Putative Role of Monoaminergic Systems in Antidepressant and Anxiolytic Effects of Naringin in Mice: An Interaction Study with Receptor Antagonists

Aim: Stress-related disorders like depression and anxiety represent one of the greatest therapeutic challenges globally. Although previous studies have revealed the antidepressant-like potentials of naringin, the neurotransmitter receptor interaction mechanisms of action have not been studied, hence, this study was carried out to evaluate the role of neurotransmitter-receptor antagonists in the antidepressant-like effects of naringin in mice. Method: Male Swiss mice were subjected to chronic unpredictable mild stress (CUMS) apart from mice in the control group. The mice were then pretreated with different neurotransmitter antagonists; metergoline (4 mg /kg i.p.), a 5-HT1 and 5-HT2 -receptor antagonist; propranolol; (0.2 Original Research Article Anyanwu et al.; JPRI, 33(47B): 661-676, 2021; Article no.JPRI.75412 662 mg/kg i.p.), β1,2-noradrenoceptor antagonist or haloperidol (0.2 mg/kg i.p.), D 2 -dopaminergic receptor antagonists prior to the administration of naringin or vehicle (10 mL/kg). The antidepressant-like and anxiolytic effects of naringin were evaluated 30 min later using the tail suspension test (TST), open-field test (OFT), sucrose preference test (SPT) and elevated plus maze (EPM) tests paradigms. Results: Administration of naringin following CUMS significantly decrease immobility time and locomotion activity in TST and OFT respectively, relative to control while increasing preference to sucrose in SPT, open arm entries as well as time spent in open arm in EPM, relative to control suggesting antidepressant-like property. Pretreatment with metergoline, propranolol, and haloperidol following CUMS increased immobility time in TST, locomotor activity in OFT and IOAA in the EPM. Reduced preference for sucrose in SPT, open arm entry and duration in EPM relative to control (p < 0.05), however, these effects were attenuated by naringin. Conclusion: These findings suggest that the antidepressant-like activity exhibited by naringin might be mediated via interactions with 5-HTergic, noradrenergic, and dopaminergic receptors, while the anxiolytic effect might involve interaction with both 5-HTergic and noradrenergic receptors.


INTRODUCTION
Stress-related disorders such as depression and anxiety represent one of the greatest therapeutic challenges in the twenty-first century. It is burdensome as it has a high lifetime prevalence rate and a high level of comorbidity with other psychiatric diseases [1]. There is a growing research interest on the treatment of these stress-related disorders, albeit findings in drug discovery research have implicated the involvement of neuroactive peptides in the pathophysiology of these disorders [2][3][4]. Depression is associated with anhedonia, low self-esteem, guilty feelings, worthlessness, social impairment, sadness and suicidal tendencies [5][6], while anxiety disorder is associated with excessive fear and panic attacks [5]. Neuropsychiatric disorders are seen as a complex disorder, the mechanisms underlying their pathogenesis remain unclear although some peptide neurotransmitters in the brain has been implicated in previous researches [7-9]. Incidentally, common findings suggest that monoamine oxidase inhibitors (MAOI) in monoamine hypotheses play a major role in depression [10], as monoamine oxidase inhibitors has been used to relieve some of the depressive symptoms and anxiety [11].
The symptoms of depression and anxiety steams from disturbance in the transmission of neuropeptide neurotransmitters like serotonin (5-HT), noradrenaline (NA) and dopamine (DA) all of which play important roles in transmission of chemical information (impulses) across the synapse [10,12]. Recent works on the treatment of depression and anxiety demonstrate that the administration of selective serotonin reuptake inhibitors, dopamine inhibitors and/or serotoninnoradrenaline reuptake inhibitors, augments the level of the neurotransmitters (serotonin, dopamine and/or noradrenaline) in the central nervous system as an important step and strategy for the treatment of this disorder [9, [13][14]. Furthermore, non-synaptic approach of a selective and moderate induction of the carriermediated release of noradrenaline and serotonin is also proposed for the treatment of depression as active antidepressants [12].
The hypothalamus-pituitary-adrenal (HPA) axis and corticosteroid receptor signaling dysregulation has been reported to be the hallmark of major depressive and anxiety disorders [15]. This dysregulation is as a result of the increased production and secretion of corticotropin-releasing hormone (CRF) [16], triggered by chronic exposure to unpredictable stress. The corticosteroid receptor hypothesis of depression has led to another line of thought and research, focusing on the brain neuropeptide receptors, and especially the CRF receptors as drug target [17]. A variety of psychopharmacological agents are used to treat patients with major depressive disorder and anxiety disorders. Foremost among these medications are the anxiolytics and antidepressants such as imipramine, iproniazid, fluoxetine, sertraline, duloxetine etc (Saltiel et al., 2015; Montoya et al., 2016) [18][19]. The most widely used antidepressant medications have traditionally been the selective serotonin reuptake inhibitors (SSRI), which act primarily by enhancing serotonin neurotransmission [20]. However, currently used antidepressants are the serotonin-norepinephrine-dopamine reuptake inhibitor (SSNDRI), also referred to as triple reuptake inhibitor (TRI). This medication act as a combined reuptake inhibitor of the monoamine neurotransmitters, serotonin, noradrenalin and dopamine [20]. Various studies have shown the role of monoamines such as 5-HTergic, noradrenergic, and dopaminergic systems in the pathophysiology of depression [21][22][23]4]. The principal therapeutic benefit of TRI lies in their ability to increase the availability and effect of the three neurotransmitters to relieve depressive symptoms and enhance cognitive balance, which act via mechanisms related to modulation of 5-HT 1,2,6 , α 1,2 and β 1,2 -noradernergic and D1,2dopaminergic receptor systems in particular [24]. The monoamine neurotransmitter (5-HT, α, β and D) receptors are exclusively distributed in specific areas in the central nervous system with high distribution in limbic and cortical areas and are connected post-synaptically to GABAergic, glutamatergic, dopaminergic, and noradrenergic interneurons [24]. Therefore, ligands of these monoamine receptors exert their pharmacological activity via interactions with each other and other neurotransmitter receptors [25]. Pool of evidence abound on the interaction of biomolecules like phenolic compounds with monoaminergic and nonmonoaminergic receptors to induce their psychopharmacological activities [25]. Considering that some biosubstances have antidepressant-like effect and anxiolytic potential, this potential could probably be due a modulation of the monoaminergic pathways via neurotransmitter-receptor interactions.
Although studies have shown that naringin enhances calcium/calmodulin-dependent protein kinase II activity in a mouse model of Alzheimer's disease [42], attenuates mitochondrial oxidative damage against aluminum-induced neurotoxicity in rats [43], modulates cholinergic and nitric oxide pathways in stressed mice [44], improved long-term memory deficits [42,43,44], and exhibit antidepressant-like effect via monoaminergic system [45], however, the neurotransmitter receptor interaction mechanism of naringin remain unexplored. Furthermore, literature search revealed that no information exists on the monoaminergic and nonmonoaminergic psychopharmacologic receptor interactions. Hence, we investigated the role of monoaminergic and nonmonoaminergic neurotransmitter receptors interaction in the antidepressant-like and anxiolytic effects of naringin in swiss mice.

Laboratory Animals
Adult male Swiss mice (20-25 g; 7 weeks old) were housed, acclimatized and maintained in plastic cages (42 × 30 × 27 cm), in controlled temperature (25 ± 1°C) with a 12-h light/ dark cycle. They were allowed to have access to standard rodent pellet food and water ad libitum throughout the experimental period.

Drug and treatments
Metergoline -MET, propranolol -PRO, haloperidol -HLP, and naringin -NAR (Cas No. 3H1/X0,6/S/16/3/3AM 12358-M3) used in this study were all purchased from Sigma-Aldrich, St. Louis MO, USA. All drugs were dissolved in normal saline immediately before use and administered intraperitoneally (i.p.). The dose of naringin (10 mg/kg, most effective dose) used in this study was based on results obtained from previous study (Ben-Azu et al., 2018b) [46]. The doses of receptor blocker(s): metergoline (4 mg/kg i.p), propranolol (1 mg/kg i.p), haloperidol (0.2 mg/kg i.p), and the routes of administration used were chosen based on documented data in [10,47,48,23]. Normal saline solution was also administered intraperitoneally in a volume of 10 mL/kg per body weight, as normal control. All receptor blocker(s) were administered 15 min prior to treatment with naringin, for the purpose of blocking different neurotransmitter receptors [23] in order to elucidate the neurochemical receptor activity behind the antidepressant-like properties, locomotor activity as well as anxiolytic effect of mice in the tail suspension test (TST), sucrose preference test (SPT), Open field test (OFT) and elevated plus maze. All behavioral tests were carried out between 8:00 -14: 00; 30 min after naringin administration [49]. In a bid to show the possible contribution of the serotonergic noradrenagic and dopaminergic systems in the antidepressant-like and anxiolytic activity of NAR in the TST, OFT, SPT and EPM, the animals were separated into 3 sets (sets A-C) of 5 treatment groups with 5 mice in each group. All the groups except group 1 in each of the 3 sets, were subjected to chronic unpredictable mild stress (CUMS), for 21 days. Twenty-four (24) hours after the last day of CUMS, group 1 was treated with 10 mL/kg of vehicle; Group 2 was left untreated to serve as reference group. Group 3 was administered naringin (10 mg/kg, i.p.) in each of the 3 sets. Group 4 was pretreated with metergoline (4 mg/kg, i.p.; a nonselective 5-HT 1 and 5-HT 2 receptor antagonist), propranolol (1 mg/kg, i.p.; a β-adrenoceptor antagonist), and haloperidol (0.2 mg/kg, i.p.; dopaminergic D2 receptor antagonist) for sets A-C respectively. Group 5 was pretreated with metergoline (4 mg/kg, i.p.), propranolol (1 mg/kg, i. p) and haloperidol (0.2 mg/kg, i.p) respectively, 15 min before the administration of naringin (10 mg/kg, i.p.). Thirty minutes later [2], all animals were evaluated in the tail suspension, open field, sucrose preference and elevated plus maze models as previously described in literature [50,51,52,53,54,4].

Tail Suspension test
This test was carried out according to the method of Steru et al.and Sherman et al., [50,51]. A new set of animals were treated with the various drugs as described earlier. Thirty minutes after treatment, mice were suspended individually on a retort stand, placed 50 cm above the floor with the help of an adhesive tape placed approximately 1 cm from the tip of the tail. The total duration of immobility was recorded during the last 4 min of the 6 min test. An animal was considered to be immobile when it did not show any movement of the body and hangs passively.

Open-field test
This was engaged as previously described by [53]. The OFT consists of a wooden box (28 × 28 × 25 cm) with visible lines drawn to divide the floor into 16 (7 × 7 cm) equal squares with a frontal glass wall, and placed in a sound free room. The animals were placed in the rear left square and left to explore it based on number of line crossings for 5 min. Thereafter, the observation chamber was cleaned with 70% ethanol to remove residual odor

Sucrose Preference Test
For the sucrose intake test, mice were trained to consume 2% sucrose solution prior to the start of the experiment. They were exposed to 2% sucrose solution for 24 h period in their home cages without any food or water available. Prior to the test, mice were food and water deprived for 12 h. after a 12-h period of food and water deprivation, mice were given two bottles containing water or 2% (wt-vol) sucrose solution. Six hours later, the volumn of water and sucrose consumed will be measured. The mice' sensitivity to reward will be calculated as the percentage of total liquid intake attributed to the 2% sucrose solution, according to the following, equation:

Elevated-plus Maze
The elevated plus maze test was used to assess for possible anxiety component of the

Chronic Unpredictable Mild Stress (CUMS) Procedure
In CUMS, the mice are chronically exposed to variable unpredictable mild stressors to induce behavioral deficit (depressive-and anxiety-like behaviors) [55]. Prior to the start of the CUMS procedure, all of the mice were given 2% sucrose water for 24 hr to avoid neophobia for sucrose consumption training [54]. Mice were exposed to the following stressors daily for 21 days: 24 hr food deprivation, 24 hr water deprivation, 7 hr cage tilt (45° inclined), 1 min tail pinch with push pine, 0.45 x 12 mm (1 cm from the end of the tail), 7 hr wet bedding in cage (200 mL water in 100 g sawdust bedding), Hypoxia [15 min inside an air-tight hypoxic transparent plastic-container (height 23 cm, diameter 10 cm), overnight illumination (12 hr), Exposure to predator (12 hr). Control (unstressed) mice were undisturbed except for necessary procedures such as routine cage cleaning. Twenty-four hrs after the last application of unpredictable stressor, the different treatments to access the involvement of serotonergic, noradrenagic and/or dopaminergic pathways were given.

Statistical Analysis
Data were expressed as mean ± S.E.M.
(standard error of mean). Data were analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test for multiple comparisons where appropriate using the Graph Pad Prism software version 5. A level of p < 0.05 was considered as statistically significant for all tests.

The Involvement of 5hydroxytryptaminergic (5-TH) Neurotransmission on the Actions of Naringin in the TST, OFT, SPT and EPM
Effects of pretreatment with metergoline on the actions of naringin in the TST, OFT, SPT and EPM neuro-behavioral activities in CUMSinduced depressed mice is shown in Fig. 2. According to the results in Fig. 2 (a- Post-hoc analysis revealed that pretreatment with MET (4 mg/kg, i.p.) prior to naringin (10 mg/kg, i.p.) following CUMS significantly (p<0.05) reversed all the CUMS-induced depressive-like symptoms when compared with CUMS group.

Effect of Noradrenergic Pathway on the Actions of Naringin in the TST, OFT, SPT, and EPM Neurobehavioral Activities
The effect of pretreatment of mice with propranolol (0.2 mg/kg, i.p.) on the actions of naringin in the TST, OFT, SPT, and EPM is shown in Fig. 2. As shown in Fig. 2a -  Interestingly, naringin treatment following CUMS was able to enhance the suppressed locomotor activity and sucrose preference in OFT and SPT respectively relative to the mice in CUMS group, albeit not significant (p> 0.01). The increase in percentage OAE and duration in EPM Naringin treatment following CUMS was also not significant when compared to the mice in both CUMS and saline treated groups.
Post-hoc analysis by the Bonferroni test showed that pretreatment with haloperidol (0.2 mg/kg, i.p.) prior to treatment with naringin (10 mg/kg, i.p.) significantly reversed the reduced preference for sucrose (in SPT and percentage open arm entry in EPM) which was induced by CUMS and HLP (0.2 mg/kg, i.p.) pretreatment, relative to the mice in CUMS group, but did not affect locomotion activities in OPT, immobility time in TST and IOAA in EPM relative to the mice in CUMS and saline treated groups.

DISCUSSION
The monoaminergic system has been implicated as the neurochemical pathway involved in the pathophysiology and treatment of depression [7,11], hence, we assessed the involvement of the serotonergic, noradrenergic and dopaminergic receptor interaction in the action of naringin on the TST, OFT, SPT and EPM behavioral activities in Swiss mice. Specific behavioral features and symptoms of anxiety and depression has been associated with deficiency or malfunction of certain monoamine neurotransmitters [56][57]. Depletion of the 5-HT, NE and/or DA is used as a model to test the involvement of monoaminergic systems in depression and anxiety. Therefore, to induce a state of depletion in the monoaminergic systems, we used enzyme-blocking agents to decrease the production of the monoamines. Metergoline was used to block 5HT 1 -and 5-HT 2receptors, propranolol was used to block β1,2nor-adrenoceptor while haloperidone was used to block D2 -dopaminergic receptor) [22][23].
Interestingly, increased 5-HT activity has been associated with certain symptoms such as fatigue, while 5-HT deficiency is common in anxiety, obsessions, and compulsions [58,59]. The involvement of the serotonergic system in the antidepressant-like and anxiolytic effects of naringin was studied using metergoline in TST, OFT, SPT and EPM paradigms. TST and SPT are validated behavioral models for assessing behavioral despair, hoplesness and anhedonic behaviors [50], for the detection of compounds with antidepressant property in rodents and elucidating their possible mechanisms of action [51,54], while the OFT and EPM are established behavioral model for assessing locomotion activities and anxiety-related behaviors respectively for the detection of compounds with anxiolytic property in rodents [52,53].
The result of this study shows that CUMS induced depression and anxiogensis in mice but pretreatment with naringin, following CUMS significantly (p < 0.05) alleviated the depressive and anxiogenic behaviors evidenced by the increased preference to sucrose and open arm entries and duration in SPT and EPM respectively (Fig. 2 c, d) in relation to control (vehicle) group (P< 0.05). It significantly (p < 0.001) reduced the immobility time in TST (Fig.  2a), locomotion activities in OFT (Fig. 2b) and index of open arm avoidance in the EPM (Fig.  2e) when compared to mice in the vehicle control group. Treatment with metergoline, a 5-HT 1 and 5-HT 2 -receptor antagonist (4 mg/kg, i.p.) significantly attenuated all the antidepressant-like effects of naringin as compared with the control (vehicle) group.
Studies have established that pretreatment of experimental animals with 5-HT 2A antagonists attenuates the antidepressant-like effects of known antidepressants like fluoxetine and imipramine [58,60]. On the contrary, 5-HT 2A receptor agonists has been shown to enhance the antidepressant-like effect of known antidepressant agents [61]. Therefore, the attenuated antidepressant-like effects such as increased immobility time in TST, decreased preference to sucrose and open arm entries and duration recorded in SPT and EPM were all reversed by naringin as shown in this study (Fig.  1a, c-e). These reversals of attenuated antidepressant and anxiolytic effects of naringin by pretreatment with metergoline, a 5-HT 2A receptor antagonist suggest the participation of 5-HT 2A receptors in the antidepressant-like and anxiolytic effects of naringin in TST, SPT and EPM. Similar observations were made by [4,23] in their works investigating the involvement of monoamine neurotransmitters in the antidepressant-like effects of morin and methyl jasmonate respectively [4,23].
The enhancement of NA neurotransmission is one of the mechanisms of action of some antidepressant drugs [56]. Several lines of evidence have linked reduced NA neurotransmission with decreased alertness, low energy, problems of inattention and concentration [62][63]. However, in this study, we use propranolol (a β1,2adrenoceptor antagonist) to evaluate the involvement of noradrenergic pathway in the antidepressant-like and anxiolytic effects of naringin in TST, OFT, SPT and EPM paradigms.
Preclinical and clinical studies have shown that decreased exploratory activity and increased behavioral despair are key features associated with depressive-like behavior [63][64][65], and antidepressant drugs are known to increase exploratory activity and also decrease behavioral despair via increase in serotonergic and noradrenergic neurotransmissions [58,62].
In the present study, we observed that CUMS induced depression and anxiogensis in mice but pretreatment with naringin, following CUMS significantly (p < 0.05) alleviated the depressive and anxiogenic behaviors evidenced by the increased percentage open arm entries and duration in EPM (Fig. 3d) and immobility time in TST (Fig. 3a). According to the results, there was no recorded significant effect on sucrose intake of the mice in SPT (Fig. 3c) when compared to mice in the vehicle control group. Most importantly, we found that treatment with naringin (10 mg/kg, i.p.) and propranolol (1 mg/kg, i.p.) a β1,2adrenoceptor antagonist, following CUMS had both significantly antidepressant-like (decreased immobility time) and anxiolytic-like (i.e., increase of open arm time) effect in TST and EPM ( Fig. 3a and d) as seen in previous works [23,48]. Furthermore, Treatment with propranolol, had no significant effect on sucrose preference in SPT (3c) as compared with the control (vehicle) group. The manner of result observed in this study is similar to observations established and recorded with some antidepressant compounds in previous literatures [22,[47][48]. This could be the evidence that there is possible involvement of β1,2adrenoceptor receptors in the antidepressant-like potential of naringin.
Previous studies have shown that excitatory neurotransmitter systems play an important role in the regulation of locomotion, and increased motor activity is suggestive of enhanced excitatory neurotransmission [4,23]. Supportive evidence has also shown that alteration in excitatory neurotransmitters (e.g., dopaminergic and glutamate) have been shown to play a prominent role in several neuropsychiatric diseases including depression, schizophrenia, Parkinson's disease among others [13, 37,66]. Thus, the ability of naringin to increase locomotor activity in the OFT based on increased number of line crossing after repeated administration suggests modulation of excitatory neurotransmitter systems. Our results demonstrated significant increase in sucrose preference (Fig. 4c) and decrease in immobility time (Fig. 4a) by naringin following CUMS. This is suggestive of depressive-like behavior. Pretreatment with haloperidol prior to naringin following CUMS attenuated the sucrose preference and enhanced immobility time as observed in TST and SPT respectively. This is suggestive of the inhibition of the antidepressantlike effect of naringin in a manner similar to previous results with other antidepressant agents [4, 23,49,75], this suggests its possible interaction with both D1 and 2-doperminergic receptors. The decrease in dopaminergic activity due to antagonism of central D2 and 2 receptors may perhaps explain the reduced sucrose preference and anti-immobility effect of naringin caused by haloperidol in this study.

CONCLUSION
This present study is of particular interest, being that, it demonstrated the involvement of monoaminergic system in the antidepressant and anxiolytic effects of naringin in mice following CUMS. Taken together, the results of our study provide evidences, which suggest that naringin produces its action in the treatment of depressive and anxiety disorders via mechanisms related to serotonergic, adrenergic and dopaminergic transmissions. Furthermore, we can affirm that there is an effect on some behaviour of mice, but to confirm the effect, a clinical study of the antidepressant study of naringin should be demonstrated.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The experimental protocols were approved by the University's College of Medicine Research Ethics Committee (COMRAC). Five animals per group were used for the study. The experiment was performed in accordance with the regulatory protocol of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). Also, efforts were made to minimize the suffering of the animals during CUMS experimental protocols and behavioural tests.