Non-steroidal anti-inflammatory drugs (NSAIDs) are the most preferred drugs in the treatment of inflammatory diseases because of their efficacy in eliminating the pain, fever, flushing and edema which develop as a result of inflammatory mediator secretion (1-3). The advantage of NSAIDs compared to narcotic analgesics and steroidal anti-inflammatory drugs is that they trigger a combination of analgesic, antipyretic and anti-inflammatory effects. Research shows that both anti-inflammatory and side-effects of NSAIDs are related to cyclooxygenase (COX) inhibition (4). It has been suggested that while COX-2 enzyme inhibition is responsible for the therapeutic (anti-inflammatory) effects of NSAIDs, COX-1 enzyme inhibition is responsible for their side-effects on the gastrointestinal system (GIS) (5). Nimesulide, celecoxib and refecoxib, which are selective COX-2 inhibitors, were developed after studies were carried out into ways of decreasing the gastrointestinal and other side-effects of NSAIDs.

These drugs produce analgesic, antipyretic and anti-inflammatory effects as efficiently as classic anti-inflammatory drugs (6). Therapeutic effects of selective COX-2 inhibitory drugs are equal to those of traditional NSAIDs, while their side-effects are fewer (7).

The anti-inflammatory effect mechanism of NSAIDs is based on inhibiting the synthesis of chemical mediators of inflammation (8). It is known that NSAIDs produce anti-inflammatory effects by inhibiting the synthesis of prostaglandins (PG), which are the products of COX (3, 9). However, some investigators have shown that anti-inflammatory doses of NSAIDs are higher than is necessary to inhibit PG synthesis (9-10). Besides, it was found that sodium salicylate, a weak COX inhibitor, is as effective in treating rheumatoid arthritis as aspirin, which is a potent COX inhibitor (10). In a previous study we demonstrated that the anti-inflammatory effects of diclofenac sodium and calcium channel blockers (CCBs), which have significant anti-inflammatory effects in intact rats, are lost in adrenalectomized rats (11).

The inability of diclofenac sodium and CCBs to prevent inflammation in adrenalectomized rats shows that the adrenal gland hormones have a role in the anti-inflammatory effect mechanism of these drugs.

The aim of our study is to investigate whether the anti-inflammatory effects of some NSAIDs are related to adrenal gland hormones, like those of diclofenac sodium, and to investigate the anti-inflammatory action mechanism of hormones found to play an anti-inflammatory role .


MATERIALS AND METHODS
Animals

In this study we used a total of 650 (190 intact and 460 adrenalectomized) male Albino Wistar rats, obtained from the Medical Experimental Research Centre, Ataturk University. The animals weighed between 210 and 230 g and were fed under normal temperature conditions (22°C) in separate groups before the experiments.

Chemicals

Thiopental sodium was purchased from IE Ulugay A.S. Istanbul, Turkey; indomethacin was purchased from Deva, Turkey; Nimesulide from Phizer, Turkey; diclofenac sodium from Deva, Turkey; ibuprofen from Abbott; tenoxicam from Biofarma Turkey; aspirin from Bayer, Turkey; adrenalin from Biofarma, Turkey; prednisolone from Fako, Turkey; propranolol from Sanofi-Synthelabo; metoprolol was purchased from AstraZeneca, Yohimbine; phenoxybenzamine and prazosin were purchased from Sigma. This study was approved by the local Ethics Committee of Ataturk University of Erzurum, Turkey.

Effects of IDINTA on carrageenan-induced inflammatory paw edema in intact rats

In this series of experiments the anti-inflammatory effects of indomethacin, diclofenac sodium, ibuprofen, nimesulide, tenoxicam, and aspirin (IDINTA) on carrageenan-induced inflammatory paw edema were studied on a total of 190 intact rats (12). Initially the rats were divided into 19 groups (n=10), 18 of which then received indomethacin (6, 12 and 25 mg/kg), diclofenac sodium (6, 12 and 25 mg/kg), ibuprofen (25, 50 and 100 mg/kg), nimesulide (25, 50 and 100 mg/kg), tenoxicam (6, 12 and 25 mg/kg) and aspirin (25, 50 and 100 mg/kg) respectively, by oral gavage. The control group received an equal volume of distilled water as vehicle. One hour after drug administration, 0.1 ml of 1% carrageenan was injected into the hind paw of each animal. Before the carrageenan injection the normal paw volumes, up to the knee joint, were measured by plethysmometry. Carrageenan-induced increase in the paw volume (paw edema) was measured four times at one hour intervals. The anti-inflammatory effects of the drugs were determined by comparing the results of the drug-treated groups with that of the control group.

Effects of IDINTA on carrageenan-induced inflammatory paw edema in adrenalectomized rats

In this experiment the anti-inflammatory effects of IDINTA on carrageenan-induced inflammatory paw edema were studied in a total of 190 rats which had previously been adrenalectomized. For this purpose the adrenal glands of the rats were removed under 25 mg/kg thiopental anesthesia (11). After surgery, the adrenalectomized rats were supported with 1% NaCl solution instead of water and pellet food for seven days. On the 8th day the rats divided into 19 groups (n=10), 18 of which then received indomethacin (6, 12 and 25 mg/kg), diclofenac sodium (6, 12 and 25 mg/kg), ibuprofen (25, 50 and 100 mg/kg), nimesulide (25, 50 and 100 mg/kg), tenoxicam (6, 12 and 25 mg/kg) and aspirin (25, 50 and 100 mg/kg) by oral gavage, respectively. The control group received an equal volume of distilled water as vehicle, and anti-inflammatory effects of the drugs were determined as described above.

Effects of IDINTA on carrageenan-induced inflammatory paw edema in adrenalectomized rats pre-treated with prednisolone and adrenalin

A total of 140 rats were adrenalectomized as described above. These rats were divided into 14 groups. The first 6 groups of adrenalectomized rats (1, 2, 3, 4, 5 and 6th groups) received prednisolone (5 mg/kg, i.p) and the second 6 groups (7, 8, 9, 10, 11 and 12th groups) received adrenalin (100 µg/kg, i.p). After 5 minutes, the stated doses of IDINTA were administered by oral gavage to the prednisolone-dosed and adrenalin-dosed rat groups, respectively. The 13th and 14th groups of rats received only prednisolone and adrenalin respectively, in the same way. The control group received distilled water as vehicle. The anti-inflammatory effects of mentioned NSAIDs were determined as described above (11).

Effects of adrenalin and prednisolone on carrageenan-induced inflammatory paw edema in adrenalectomized rats pre-treated with prazosin, yohimbine and phenoxybenzamine

In this series of our experiments, a total of 70 rats were adrenalectomized as described above. These rats were divided to 7 groups. The first two groups of adrenalectomized rats (1st and 2nd groups) received prazosin (4 mg/kg), the second two groups (3rd and 4th groups) received yohimbine (10 mg/kg) and the last two groups (5th and 6th groups) received phenoxybenzamine (20 mg/kg) by intra-peritoneal injection. Thirty minutes after the prazosin and yohimbine injections and two hours after the phenoxybenzamine injection, prednisolone (5 mg/kg) and adrenalin (100 µg/kg) were administered to the rat groups, respectively, by intra-peritoneal injection. The control group received distilled water as vehicle. Effects of prednisolone and adrenalin on carrageenan-induced paw edema were then determined as described above.

Effects of adrenalin and prednisolone on carrageenan-induced inflammatory paw edema in adrenalectomized rats pre-treated with propranolol and metoprolol

A total of 50 rats were adrenalectomized as described above. These rats were divided into 5 groups. The first two groups of adrenalectomized rats (1st and 2nd groups) received propranolol (40 mg/kg) and the second two groups (3rd and 4th groups) received metoprolol (50 mg/kg) by oral gavage. One hour after drug administration, prednisolone (5 mg/kg, ip) and adrenalin (100 µg/kg, ip) were injected into the propranolol-treated and metoprolol-treated rat groups respectively. The control group received distilled water as vehicle. The anti-inflammatory effects of prednisolone and adrenalin were determined as described above.

Statistical analyses

All results were shown as means± SE. One-way analysis of variance was used to evaluate the results; p<0.05 was considered significant.

The present study was approved by the Ethics Committee of Ataturk University, Faculty of Medicine.


RESULTS
Effects of IDINTA on carrageenan-induced inflammatory paw edema in intact rats

As seen in Fig. 1A and 1B, four hours after carrageenan injection IDINTA inhibited carrageenan-induced paw edema with ID50 of 9.82, 10.81, 95.21, 75.23, 8.21 and 61.84 mg/kg respectively in intact rats.

Fig.1A. Dose-activity relationship of indomethacin, diclofenac Na and tenoxicam in intact rats determined after different experimental procedures: administration of indomethacin (6, 12 and 25 mg/kg), diclofenac sodium (6, 12 and 25 mg/kg) and tenoxicam (6, 12 and 25 mg/kg) respectively, to intact rats. (n=10 in each group) After 1 hour 0.1 ml of 1% carrageenan injection into the hind paw of each animal. And the increase in the paw volume was measured four times at one hour intervals.

Fig.1B. Dose-activity relationship of ibuprofen, nimesulide and aspirin in intact rats determined after different experimental procedures: administration of ibuprofen (25, 50 and 100 mg/kg), nimesulide (25, 50 and 100 mg/kg) and aspirin (25, 50 and 100 mg/kg) respectively, to intact rats. (n=10 in each group) After 1 hour 0.1 ml of 1% carrageenan injection into the hind paw of each animal. And the increase in the paw volume was measured four times at one hour intervals.

Effects of IDINTA on carrageenan-induced inflammatory paw edema in adrenalectomized rats

By the fourth hour of inflammation, the stated doses of IDINTA decreased carrageenan-induced paw edema with ID50 of 152.97, 188.17, 1275.0, 433.67, 188.16 and 1028.17 mg/kg respectively, in adrenalectomized rats (Fig. 2A and 2B).

Fig.2A. Dose-activity relationship of indomethacin, diclofenac Na and tenoxicam in adrenalectomized (ADX) rats determined after different experimental procedures: All of the rats have been adrenalectomized 1 week prior to the experiment. Administration of indomethacin (6, 12 and 25 mg/kg), diclofenac sodium (6, 12 and 25 mg/kg) and tenoxicam (6, 12 and 25 mg/kg) respectively, to ADX rats. (n=10 in each group) After 1 hour 0.1 ml of 1% carrageenan injection into the hind paw of each animal. And the increase in the paw volume was measured four times at one hour intervals.

Fig.2B. Dose-activity relationship of ibuprofen, nimesulide and aspirin in adrenalectomized (ADX) rats determined after different experimental procedures: All of the rats have been adrenalectomized 1 week prior to the experiment. Administration of ibuprofen (25, 50 and 100 mg/kg), nimesulide (25, 50 and 100 mg/kg) and aspirin (25, 50 and 100 mg/kg) respectively, to ADX rats. (n=10 in each group) After 1 hour 0.1 ml of 1% carrageenan injection into the hind paw of each animal. And the increase in the paw volume was measured four times at one hour intervals.

Effects of IDINTA on carrageenan-induced inflammatory paw edema in adrenalectomized rats pre-treated with prednisolone and adrenalin

Indomethacin + prednisolone, indomethacin + adrenalin, diclofenac + prednisolone, diclofenac + adrenalin, ibuprofen + prednisolone, ibuprofen + adrenalin, nimesulide + prednisolone, nimesulide + adrenalin, tenoxicam + prednisolone, tenoxicam + adrenalin, aspirine + prednisolone and aspirine + adrenalin, at the doses stated above, decreased carrageenan-induced paw edema by 90.3% (p<0.0001), 82.7% (p<0.0001), 93.5% (p<0.0001), 84.8% (p<0.0001), 94.6% (p<0.0001), 80.5% (p<0.0001), 91.4% (p<0.0001), 79.4% (p<0.0001), 93% (p<0.0001), 97.8% (p<0.0001), 89.2% (p<0.0001) and 76.1% (p<0.0001), respectively, in adrenalectomized rats. The same doses of prednisolone and adrenalin suppressed carrageenan-induced inflammation by 95.7% (p<0.0001) and 78.3% (p<0.0001), respectively, when used alone (Table I).

Table I. Effects of IDINTA on carrageenan-induced inflammatory paw edema in prednisolone and adrenalin given adrenalectomized rats.

Effects of adrenalin and prednisolone on carrageenan-induced inflammatory paw edema in adrenalectomized rats pre-treated with prazosin, yohimbine and phenoxybenzamine

Prazosin + prednisolone, prazosin + adrenalin, yohimbine + prednisolone, yohimbine + adrenalin, phenoxybenzamine + prednisolone, phenoxybenzamine + adrenalin, decreased carrageenan-induced paw edema by 93.9% (p<0.0001), 97% (p<0.0001), 92.9% (p<0.0001), 98% (p<0.0001), 87.8% (p<0.0001), 98% (p<0.0001) in adrenalectomized rats (Table II).

Table II. Effects of adrenalin and prednisolone on carrageenan-induced inflammatory paw edema in prazosin, yohimbine and phenoxybenzamine given adrenalectomized rats.

Effects of adrenalin and prednisolone on carrageenan-induced inflammatory paw edema in adrenalectomized rats pre-treated with propranolol and metoprolol

In the adrenalectomized rat groups which received propranolol+prednisolone, propranolol+adrenalin, metoprolol+prednisolone and metoprolol+adrenalin, carrageenan-induced paw edema was inhibited by 36.2% (p<0.05), 0% (p>0.05), 87.5% (p<0.0001) and 72.3% (p<0.0001), respectively (Table III).

Table III. Effects of adrenalin and prednisolone on carrageenan-induced inflammatory paw edema in propranolol and metoprolol given adrenalectomized rats.

DISCUSSION
This study investigated whether or not the anti-inflammatory effects of indomethacin, diclofenac sodium, ibuprofen, nimesulide, tenoxicam and aspirin (IDINTA) are related to adrenal gland hormones. Additionally, we also investigated the mechanism of the anti-inflammatory action of certain hormones whose anti-inflammatory role has been determined.

In our first series of experiments, the anti-inflammatory effects of IDINTA on carrageenan-induced inflammatory paw edema were investigated in intact rats. In a parallel experiment, the anti-inflammatory effects of IDINTA on carrageenan-induced inflammatory paw edema were also investigated in adrenalectomized rats.

It is known that IDINTA significantly inhibits carrageenan-induced inflammation in intact rats (11, 13-16). The reason we re-studied these anti-inflammatory effects was to compare the anti-inflammatory properties of IDINTA in the same season, time of day and environmental conditions, between intact and adrenalectomized rats. Our experimental results showed a significant difference between the anti-inflammatory effects of IDINTA in adrenalectomized rats and in intact rats. The loss of anti-inflammatory effects of IDINTA in adrenalectomized rats shows that the adrenal gland hormones have a role in the anti-inflammatory effects of these drugs. In one of our previous studies we showed that diclofenac sodium produced insignificant anti-inflammatory effects in adrenalectomized rats, but the role of this adrenal gland hormone has not yet been explored in terms of its anti-inflammatory action mechanism (11). Valle et al. showed that the anti-inflammatory effects of indomethacin and piroxicam decreased in adrenalectomized rats and were completely absent in diabetic-adrenalectomized rats (17).

In the second series of experiments we investigated whether the anti-inflammatory effects of IDINTAs are related to cortisol (corticosterone in rats) and adrenalin. Prednisolone was used instead of cortisol in all experiments. For this purpose, the effects of IDINTA on carrageenan-induced inflammation were determined in prednisolone and adrenalin-dosed adrenalectomized rats. The results of these trials showed that IDINTA significantly prevented carrageenan-induced inflammation in adrenalectomized rats pre-treated with prednisolone and adrenalin. The difference between the anti-inflammatory action in rat groups which received adrenalin alone, and prednisolone alone, or adrenalin and prednisolone + IDINTA, was statistically insignificant. Thus, these results show that both adrenalin and prednisolone have a role in the anti-inflammatory effect mechanism of IDINTA. It is known that the most important anti-inflammatory effect mechanism of glucocorticoids is to break up arachidonic acid metabolism (prostaglandins, tromboxans, leukotrienes) by inhibition of phospholipase A2 (18-19). It has also been reported that glucocorticoids and catecholamines inhibit the production of pro-inflammatory cytokines (IL-12, TNF-, INF-), whereas they stimulate the production of anti-inflammatory cytokines (IL-10, IL-4) (20). Pettipher et al. demonstrated the role of adrenalin in suppression of TNF-.(21). It has also been reported that clenbuterol, a ß2-receptor agonist, inhibits lipopolysaccharide-stimulated TNF- and IL-6 production (22).

In this study we show that adrenalin, as IDINTA, inhibits carrageenan-induced inflammation. The anti-inflammatory effects of adrenalin have already been reported in the literature, but there is no information on the anti-inflammatory effect of adrenalin on carrageenan-induced inflammation.

In the third series of our experiments, we investigated whether or not prednisolone and adrenalin stimulate anti-inflammatory effects via the 1 and 2-receptors. Our results show that prednisolone and adrenalin significantly prevent carrageenan-induced inflammation in rat groups pre-treated with prazosin, yohimbine and phenoxybenzamine. According to this data we can say that the anti-inflammatory effects of prednisolone and adrenalin are not related to the a1 and a2-receptors. Prazosin is a selective 1-blocker, and yohimbine is a selective 2-receptor blocker (3, 23). Phenoxybenzamine is an irreversible blocker of both 1 and 2-receptors (3).

There are some studies which show that a-adrenergic activation stimulates arachidonic acid metabolism and increases eicosanoid production (24). The finding that phentolamine, an 1-receptor blocker, prevents carrageenan-induced inflammation (25) is consistent with our results. Namely the anti-inflammatory effects of prednisolon and adrenaldin were not lost in the rats given an 1-receptor blocker.

In the next series of experiments we investigated whether or not the anti-inflammatory effects of prednisolone and adrenalin are related to the b1 and b2-receptors. Results show that prednisolone and adrenalin produce anti-inflammatory effects via the ß2-adrenergic receptors. Prednisolone and adrenalin prevented carrageenan-induced inflammation in rats pre-treated with metoprolol, but not in those pre-treated with propranolol. It is known that metoprolol is a selective blocker of ß1-receptors and propranolol is a non-selective blocker of ß1 and ß2-receptors (3). This data, obtained from our study and literature, confirms that the ß2-adrenergic receptors have a role in inflammation, and stimulation of these receptors causes inhibition in inflammation.

In conclusion, 1) It has been determined that anti-inflammatory effects of IDINTA are lost in adrenalectomized rats; 2) Adrenaline and prednisolone is shown to have a role in the anti-inflammatory effect mechanism of IDINTA; 3) It was also shown for the first time that adrenalin (totally) and prednisolone (partially) triggered their anti-inflammatory effects via the ß2-receptors but not via the 1, 2 and ß1-receptors; 4) It was therefore concluded that the ß2-adrenergic receptors have an indirect role in the anti-inflammatory mechanism of the particular NSAIDs we used; 5) Results suggested that ß2 adrenergic receptor agonists can be tried as a systemic anti inflammatory drug like current NSAIDs.

This research was conducted in Laboratory of Pharmacology at Ataturk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum/Turkey.

Conflicts of interest statement: None declared.


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