Recently anti-inflammatory effects of antidepressant drugs have been demonstrated. Venlafaxine belongs to newer antidepressants with serotonin norepinephrine reuptake inhibition property.
The pain alleviating properties of venlafaxine in different pain models such as neurogenic pain, diabetic neuropathy and fibromyalgia have been demonstrated.
Anti-inflammatory effects of venlafaxine and also its underlying mechanisms remain unclear. The present study was designed to evaluate the anti-inflammatory effects of venlafaxine and determine possible mechanisms underlying this effect. Thus we have examined the anti-inflammatory effects of intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of venlafaxine in the carrageenan-induced paw edema in rats.
Our results showed that both i.p. (50 and 100 mg/kg) and i.c.v. (50 and 100 μg/rat) injection of venlafaxine inhibited carrageenan-induced paw edema. We also found that both i.p. and i.c.v. administration of venlafaxine significantly decreased myeloperoxidase (MPO) activity, interleukin (IL)-1β and tumor necrosis factor (TNF)-α generation.
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Finally, we tried to reverse the anti-inflammatory effect of venlafaxine by yohimbine (5 mg/kg i.p.) an alpha2-adrenergic antagonist. Our results showed that applied antagonists failed to change the anti-inflammatory effect of venlafaxine.
These results demonstrated the anti-inflammatory effects of systemic and central venlafaxine injections and showed that these effects mediated mostly through the inhibition IL-1β and TNF-α generation and decreased MPO activity in the site of inflammation.
Key words: Venlafaxine, Carrageenan, Inflammation, Rat
Recently the role of antidepressants, particularly conventional tricyclic antidepressants (TCAs) such as amitriptyline, nortriptyline, and doxepin for alleviating various types of pain, such as inflammatory and neuropathic pain have been shown.
Moreover it has been reported that fluoxetine, paroxetine and sertraline can modulate the ability of microglia to produce the proinflammatory-cytokine such as tumour necrosis factor-a (TNF-a) and the free radical nitric oxide (NO). (Hashioka et al., 2007, 2009; Horikawa
et al., 2010; Hwang et al., 2008).
Venlafaxine is a structurally novel phentylethylamine which belongs to newer antidepressant drug that blocks the synoptosomal uptake of noradrenaline and serotonin (Lloyd 1992).
The pain alleviating properties of venlafaxine in different pain models such as neurogenic pain, diabetic neuropathy and fibromyalgia have been demonstrated (3-5).
In addition venlafaxine does not induce the usual tricyclic antidepressants (TCAs) side-effects caused by their anticholinergic, anti-histaminic and alpha-1 adrenergic antagonistic properties (Ellingrod et al, 1994).
Thus this drug could be a novel and promising treatment in inflammatory pains. But the anti-inflammatory effect of venlafaxine and also underlying mechanisms which may be involved in this effect has not been fully examined. The aims of the present study were to (a) determine the effect of systemic venlafaxine injections on the carrageenan-induced paw edema, (b) examine the possible involvement of central mechanism in the anti-inflammatory activity of venlafaxine, (c) determine the effect of venlafaxine on the myeloperoxidase activity in the site of inflammation, (d) evaluate the effect of venlafaxine on inflammatory cytokines such as IL-1β and TNF-α generation and (e) investigate the potential role of alpha2-adrenergic receptors in this effect of venlafaxine.
2. Materials and Methods
2.1. Animals and housing conditions
The experiments were performed on male Sprague–Dawley rats (200–250 g). They were housed four per cage, in a room under controlled temperature (23±2 °C), humidity (50%) and lighting (12/12 h light/dark cycle), with food and water available ad libitum.
Venlafaxine was obtained from Darupakhsh pharmaceutical Co., Iran. Carrageenan (lambda) was obtained from Fluka Chemical (Switzerland) and dissolved in saline solution. Aprotinin A, Hexadecyl trimethylammonium bromide (HTAB), phenylmethylsulfonyl fluoride, bovine serum albumin, ethylenediaminetetraacetic acid (EDTA), benzethonium chloride, and Tween 20 were all purchased from Sigma Chemical Company (St. Louis, MO, USA). IL-1β (ALPCO, USA), and TNF-α (R&D Company,USA) kits were used for measurement the cytokines levels.
2.3. Surgical procedure
The rats were anesthetized with i.p. injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). Then an i.c.v. cannula was implanted with stereotaxic coordinates: AP, −0.8 mm; L, 1.4 mm; and V, 3.3 mm, based on Paxinos and Watson atlas (Budantsev et al., 1993). The rats were handled daily for additional five days before the experiments to familiarize them to manipulation and lessen nonspecific stress responses. Rats with the i.c.v. cannulas were sacrificed at the end of the experiments, and their brains were removed and examined to confirm the correct inserting of the cannula.
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2.4. Carrageenan-induced paw edema
100 μl of a 1% (w/v) suspension of carrageenan lambda was injected subplantar in the right hind paw (Winter et al.,1962). Immediately before carrageenan injection and then at 4 h after that the volume of the paw was determined by a Plethysmometer (Ugo Basile, Italy). The data were expressed as the difference in the paw volume (ml) and were compared to pre-injection values.
2.5. Experimental design
All doses that applied in this experiment were chosen according to previous studies (Hajhashemi et al., 2010b).
In the first series of experiments, the effect of venlafaxine (50 and 100 mg/kg, i.p. n=6) on paw edema was examine. Venlafaxine was injected 30 min before subplantar injection of carrageenan. Paw volumes (ml) were measured before carrageenan injection, and then again, at 4 h after that to determine the difference in paw volume. Control group received only vehicle (5ml/kg i.p.; n=6). At the end of the experiments, animals were sacrificed, and the inflamed paw tissues were collected for cytokines and myeloperoxidase activity measurement.
In the second series, we used the i.c.v. route to determine the involvement of supraspinal levels in the anti-inflammatory effect of venlafaxine (Hajhashemi et al., 2010b).
Venlafaxine was injected smoothly for 1 min through the i.c.v. cannula (50 and 100 μg/rat, n=6) 30 min before carrageenan injections and the paw volumes were measured. The control group received vehicle (i.c.v.; 5 μl; n=6). At the end of the experiments for cytokine and myeloperoxidase activity measurement animals were sacrificed, and the paw tissues were collected.
Finally, in order to evaluate the possible involvement the alpha2- adrenergic receptors in the inhibitory effect of venlafaxine on carrageenan-induced inflammation, animals were pretreated with yohimbine (5 mg kg-1, i.p.,) in co-administrated with venlafaxine.
2.6. Myeloperoxidase Activity Assay.
MPO activity, an index of polymorphonuclear leukocyte accumulation, was measured in the inflamed paw. . Paw tissue was chopped and homogenized in potassium phosphate buffer containing 0.5% HTAB (hexadecyltrimethylammonium bromide). Then, the homogenate was sonicated in an ice bath. After that, the suspensions were centrifuged at 15,000 rpm for 15 min at 4â-¦C and then 2.9mL of 50mM potassium phosphate buffer (pH 6.0) containing O-dianisidine dihydrochloride (0.167 mg/mL) and 0.005% hydrogen peroxide was added to the supernatant. The absorbance of the reaction mixture was measured at 450nm using a UV-Vis spectrophotometer. MPO activity was expressed in units (U) per gram of wet tissue weight.
2.7. Measurement of the IL-1β and TNF-α level in the rat paw
TNF-α and IL-1β levels in the whole skin of inflamed paws were measured (Nacife et al., 2004). The tissue samples were weighed; snap frozen on liquid nitrogen and stored at −70 °C. Tissue was homogenized in phosphate buffered saline (PBS; pH=7.4) containing Tween-20, 0.5%, 0.4 M NaCl, 0.05%, 0.1 mM phenylmethylsulfonyl fluoride, bovine serum albumin, aprotinin A 20 KI, 0.1 mM benzethonium chloride, and 10 mM EDTA. The homogenates were centrifuged at 12,000×g for 30 min at 4 °C, and then ELISA kits were used to measure the levels of IL-1β and TNF-α in the supernatants.
2.8. Statistical analysis
The data are expressed as the means±S.E.M. Data were compared by one-way analysis of variance (ANOVA) followed by Fisher LSD post-hoc test for multiple comparisons. The probability of P<0.05 was considered to show significant differences for all comparisons made.
3.1. Effect of i.p. injection of venlafaxine on carrageenan-induced paw edema
As shown in Fig. 1, i.p. injection of venlafaxine at doses of 50 and 100 mg/kg significantly decreased the development of paw edema as compared to the control group.
3.2. Effect of i.c.v. injection of venlafaxine on carrageenan-induced paw edema
As illustrated in Fig. 2, i.c.v. administration of venlafaxine (50 and 100 μg/rat) produced an attenuate paw edema formation as compared to the control group.
3.4. Effect of venlafaxine on the MPO activity
The MPO activity of paw tissue was significantly increased at 4 h after injection of carrageenan. As shown in Fig. 3, venlafaxine (50 and 100 mg/kg i.p.) significantly reduced MPO activity in the paw. The i.c.v. administration of venlafaxine (50 and 100 μg/rat) also decreased the MPO activity, compared to control group.
3.5. Effect of venlafaxine on IL-1β concentration
As illustrated in Fig. 4, carrageenan injections significantly increased IL-1β concentration in the hind paw. Venlafaxine (50 and 100 mg/kg i.p.) significantly reduced IL-1β level. The i.c.v. administration of venlafaxine (50 and 100 μg/rat) also significantly attenuates production of IL-1β in the carrageenan-injected paws.
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3.6. Effect of venlafaxine on TNF-α concentration
As shown in Fig. 5, i.p. administration of venlafaxine (50 and 100 mg/kg) significantly reduced TNF-α generation. The i.c.v. injection of venlafaxine (50 and 100 μg/rat) also inhibited the generation of TNF-α in the carrageenan-injected paw.
3.7. Effect of yohimbine on the anti-inflammatory effect of venlafaxine
Pretreatment with yohimbine (5 mg kg-1, i.p.) 30 min prior to venlafaxine (50 mg kg -1) did not alter the anti-inflammatory effect of venlafaxine (Fig. 6).
In the present study at first, we demonstrated that i.p. and i.c.v. administration of venlafaxine exhibit a significant anti-inflammatory effect in the carrageenan-induced paw edema in rats.
Our results showed that venlafaxine was effective in attenuating paw edema in the inflammation induced by carrageenan.
Carrageenan-induced paw edema is one of the most frequently used models for the study of inflammation and inflammatory pains and is broadly used for evaluation the anti-inflammatory activity of different compounds. Release of nitric oxide and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, and interleukin-1β (IL-1) are happen following carrageenan injections in to the hind paw (Halici et al., 2007; Nacife et al., 2004).
In the present study the interference of venlafaxine with the PMN cells migration has examined. Our results showed that both i.p. and i.c.v. administration of venlafaxine caused a noticeable reduction in the infiltration of PMN leucocytes into the site of inflammation.
As mentioned above carrageenan injection also aggravates the release pro-inflammatory cytokines such as TNFα and IL-1β (Halici et al., 2007;
Nacife et al., 2004). Cytokines have a vital role in the generation and deterioration of a various types of inflammatory disease including asthma, arthritis, and inflammatory bowel disease (Codarri et al., 2010; Feldmann and Maini, 2008; Ichinose and Barnes, 2004; Papadakis and
Targan, 2000). Interestingly production of pro-inflammatory cytokines may be implicated in the pathogenesis of major depression (Leonard, 2001; Maes et al., 1995). Therefore, the present study was designed to determine whether the anti-inflammatory effect of venlafaxine could influence the TNF-α and IL-1β generation in the inflamed site. Our results showed that i.p. and i.c.v. injection of venlafaxine significantly reduced the IL-1β levels in the carrageenan-injected paw tissues. Systemic and central injection of venlafaxine also decreased concentration of TNF-α.
Multiple lines of studies also have shown that central mechanisms modulate peripheral inflammation.
[28–30]. It is also well known that venlafaxine cause alterations in central norepinephrine and
[31–33]. Our results showed that i.c.v. administration of venlafaxine attenuates the development of paw edema.
Thus the effect of venlafaxine on central nerves system to alter neuroimmune interactions and/or sympathetic nervous system activity that affect the function of the immune system could be considered as one of the possible mechanisms of venlafaxine in attenuation of inflammation.
. Also it is possible that venlafaxine changes the activity of descending neuronal pathways, projecting from the brain to the spinal cord, leading to inhibition of the peripheral nerve activity and dorsal root reflexes associated with the neurogenic component of inflammation [35, 36].
Body of evidences showed the connection between the inflammation and the generation of pain (Marchand et al., 2005). Previous studies have demonstrated that the inhibition of pro-inflammatory cytokines generations, attenuates the hyperalgesia induced in different inflammatory pains (Ren and Dubner, 2010; Abbadie, 2005). Therefore, it seems possible that the effect of venlafaxine on PMN cells migration, TNF-α and IL-1β production may participates in its analgesic effect.