrated and cumulative measure of this effect. To compare the course of the edema of all groups, we integrated all curves to obtain the cumulative areas. In this way, one value for each area under the entire curve was obtained. Statistical analysis of these values was performed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
Some female rats from selected groups were killed at 3 h or 5 h after carrageenan injection, and the subplantar edematous area from paw was excised and prepared for further determinations (see below).
MPO assay in inflamed paws
Subplantar tissue obtained from five female animals per group, killed 3 h after carrageenan or saline injection, was homogenized in 1 ml hexadecyltrimethylammonium bromide buffer (Sigma) using a Polytron homogenizer (3 cycles of 10 sec at maximum speed). After centrifugation of homogenates at 13,000 rpm for 2 min, supernatant fractions were assayed for MPO activity using the method described by Bradley et al. (23). Briefly, samples were mixed with phosphate buffer containing 1 mM O-dianisidine dihydrochloride and 0.001% hydrogen peroxide in a microtiter plate reader. Absorbance was measured at 450 nm, taking three readings at 30-sec intervals. Units of MPO were calculated considering that 1 U MPO = 1 μmol H2O2 split, and 1 μmol gives a change in absorbance of 1.13 x 10–2 nm/min. MPO activity was expressed as units of MPO per milligram of protein.
Western blot analysis
Subplantar tissue (0.1 g), obtained from female rats killed 5 h after carrageenan or saline injection, was disrupted by homogenization on ice in lysis buffer (HEPES 20 mM, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2; 1 mM EGTA; 0.2 mM EDTA; 25% (vol/vol) glycerol; 0.5% Nonidet P-40; 0.5 mM phenylmethylsulfonyl fluoride; 1.5 μg/ml trypsin inhibitor; 3 μg/ml pepstatin A; 2 μg/ml leupeptin; 0.1 mM benzamidine; and 0.5 mM dithiothreitol). After 1 h, tissue lysates were obtained by centrifugation at 100,000 x g for 15 min at 4 C. Protein concentrations were estimated by the Bio-Rad protein assay (Bio-Rad Laboratories, Segrate, Milan, Italy) using BSA as standard.
For Western blot analysis, 25 or 40 μg protein of lysates was dissolved in Laemmli’s sample buffer, boiled for 5 min, and subjected to SDS-PAGE (8% polyacrylamide). The blot was performed by transferring proteins from a slab gel to nitrocellulose membrane at 240 mA for 40 min at room temperature. The filter was then blocked with 1x PBS, 5% nonfat dried milk for 40 min at room temperature and probed with specific monoclonal antibodies against iNOS (BD Biosciences Transduction Laboratories, Lexington, KY; 1:2,000), or COX-2 (Cayman Chemical, Ann Arbor, MI; 1:1500), or PPAR- (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100), or hsp72 (Stressgen Bioreagents, Victoria, British Columbia, Canada; 1:15,000) in 1x PBS, 5% nonfat dried milk, 0.1% Tween 20 at 4 C, overnight. The secondary antibody (antimouse IgG, or antirabbit IgG, or antigoat IgG peroxidase conjugate) was incubated for 1 h at room temperature. Subsequently, the blot was extensively washed with PBS, developed using enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturer’s instructions, and exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY). -Tubulin protein (Sigma; 1:1000) Western blot was performed to ensure equal sample loading.
The protein bands of iNOS (130 kDa), COX-2 (70 kDa), PPAR- (55 kDa), or hsp72 (72 kDa) on x-ray film were scanned and densitometrically analyzed with a model GS-700 imaging densitometer (Bio-Rad Laboratories).
Carrageenan-induced pleurisy
Carrageenan pleurisy was induced in other SHAM or OVX groups of rats (n = 6 in each group). A group of OVX animals was treated by gavage with RAL at 3 mg/kg·d in 17.5% hydroxypropyl-?-cyclodextrin, as described above. Another group of OVX rats received E2 (25 μg/kg, sc, twice a week) as reference drug. SHAM and OVX untreated rats received vehicle in the same conditions. All treatments were initiated on d 2 of the surgery and continued up to 7 wk.
Animals were anesthetized with isoflurane, and pleurisy was initiated by injection, into the pleural cavity, of 200 μl of a 1% wt/vol carrageenan solution in saline. After 4 h, the rats were killed. Pleural exudates were harvested by rinsing the pleural cavity with 2 ml saline solution. Exudates with blood contamination were rejected. The exudate volumes were measured, the samples were centrifuged at 800 x g for 10 min, and the cell pellet was resuspended in saline solution for cell count. Leukocyte number was estimated after Turk solution staining with the Neubauer counting chamber.
Measurement of circulating estradiol
After the experiments, serum estradiol level was determined by an ELISA kit (Abbott Laboratories, Abbott Park, IL) to determine the hypoestrogenism induced by ovariectomy and its level in E2 or RAL-treated animals.
Statistical analysis
All data were presented as mean ± SEM. Statistical analysis was performed by ANOVA test for multiple comparisons followed by Bonferroni’s test. Statistical significance was set at P < 0.05.
Results
Effects of RAL and E2 on the course of the carrageenan-induced paw edema in OVX and male rats
Ovariectomy significantly reduced estradiol serum levels (P < 0.05), and this decrease was reversed by E2 replacement but not by RAL treatment (data not shown).
As reported in Fig. 1, hypoestrogenism amplified the acute inflammatory process induced by intraplantar injection of carrageenan. In fact, ovariectomy had a strong proinflammatory effect seen by an increase in rat paw edema, evaluated as AUC and expressed in milliliters of edema per hour (P < 0.001). This effect was inhibited by E2 or RAL treatment. Moreover, RAL acts in a dose-dependent manner, and significantly, at 3 and 10 mg/kg (P < 0.05 and P < 0.01, respectively). Figure 2 shows the time course of edema, highlighting which mediators are modified the most by drug treatment. E2 and RAL (10 mg/kg) reduced paw edema every hour, whereas RAL (3 mg/kg) treatment showed a significant effect only 3 and 5 h after carrageenan injection, the endpoints chosen for the determination of MPO activity and protein expression.
FIG. 1. Antiinflammatory effect of 7-wk RAL treatment (RAL 1, 3, or 10 mg/kg) on paw edema of OVX rats. The SHAM animals were subjected to the same general surgical procedure as OVX groups, except ovarian excision. E2 (25 μg/kg) was used as reference drug. The inflammatory effect was evaluated as AUC and expressed in milliliters of edema per hour. Values are means ± SEM from n = 10 rats for each group. ***, P < 0.001 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
FIG. 2. Time-dependent development of paw edema elicited by carrageenan in SHAM and OVX rats. Seven weeks of RAL (1, 3, or 10 mg/kg) and E2 (25 μg/kg) treatments significantly inhibited edema formation at the indicated time points. Data are means ± SEM of 10 animals for each group. **, P < 0.01; ***, P < 0.001 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
To confirm the antiphlogistic effect of RAL, we induced paw edema in male rats, where the estrogen serum levels are not influenced by the estrous cycle. The animals were treated with a single administration (60 min before carrageenan injection) of E2 (50 μg/kg) or RAL (3, 10, and 30 mg/kg). In Fig. 3, RAL showed a reduction of edema at the highest dose used (P < 0.05), and E2 (50 μg/kg) had a considerable antiinflammatory effect, confirming the protective effect of estrogen in another carrageenan-induced tissue injury (5, 6).
FIG. 3. Antiinflammatory effect of acute administration of RAL (3, 10, or 30 mg/kg) or E2 (50 μg/kg) on carrageenan paw edema of male rats. Control animals (CON) received drug vehicle. Data are means ± SEM of five animals for each group. *, P < 0.05; ***, P < 0.001 vs. CON.
The control AUC value of edema in male rats is slightly higher than in female rats. This is because male and female rats were the same age but had different weights. This difference reflects the measurement of basal paw volume before carrageenan. In fact, male vol was 0.64 ± 0.04 ml, whereas female vol was 0.39 ± 0.04 ml (P > 0.05). This difference was due to the size of the paws and not to sex.
RAL and E2 effect on some inflammatory parameters of carrageenan-induced paw edema
MPO activity, a marker of polymorphonuclear cell (PMN) accumulation in subplantar areas, was determined in control and inflamed tissues (Fig. 4). In the paws of OVX rats, a slight, but not significant, increase of MPO activity (127.3 ± 14.37 vs. 101.1 ± 10.01 SHAM values) was revealed, whereas it was undetectable in saline control paws of animals treated with vehicle or drugs (data not shown). In carrageenan-injected animals, RAL treatment significantly reduced the activity of MPO, in a dose-dependent manner, vs. OVX animals. The effect of E2 was higher and significant both vs. SHAM and OVX rats.
FIG. 4. Dose-dependent effect of RAL on MPO activity. MPO activity was measured in subplantar paw homogenates of SHAM, 7-wk OVX, and OVX rats treated with RAL (1, 3, or 10 mg/kg) or E2 (25 μg/kg). The animals were killed 3 h after carrageenan paw injection. Data are means ± SEM of five animals for each group. **, P < 0.01 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
To evaluate the effect of the drugs on inflammatory enzymes, Western blot analysis of COX-2 and iNOS was performed on lysates of subplantar tissues from animals killed 5 h after edema induction. We chose 5 h because this is the time point where these proteins are overexpressed in a very significant manner. In saline-injected paws, both these inducible enzymes were not expressed (data not shown). Injection of carrageenan induced the expression of these proteins in all groups studied, and ovariectomy clearly increased their expression (Figs. 5A and 6A). E2 (25 μg/kg) and RAL (3 mg/kg) significantly reduced COX-2 and iNOS expression to SHAM level as evidenced by densitometric analysis (Figs. 5B and 6B).
FIG. 5. Western blot analysis showing the increase of induction of COX-2 expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Seven weeks of RAL (3 mg/kg) or E2 (25 μg/kg) treatment significantly reverted the increase of COX-2 expression induced by hypoestrogenism. This effect was revealed by densitometric analysis of bands. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates obtained from two animals per group is shown, and densitometric analysis of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
FIG. 6. Western blot analysis showing the increase of induction of iNOS expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Seven weeks of RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reverted the increase of iNOS expression induced by hypoestrogenism. This effect was revealed by densitometric analysis of bands. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates from two animals per group is shown, and densitometric analysis of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
Moreover, to elucidate whether the protective effect of RAL was related to the modulation of PPAR- expression, we examined the modification of this receptor in the same subplantar tissue lysates. The basal expression of PPAR-, evaluated in saline-injected paw tissues, was enhanced in inflammatory conditions, whereas 7-wk ovariectomy induced a clear reduction of this specific receptor protein band (Fig. 7A). Both hormonal replacement and RAL treatment reversed this reduction, as revealed by densitometric analysis (Fig. 7B).
FIG. 7. Western blot analysis showing the decrease of PPAR- expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Control saline-injected paw tissue lysate is also reported (Saline). Seven weeks of RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reversed this reduction, as seen by densitometric analysis. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates from two animals per group is shown, and densitometric analysis of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
Moreover, carrageenan injection was associated with increased hsp72 compared with protein expression observed in lysates from tissues collected from saline-treated rats. In OVX animals, 5 h after carrageenan, a decreased hsp72 expression was also detected by immunoblotting analysis compared with SHAM rats (Fig. 8). In contrast, both drug treatments reverted the OVX effect, inducing an increase of protein until SHAM levels.
FIG. 8. Western blot analysis (A) showing the decrease of hsp72 expression in subplantar tissue lysates from 7-wk OVX rats vs. SHAM. Control saline-injected paw tissue lysate is also reported (Saline). Seven weeks of RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reversed this reduction, as revealed by densitometric analysis. The animals were killed 5 h after carrageenan paw injection. A representative blot of lysates from two animals per group is shown, and densitometric analysis (B) of all animals is reported (n = 5 rats from each group). Equal loading was confirmed by -tubulin staining.
Further antiinflammatory effect of RAL and E2: carrageenan-induced pleurisy in OVX rats
Ovariectomy amplified the acute pleurisy induced by carrageenan injection, producing an increase of turbid exudates (Fig. 9, upper panel, 2.0 ± 0.1 vs. 1.4 ± 0.1 ml/rat, OVX vs. SHAM) that contained a larger amount of polymorphonuclear leukocytes (Fig. 9, lower panel, 117.4 ± 10.4 vs. 69.7 ± 2.0 x 106cell/rat in SHAM). E2 and RAL treatment significantly reduced exudate formation (43.6% and 20.1%, 1.1 ± 0.1 and 1.6 ± 0.1, respectively) and polymorphonuclear leukocyte infiltration in the pleural cavity (47.5% and 30%, 61.5 ± 6.8 and 82.1 ± 5.3, respectively).
FIG. 9. Effect of RAL and E2 on carrageenan-induced pleurisy in OVX rats. Volume exudate (upper panel) and accumulation of PMNs (lower panel) in pleural cavity at 4 h after carrageenan injection. Both 7-wk RAL (3 mg/kg) and E2 (25 μg/kg) treatments significantly reduced pleural exudation and leukocyte infiltration. Data are means ± SEM from six animals per group. ***, P < 0.001 vs. SHAM; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. OVX.
Discussion
Here we demonstrated that RAL, a lipolytic SERM with tissue-specific agonist activity, plays a protective antiinflammatory role in the development of carrageenan-induced edema and pleurisy.
Rat paw edema has been well characterized and described (24), and most nonsteroidal antiinflammatory drugs have been shown to be active in this inflammatory model. Moreover, the model is useful to assess the contribution of mediators involved in vascular changes associated with acute inflammation. This study used OVX animals, and the results indicate that the estrogen state of rats is a critical issue. Indeed, the OVX animals have a more consistent inflammatory response to carrageenan (increased edema) than SHAM rats.
In several experimental models, estrogens display antiinflammatory activity: arthritis (25), uveitis (26), encephalomyelitis (27), and pleurisy (5, 6). The mechanism behind estradiol’s antiinflammatory effect may be multifactorial: the hormone-dependent blockade of cytokines such as IL-6, IL-1, and TNF-; the inhibition of macrophage infiltration in the damaged tissues; and the decrease of inflammatory mediators.
Growing evidence indicates a role for proinflammatory factors in the pathogenesis of coronary artery disease, among them: liver-derived acute-phase reactant proteins, such as C-reactive protein (CRP), fibrinogen, and albumin, as well as cytokines (28). However, in clinical studies, estrogen alone (29) or hormonal replacement therapy (HRT) (30, 31) using estrogen and progestin combination has different effects on markers of inflammation. In fact, proinflammatory effects of HRT have been demonstrated for CRP, albumin, metalloproteinase, and factor VII. It is important to consider that these effects may be modified by obesity and that the route of administration also appears to play an important role, because oral formulation of estrogens may produce a strong first-pass liver effect.
Although the impact of RAL on the venous system is generally similar to that of HRT, its effect on the arterial system and subsequent risk of atherosclerotic events appears to be either neutral or slightly favorable, based on the limited clinical data (32). RAL has a neutral effect on CRP (33) and a favorable effect on low-density lipoprotein fibrinogen and lipoprotein(a) (34).
Using OVX rats, we recently demonstrated that 7 wk of RAL treatment modified the lipid profile by reducing cholesterol and low-density lipoprotein levels. This drug also shows an estrogen agonist effect on fat mass homeostasis and weight gain (35). Seven-week ovariectomy induced in rats a mild obesity that mimics estrogen insufficiency in humans and creates a useful model to study the mechanism by which hypoestrogenism influences hormonal status and metabolic processes. On the basis of these previous data, we used the same E2 and RAL doses and route of administration, beginning the treatment on d 2 to prevent the early modifications of estrogen loss.
E2 treatment is known to reduce the carrageenan-induced inflammatory process, especially in pleurisy. Our present results confirm these findings; in fact, the pharmacological dose of E2 (25 μg/kg, a higher dose than used in replacement therapy) dramatically reduced paw edema. All of the parameters examined revealed that RAL had a significant dose-dependent, but minor, effect compared with the exogenous hormone. E2 more significantly reduced MPO activity, COX-2, and iNOS expression and increased protective parameters (hsp72 and PPAR-) but also reduced exudates and cell migration in the pleural cavity.
RAL may be antiinflammatory by reducing inducible proinflammatory enzymes (COX-2 and iNOS) and decreasing infiltration of PMNs into the inflamed paw and pleural cavity. This is revealed by the declining MPO activity after 7 wk of RAL treatment. MPO enzyme, contained in azurophilic granules of PMNs, is involved in the formation of reactive oxygen species and oxidation of biological materials responsible for tissue damage. In fact it is undetectable in noninflamed control paws, evident in SHAM and OVX inflamed tissue, and significantly reduced by E2, and dose-dependently by RAL.
The inducible isoform of COX appears to be responsible for prostaglandin production in edema 1 h after carrageenan injection (8, 36). COX-2 is usually absent, or present only in a small amount, in cells under basal conditions but is overexpressed in the immune cells or tissue after phlogogenic stimuli or cytokine induction. More recently, the substained late phase (1–6 h) of carrageenan paw edema has been attributed to the induction of COX-2 in the tissue (10). Inhibitors of NOS activity reduce the development of carrageenan-induced inflammation and support a role for NO in the pathophysiology associated with this model of inflammation (13). Although NO itself is a weak oxidant, biochemical studies have shown that NO rapidly interacts with superoxide anion to yield peroxynitrite, which then decomposes to form highly reactive oxidant species responsible for lipid peroxidation and tissue damage.
The NOS and COX pathways appear to cooperate in amplifying the inflammatory response. This is achieved by a synergistic interaction between NO and prostaglandins on blood flow and microvascular permeability (37) as well as by NO-driven COX activation, leading to the exaggerated production of prostaglandins (13, 15, 17).
We showed that ovariectomy and decreasing estrogen levels can increase both COX-2 and iNOS expression. This study provides the first evidence that RAL causes a substantial reduction of acute inflammation in the rat by reducing tissue expression of these proinflammatory enzymes.
Moreover, here we demonstrate that RAL is able to modulate PPAR- expression in inflamed paw tissue. PPARs are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, which includes the classical steroid, thyroid, and retinoid hormone receptors as well as many orphan receptors. Among three related PPAR isotypes, PPAR- has been attracting considerable attention as a regulator of adipogenesis and expression of adipocyte genes involved in lipid metabolism (38). Apart from its metabolic effects, agonists of PPAR- have been demonstrated to modulate the inflammatory responses of several cells by inhibiting the expression of proinflammatory cytokines (20, 39), iNOS (40), and COX-2 (41). On the other hand, it has also been reported that cytokines, such as TNF-, antagonize the synthesis of PPAR-, block adipocyte differentiation, and contribute to insulin resistance (39). In our experimental model, RAL or E2 treatment counteracted the inhibition of PPAR- expression caused by ovariectomy, restoring this receptor protein expression to SHAM levels and identifying a possible PPAR-dependent antiinflammatory effect of these drugs. Multiple functions have been proposed for PPAR- in inflammation, but how PPAR- signaling pathway may affect the development of acute inflammation remains unclear. It is known that activated PPAR- could down-regulate activator protein-1, nuclear factor-B (NF-B), and signal transducer and activator of transcription activity (42). Among these, NF-B has been shown to activate the genes encoding chemotactic and inflammatory cytokines, cell adhesion molecules, iNOS and COX-2; and both enzymes are inhibited in inflamed paw tissues from RAL or E2-treated rats. However, it cannot be excluded that NF-B inhibition and other mechanisms participate in establishing an antiinflammatory effect of RAL and E2.
Moreover, we were the first to show a modulation of PPAR- expression after inflammation in vivo. This receptor was more expressed in inflamed paws and reduced by ovariectomy. Exogenous E2 or RAL treatment restores PPAR- expression, suggesting a contribution to their antiinflammatory effect. In addition, treatment of OVX rats with RAL or E2 caused an increase of cytoprotective hsp72 protein expression. In the case of inflammation, a protective role of hsp has been shown in a variety of experimental models (43, 44, 45), and it was recently demonstrated that carrageenan causes HSF-1-induced hsp72 expression in inflamed paw tissues and that activation of the heat shock response seems to be closely associated with the remission of the inflammatory reaction (22). The effect of these two drugs on PPAR- and hsp72 expression was evident only in inflammatory conditions, after carrageenan injection, whereas both treatments did not significantly modify their basal expression in saline-injected paws (data not shown).
We also confirmed RAL antiinflammatory activity in male rats after acute drug administration, to avoid estrogen serum level variability due to the different stages of the estrous cycle. As seen in the acute edema experiment, RAL effect was significant at the highest dose (30 mg/kg; P < 0.05) compared with effective doses in OVX rats (3 mg/kg, P < 0.05; and 10 mg/kg, P < 0.01).
Leukocyte migration at the site of inflammation is fundamental in the inflammatory process. RAL exerted a marked inhibition on leukocyte infiltration and pleural exudate in carrageenan-induced pleurisy.
All of these findings support the view that RAL attenuates the carrageenan acute inflammation in the rat in a manner similar to estrogen. E2 was more potent than RAL in inhibiting inflammation induced in OVX rats. This different potency could be due to several factors. In fact, the effect of orally dosed RAL was lower than sc injected E2, which inhibits inflammation more efficiently. This may reflect differences in the bioavailability of these agents, depending on the route of administration, the dosing time protocol, and the differences in metabolism, that is more marked in oral administration of RAL. RAL was used by oral route of administration as normally used in therapy. Overall the antiinflammatory activity profile of these two drugs is consistent with previous observations that evidence the major potency of E2 vs. RAL in lowering serum cholesterol (46) and in preventing bone loss (47). Furthermore the pharmacology of SERMs is very complex. In particular, RAL is a mixed agonist/antagonist and possesses sufficient intrinsic activity to act as an agonist in bone and liver, as well as in modulating the inflammatory process, but it is a relatively pure antagonist in uterine tissue.
Here we have focused our attention on the antiinflammatory effect of RAL, examining only some specific inflammatory or antiinflammatory parameters. However, it is well known that the inflammatory response is articulate and that other mechanisms and mediators might be differently regulated by the activity of RAL.
It is of great interest to further investigate the precise contribution of intracellular receptors, ER and ER?, in mediating these effects. Crystal structures of ER have been made with E2 and RAL in the ligand-binding site; and from the differences between them, some conclusions have been drawn: RAL deforms the binding site for coactivators on ER, thereby producing a weaker activation than E2 (48).
Acknowledgments
The authors thank Lilly Research Laboratories for providing RAL, and Dr. Joseph Sepe for editorial assistance with the manuscript.
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