Introduction

Depression is a common mental disorder worldwide, with an estimated 3.8% of the population affected, including 5.0% of young adults and 5.7% of people around 60 years old. Depressed patients typically experience symptoms such as sadness, anhedonia, fatigue, insomnia, concentration problems, and suicidal ideation (WHO 2021). According to the Global Burden of Disease database, the number of depression cases worldwide has increased from 172 million in 1990 to 250.8 million in 2017, representing a 49.86% increase (Liu et al. 2020).

The understanding of depression pathophysiology has progressed considerably, but no single model or mechanism can explain all the aspects of this mental disorder. Different theories related to the pathogenesis of depression are cited in the literature, the most common among them being the monoamine hypothesis, understood as the reduction of serotonin, noradrenaline, and dopamine concentrations in the synaptic clefts (Malhi and Mann 2018). An increasing amount of evidence shows that depression is characterized by a dysfunction of the neurotransmission of serotonin, also known as 5-hydroxytryptamine (5-HT). The biosynthesis of serotonin involves tryptophan, an amino acid supplied by food. In the central nervous system (CNS), serotonin acts on the serotonergic receptors, being then reuptaken into the presynaptic neuron and degraded by the enzyme monoamine oxidase (MAO) (David and Gardier 2016). Consequently, molecules that act on serotonin receptors or those that block serotonin reuptake or inhibit MAO activity, increasing the availability of this neurotransmitter in the synaptic cleft, are clinically effective as antidepressants (Malhi and Mann 2018).

Despite the undeniable progress achieved regarding the selectivity and the risk/benefit ratio of antidepressants, some drawbacks remain, such as their limited effectiveness (about 30% of depressed patients do not respond to current antidepressants), relapses and recurrences, besides the long time necessary to show clinical benefits (David and Gardier 2016). Therefore, the search for new drugs that may be effective for the treatment of depression is of great importance.

In this sense, nitrogen-containing heterocyclic compounds have shown great potential in terms of biological effects (Sharma et al. 2010, 2013). The Food and Drug Administration (FDA) databases revealed the importance of nitrogen-containing heterocyclics in drug development, especially indole and its derivatives, such as indolizine (Vitaku et al. 2014). Indolizine derivatives, presenting a nitrogen-containing heterocyclic ring, have shown anti-inflammatory, antibacterial, and antioxidant effects and improvement in mood disorders, among other promising pharmacological properties (Li et al. 2021).

Simultaneously, selenium-containing compounds present important pharmacological properties (Jain and Priyadarsini 2018; Lenardão et al. 2018; Nogueira et al. 2021; Birmann et al. 2022). In addition, studies demonstrate possible links between low concentrations of selenium in the body and the occurrence of depressive symptoms. Given its importance, promising evidence has been found to suggest that organic selenium compounds have an antidepressant-like effect (Sousa et al. 2018, 2019; Birmann et al. 2019; Besckow et al. 2020; Garcia et al. 2022).

Therefore, based on the above considerations, the objective of this study was to investigate whether 1-(phenylselanyl)-2-(p-tolyl)indolizine (MeSeI), an indolizine derivative containing selenium, presents an antidepressant-like effect, as well as to evaluate the possible involvement of the serotonergic system in the antidepressant activity of MeSeI in mice. Furthermore, the interaction of MeSeI with the serotonergic system was evaluated by molecular docking and the compound acute toxicity profile was investigated in female mice.

Materials and methods

Animals

Adult male Swiss mice, provided by the Animal Facility of the Federal University of Pelotas, weighing between 25 and 30 g, were used for the behavioral tests. The animals were maintained under controlled temperature (22 ± 1 °C), a regular 12-h light/dark cycle (light on at 7:00 a.m.) and water and food ad libitum, except during the experimental protocol. The use of male mice to investigate the pharmacological properties of MeSeI was based on the literature that demonstrates behavioral tests being performed more frequently in male mice and showing good reproducibility (Anjomshoa et al. 2020; Casaril et al. 2020; Rech et al. 2021; Garcia et al. 2021, 2022; Lu et al. 2022).

The experiments were performed between 09:00 a.m. and 05:00 p.m., and each mouse was used only once. Before the start of treatment, the animals were acclimatized for 1 h. Each animal received a specific treatment, and later the open field test (OFT), the forced swim test (FST), and tail suspension test (TST) were performed. Separate groups of animals were used to perform the FST and TST. To prevent evaluator bias, observers were unaware of the treatment condition of animals in all the protocols.

The experimental protocol was approved by the Ethics Committee on the Use of Animals of the Federal University of Pelotas, Brazil (CEUA 16,057–2021) and is according to the National Institutes of Health Guide for the care and use of laboratory animals (NIH publications Nº 8023, revised 1978).

Drugs

MeSeI (Fig. 1) was prepared and characterized by the Clean Organic Synthesis Laboratory of the Federal University of Pelotas according to the procedure described by Penteado et al. (2019), and the purity (99.9%) and the structure of the compound were determined by gas chromatography-mass spectrometry (GC–MS) and by 1H, 13C, and 77Se nuclear magnetic resonance (NMR) analysis, respectively. The obtained data were in agreement with the compound (see supplementary methods for more details). MeSeI was dissolved in canola oil and administered intragastrically (i.g.) at a constant volume of 10 mL/kg body weight.

Fig. 1
figure 1

Chemical structure of 1-(phenylselanyl)-2-(p-tolyl)indolizine (MeSeI)

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The following drugs were used: fluoxetine, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2pyridinylcyclohexanecarboxamide (WAY100635), ketanserin ( +)-tartrate salt, ondansetron hydrochloride, 1-[2-[(methylsulfonyl)-amino]ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate (GR113808) and p-chlorophenylalanine methyl ester (p-CPA). Drugs were administered intraperitoneally (i.p.), except WAY100635 which was administered subcutaneously (s.c.). Fluoxetine and the antagonists WAY100635, ketanserin, ondansetron, and p-CPA were diluted in 0.9% saline. The GR113808 antagonist was suspended in 0.5% carboxymethyl cellulose (CMC) with 0.1% tween 80. All drugs were administered at a constant volume of 10 mL/kg of body weight and obtained from Sigma-Aldrich (St. Louis, MO, USA), except fluoxetine, which was acquired from local sellers. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes were measured using the Labtest kit (LABTEST®, Diagnostic S.A.), while urea was measured using the Bioclin kit (BIOCLIN 100®) all acquired from local sellers.

Experimental design

Dose–response curve in the antidepressant-like effect of MeSeI in mice

To elucidate the dose–response curve of the antidepressant-like action of MeSeI, mice were treated with MeSeI at doses of 0.5, 1, 10, and 50 mg/kg (i.g.), fluoxetine 20 mg/kg (i.p.) or canola oil (control, 10 mL/kg, i.g.) 30 min before FST or TST. The OFT was performed 4 min before the TST to check the locomotor activity of the animals (Fig. 2a). Mice were randomly selected with n = 8/9 animals by group. The doses were selected according to previous studies on the antidepressant-like action of organic selenium compounds (Besckow et al. 2020; Gall et al. 2020; Garcia et al. 2022). Fluoxetine was used as the reference antidepressant drug.

Fig. 2
figure 2

Experimental design illustration of drug administration followed by behavioral testing in mice. a Dose–response curve design of MeSeI treatment. Fluoxetine was used as a positive control. b Experimental design of the effect of serotonin depletion on the antidepressant-like effect of MeSeI. c Experimental design to elucidate the involvement of the serotonergic system in the antidepressant-like action of MeSeI. Abbreviations: MeSeI: 1-(phenylselanyl)-2-(p-tolyl)indolizine; i.g., intragastric; i.p., intraperitoneal; p-CPA, p-chlorophenylalanine; FST, forced swim test; TST, tail suspension test; OFT, open field test

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Involvement of the serotonergic system in the antidepressant-like effect of MeSeI

The FST was used to investigate a possible contribution of the serotonergic system in the antidepressant-like action of MeSeI. The dose of 50 mg/kg of MeSeI was selected once it was the dose that showed the most promising antidepressant-like effect in the screening tests (FST and TST). Mice were separated into different groups (n = 7–9 animals/group) and were pre-treated with p-CPA (100 mg/kg, i.p.) or its vehicle (10 mL/kg, i.p.), once a day, for 4 consecutive days. Twenty-four hours after the last p-CPA injection, animals were treated with MeSeI (50 mg/kg, i.g.) or canola oil (vehicle, 10 mL/kg, i.g.) and tested in the FST 30 min later (Fig. 1b).

The mice were also pre-treated with 5-HT1A receptor antagonist (WAY100635, 0.1 mg/kg, s.c.), 5-HT2A/2C receptor antagonist (ketanserin, 1 mg/kg, i.p.), 5-HT3 receptor antagonist (ondansetron, 1 mg/kg, i.p.) and 5-HT4 receptor antagonist (GR113808, 0.1 mg/kg, i.p.). After 15 min of administration of WAY100635, ketanserin, and ondansetron, or 30 min after administration of GR113808, the mice received MeSeI (50 mg/kg, i.g.) or canola oil (vehicle, 10 mL/kg, i.g.). After 30 min of administration of the compound or its vehicle, the animals were subjected to FST (Fig. 2c). The OFT was performed 4 min before the FST to verify the animal’s locomotor activity. Doses and treatment times with serotonergic receptor antagonists and p-CPA were based on previous studies (Redrobe et al. 1998; Eckeli et al. 2000; Gavioli et al. 2004; Jesse et al. 2010; Castello et al. 2018; Gall et al. 2020). p-CPA administration reduces the brain levels of 5-HT by about 60% in mice (Redrobe et al. 1998) and the doses and regimen of administration employed in this study were previously shown to completely prevent the antidepressant-like effect of fluoxetine in the mouse FST (Eckeli et al. 2000; Gavioli et al. 2004).

Behavioral tests

Open field test

The locomotor and exploratory activities of the animals were evaluated in the OFT according to Walsh and Cummins (1976) to verify if MeSeI or the pharmacological combination of the compound with the antagonists influenced these functions. Mice were placed individually in the center of a box (30 × 30 × 15 cm) divided into nine equal squares and were observed over a period of 4 min. During this time, locomotor activity was evaluated through the number of segments crossed with the four paws, and the exploratory activity expressed by the number of times rearing on the hind limbs.

Antidepressant-like behavior

Both tests, FST and TST, make it possible to assess whether the compound has an antidepressant-like effect through the analysis of the immobility of each animal. The decrease in total immobility time and the increase in time to the first episode of immobility are indicatives that there is an antidepressant-like effect of the tested compound. FST is a broad-spectrum method for testing the efficiency of antidepressants with strong predictive validity and TST is considered a supplementary test to the FST (Hao et al. 2019).

Forced swim test

The test was performed according to the methodology described by Porsolt et al. (1977). In this test, mice were forced to swim individually in an open cylindrical container (10 cm in diameter × 25 cm in height), containing 1.5 L of water at 25 ± 1 °C, for 6 min, timing the first episode of immobility (latency) and the total immobility time of the animal. Animals were considered immobile when they stopped fighting and remained floating in the water, making only the necessary movements to keep their heads above the water.

Tail suspension test

To perform this test, each mouse was individually suspended 50 cm above the floor with the adhesive tape placed approximately 1 cm from the tip of the tail. The animal was observed for 6 min, during which the total immobility time and the time to the first episode of immobility were recorded. Mice were considered immobile when they did not exhibit escape attempt behavior (Steru et al. 1985).

Molecular docking studies

A virtual screening with a molecular docking tool was performed to check for docking scores among MeSeI towards the 5-HT2A, 5-HT2C, and 5-HT4 receptors. The 2D structure of MeSeI was drawn using ChemDraw and converted to 3D using the software Avogadro 0.9.4, along with the General Amber Force Field (GAFF) method to optimize the compound (Hanwell et al. 2012). The 3D X-ray Crystal structures of human 5-HT2A and 5-HT2C serotonin receptors were used, acquired from the Protein Data Bank (5-HT2A PDB ID: 6A94; 5-HT2C PDB ID: 6BQH) (Peng et al. 2018; Kimura et al. 2019). For the development of the 5-HT4 receptor model, the homology modeling program SWISS-MODEL (Kopp and Schwede 2004) with an β2-adrenergic receptor crystal structure was used (Dubost et al. 2012).

The CHIMERA 1.5.3 software was used to remove ions, water, and ligands from the protein. With the software AutoDockTools 1.5.4 hetero groups were fixed, structures were optimized using Gasteiger charges with 500 steps of minimization, all rotatable bonds of ligands were allowed to rotate freely, and receptors were set to be considered rigid (Pettersen et al. 2004).

A molecular docking simulation was performed with AutoDock Vina (Trott and Olson 2010), with a grid box around the interaction sites of 5-HT2A (Gandhimathi and Sowdhamini 2016), 5-HT2C (Wang et al. 2019), and 5-HT4 (Dubost et al. 2012), as based on the literature. The protein–ligand interactions were analyzed with the software Discovery Studio Visualizer 2016.

Acute oral toxicity

The acute toxicity of the compound in the animals was examined to investigate a possible future application of MeSeI as a new antidepressant drug. The experimental protocol was conducted according to the Organization for Economic Cooperation and Development Guidelines (OECD 2001) using female Swiss mice, because they are generally slightly more sensitive than males. The experimental protocol was divided into two sets and for each set were used 3 female mice (control, 10 mL/kg × MeSeI, 300 mg/kg). The animals were acclimated 5 days before the experiment. On the day of the experiment, mice were deprived of food and water for 3 h before receiving the treatment. Each animal received the 300 mg/kg dose (i.g.) of MeSeI (3 doses of 100 mg/kg 30 min apart) or its vehicle (canola oil, 10 mL/kg). After treatment, the animals were observed individually every 30 min for the first 4 h to assess signs of general toxicity (salivation, diarrhea, piloerection, lethargy, ptosis, tremors/seizure, weight, and mortality) and periodically during the first 24 h. Subsequently, during the 14-day period, general signs of toxicity continued to be monitored, as well as body weight, food, and water consumption at 09:00 a.m. On the 14th day, the animals were subjected to OFT to assess locomotor and exploratory activity (protocol explained in item 2.4.1) and later euthanasia with isoflurane. A cardiac puncture was performed in each female mice to obtain heparinized blood for biochemical measurements.

Plasma samples were obtained by centrifuging blood for 10 min at 700 g to determine AST and ALT enzymatic activity and plasma urea levels. Urea levels were measured to assess renal function and were expressed as mg/dL. AST and ALT levels were measured to analyze the hepatic function and were expressed as IU/L. These parameters were performed by enzymatic colorimetric methods using commercial kits following the manufacturer’s instruction (LABTEST®, Diagnostic S.A., Minas Gerais, Brazil). All tests were performed in duplicate.

Statistical analysis

Firstly, data normality was assessed using the D’Agostino and Pearson test. Statistical analysis for comparisons between experimental and control groups was performed by one-way (MeSeI dose–response curves) or two-way ANOVA (p-CPA, WAY100635, ketanserin, ondansetron, and GR113808 effects) followed by the Newman-Keuls test for post hoc comparison when appropriate. The unpaired t-test was used to analyze the action of fluoxetine in relation to the control and for the acute oral toxicity tests. Experimental results were expressed as the mean ± standard error of the mean (S.E.M.). Probability values lower than 0.05 (p < 0.05) were considered statistically significant. Statistical analysis was performed using GraphPad Prism version 8.0.2 for Windows, GraphPad Software (San Diego, CA, USA).

Results

Antidepressant-like action of MeSeI in mice subjected to FST

The FST was used to assess the antidepressant-like effect of MeSeI in mice. Figures 3a and b demonstrate the latency time for the first episode of immobility and the total immobility time, respectively. Treatment with MeSeI at a dose of 50 mg/kg (i.g.) (F(4, 38) = 8.323, p < 0.0001) increased the latency time for the first episode of immobility compared with the control group, as well as fluoxetine was also able to increase this time (df = 15, t = 5.219, p = 0.0001, Fig. 3a). Post hoc statistical analysis evidenced that animals treated with MeSeI at doses of 1, 10, and 50 mg/kg, i.g., showed a decrease in immobility time in the FST (F(4, 38) = 9.403, p < 0.0001) compared with the control group. Furthermore, as expected, fluoxetine (20 mg/kg, i.p.) also reduced immobility time in the behavioral test (df = 15, t = 5.728, p = 0.0001) when compared with the control group (Fig. 3b).

Fig. 3
figure 3

Effect of dose–response curve of MeSeI (0.5–50 mg/kg, i.g.) and fluoxetine (20 mg/kg, i.p.) treatment on FST, TST, and OFT in mice. a Latency time for the first episode of immobility and b total immobility time in the FST. c Latency time for the first episode of immobility and (d) total immobility time in the TST. e Total number of crossings and f total number of rearings in the OFT. Fluoxetine was used as a reference drug. Values were expressed as mean ± standard error of the mean (S.E.M.) (n = 8–9 animals/group). (**) p < 0.001 and (***) p < 0.0001 when compared with the control group. Data were analyzed by One-way Analysis of Variance (ANOVA) followed by the Newman-Keuls test (control and MeSeI treatments) and unpaired t test (control and fluoxetine). Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; C, control; Flx, fluoxetine; s, seconds; i.g., intragastric; i.p., intraperitoneal; FST, forced swim test; TST, tail suspension test; OFT, open field test

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Antidepressant-like action of MeSeI in mice subjected to TST

MeSeI demonstrated antidepressant-like action in mice in the TST. According to the results, the 50 mg/kg (i.g.) dose of MeSeI increased the time to the first episode of immobility (F(4, 36) = 7.895, p = 0.0001, Fig. 3c), and at doses of 1, 10, and 50 mg/kg (i.g.), it reduced the total immobility time in the TST in mice (F(4, 36) = 8.370, p < 0.0001, Fig. 3d) compared with the control group. In addition, fluoxetine also caused similar effects on latency and total immobility time in the TST when compared with the control group (df = 15, t = 5.195, p = 0.0001, Fig. 3c and df = 15, t = 4.379, p = 0.0005, Fig. 3d).

Effect of MeSeI on spontaneous locomotor activity in mice

MeSeI, administered i.g., produced no change in the number of crossings (F(4, 38) = 0.3718, p = 0.8272, Fig. 3e) and rearings (F(4, 38) = 0.1029, p = 0.9808, Fig. 3f) in mice in the OFT at all doses tested (0.5–50 mg/kg) when compared to the control group, demonstrating that, in the present study, the compound did not cause any psycholocomotor changes. The administration of fluoxetine (20 mg/kg, ip) did not change the behavior of mice in the OFT when compared with the control group (df = 15, t = 0.8468, p = 0.4104, Fig. 3e, and df = 15, t = 0.5935, p = 0.5617, Fig. 3f).

Blockade of the antidepressant-like effect of MeSeI by serotonergic depletion

The results described in Fig. 4 demonstrate the impact of serotonin depletion by p-CPA treatment in the antidepressant-like effect of MeSeI in mice. Pretreatment with p-CPA (selective tryptophan hydroxylase inhibitor, once daily for 4 consecutive days) prevented the antidepressant-like effect of MeSeI (50 mg/kg, i.g.) in the mouse FST. Two-way ANOVA statistical analysis elucidated a main effect of MeSeI treatment on latency time to the first episode of immobility (Fig. 4a, F(1, 30) = 15.06, p = 0.0005) in the mouse FST. In addition, p-CPA blocked the reduction in total immobility time (Fig. 4b, F(1, 30) = 4.574, p = 0.0407) induced by MeSeI. As seen in Table 1, treatment with p-CPA and MeSeI caused no change in the number of crossings (F(1, 30) = 1.748, p = 0.1972) and rearings (F(1, 30) = 0.07973, p = 0.7797) in OFT.

Fig. 4
figure 4

a, b Effect of pretreatment with p-CPA (100 mg/kg, i.p) on the antidepressant-like effect of MeSeI (50 mg/kg, i.g.) in the mouse FST. Values were expressed as mean ± standard error of the mean (S.E.M.) (n = 7–9 animals/group). Data were analyzed by two-way Analysis of Variance (ANOVA) followed by the Newman-Keuls test. (**) p < 0.001 compared to the control group; (##) p < 0.001 compared with the MeSeI group. Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; s, seconds; i.g., intragastric. i.p., intraperitoneal; p-CPA, p-chlorophenylalanine; FST, Forced swim test

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Table 1 The influence of MeSeI and/or 5-HT antagonists (WAY100635, Ketanserin, Ondansetron, and GR113808) or p-CPA administration on the OFT behavioral parameters in mice
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Contribution of 5-HT2A/2C and 5-HT4 receptors in the MeSeI antidepressant-like effect in mice subjected to FST

The results described in Fig. 5 show the contribution of the serotonergic system to the antidepressant-like effect of MeSeI in the mouse FST. Figures 5a and Fig. 5b demonstrate that pretreatment with WAY100635 (a selective 5-HT1A receptor antagonist) did not block the effect of MeSeI (50 mg/kg, i.g.). Two-way ANOVA revealed a main effect of MeSeI treatment on latency time to the first episode of immobility (F(1, 30) = 9.316, p = 0.0047) and the total immobility time (F(1, 30) = 33.38, p < 0.0001). There was no significant difference in locomotor (F(1, 30) = 1.592, p = 0.2167) and exploratory (F(1, 30) = 0.3000, p = 0.5879) activity in mice that received treatment with WAY100635 and MeSeI (Table 1).

Fig. 5
figure 5

Effect of pretreatment with WAY100635 (0.1 mg/kg, s.c.) (a and b), ketanserin (1 mg/kg, i.p.) (c and d), ondansetron (1 mg/kg, i.p.) (e and f), and GR113808 (0.1 mg/kg, i.p.) (g and h) on the antidepressant-like effect of MeSeI (50 mg/kg, i.g.) in mice on FST. Values were expressed as mean ± standard error of the mean (S.E.M.) (n = 7–9 animals/group). Data were analyzed by two-way Analysis of Variance (ANOVA) followed by the Newman-Keuls test. (*) p < 0.05, (**) p < 0.001 and (***) p < 0.0001 compared to the control group; (#) p < 0.05, (##) p < 0.001 and (###) p < 0.0001 compared with the MeSeI group. Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; s, seconds; i.g., intragastric. i.p., intraperitoneal; s.c., subcutaneous; FST, Forced swim test

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On the other hand, ketanserin pretreatment (a 5-HT2A/2C receptor antagonist) blocked the increase in latency time (Fig. 5c) and the decrease in total immobility time (Fig. 5d) induced by MeSeI (50 mg/kg, i.g.) in FST. Two-way ANOVA revealed a significant interaction between ketanserin pretreatment and MeSeI treatment in latency time to the first episode of immobility (F(1, 39) = 5.399, p = 0.0254) and the total immobility time (F(1, 39) = 11.92, p = 0.0014). As seen in Table 1, the treatment with ketanserin and MeSeI did not produce any change in the number of crossings (F(1, 39) = 0.03543, p = 0.8517) and rearings (F(1, 39) = 0.7470, p = 0.3927) in OFT in mice.

In Figs. 5e and f, the results obtained from the experiment set with ondansetron are described (a 5-HT3 receptor antagonist). Pretreatment with ondansetron did not block the antidepressant-like effect of MeSeI (50 mg/kg, i.g.) on FST in mice. Two-way ANOVA statistical analysis elucidated a main effect in MeSeI treatment in latency time for the first episode of immobility (F(1, 30) = 13.59, p = 0.0009) and in the total immobility time (F(1, 30) = 25.82, p < 0.0001) in FST. No significant differences were observed in the number of crossings (F(1, 30) = 0.2617, p = 0.6127) and rearings (F(1, 30) = 0.1696, p = 0.6834) in mice that received ondansetron and MeSeI treatment (Table 1).

Finally, as shown in Figs. 5g and h, pretreatment of mice with GR113808 (a 5-HT4 receptor antagonist) prevented the antidepressant-like action of MeSeI (50 mg/kg, i.g.). Two-way ANOVA demonstrated a significant interaction in the GR113808 and MeSeI treatment for the first episode of immobility (F(1, 27) = 8.412, p = 0.0073) and for the total immobility time (F(1, 27) = 7.173, p = 0.0124) in the FST. There was no significant difference in locomotor (F(1, 27) = 1.982, p = 0.1706) and exploratory (F(1, 27) = 0.1185, p = 0.7334) activities in mice treated with GR113808 and MeSeI (Table 1).

Molecular docking reveals a possible interaction of MeSeI with 5-HT2A, 5 -HT2C, and 5-HT4 receptors

As the antagonists of 5-HT2A/2C and 5-HT4 receptors prevented the antidepressant-like effect of MeSeI, it was performed a molecular docking study to identify interactions among MeSeI and these receptors.

The results demonstrated that MeSeI presents a high docking score towards the 5-HT2C receptor (− 9.7 kcal/mol). Amino acids such as Ala222, Ser219, Val215, Leu209, Asp134, Phe328, Trp324, Ile142, and Phe223 seem to be linked to this strong affinity, as shown in Fig. 6a. Figure 6b shows that MeSeI and 5-HT2A amino acid residues mostly perform hydrophobic interactions, as follows: Pi-Sigma, Pi-Pi-Stacked, and Alkyl e Pi-Alkyl (Fig. 7b). The docking score of MeSeI with the 5-HT2A receptor was not so pronounced as with the 5-HT2C one, presenting a value of − 6.9 kcal/mol (Fig. 7a).

Fig. 6
figure 6

Binding energy between MeSeI and 5-HT2A (PDB ID: 6A94) (A). Interaction details between MeSeI and 5-HT2A (B). Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; 5-HT, 5-hydroxytryptamine

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Fig. 7
figure 7

Binding energy between MeSeI and 5-HT2c (PDB ID: 6BQH) (A). Interaction details between MeSeI and 5-HT2c (B). Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; 5-HT, 5-hydroxytryptamine

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The interaction of MeSeI towards the 5-HT4 receptor presented a docking score of − 7.9 kcal/mol. Amino acid residues Val44, Cys45, Phe64, Trp146, Val60, and Leu142 e Tyr57 seem to be linked to this interaction (Fig. 8a). Most intermolecular interactions observed in this affinity are hydrophobic, such as Pi-Sigma, Pi-Pi-Stacked, Alkyl, Pi-Alkyl, and Pi-Pi T-Shaped (Fig. 8b).

Fig. 8
figure 8

Binding energy between MeSeI and 5-HT4 (PDB ID: 5X7D) (A). Interaction details between MeSeI and 5-HT4 (B). Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; 5-HT, 5-hydroxytryptamine

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MeSeI at a high dose has low potential to cause acute toxicity to the mice

The administration of MeSeI (300 mg/kg, i.g.) did not cause the death of any animal during the 14 days of the experimental protocol, as well as it did not induce the other signs of general toxicity (salivation, diarrhea, piloerection, lethargy, ptosis, and tremors/seizure) in the first 4 h after treatment (data not shown). Both the control and treated groups gained weight, although it was less pronounced in the treated group (Fig. 9a, df = 10, t = 4.398, p = 0.0013). However, regarding food consumption (Fig. 9b, df = 2, t = 2.761, p = 0.1100) and water consumption (Fig. 9c, df = 2, t = 0.4084, p = 0.7226), there was no statistical difference between both the animal groups. In the OFT, it was observed that locomotor activity remained unchanged compared with the control group (Fig. 9d, df = 10, t = 0.7056, p = 0.4965), but there was an increase in the exploratory activity of these animals (Fig. 9e, df = 10, t = 3.361, p = 0.0072). Regarding the biochemical parameters, treatment with MeSeI (300 mg/kg, i.g.) did not change the enzymatic activities of AST (Fig. 9f, df = 10, t = 0.2855, p = 0.7811) and ALT (Fig. 9g, df = 10, t = 1.576, p = 0.1462), as well as plasma urea levels (Fig. 9h, df = 10, t = 0.5105, p = 0.6208) compared with the control group.

Fig. 9
figure 9

Effect of MeSeI treatment (300 mg/kg, i.g.) on acute oral toxicity parameters in female mice. a Weight gain percentage, b food intake, and c water intake for 14 days. Food and water intake represents data obtained from 2 cages/group containing 3 animals in each cage. d Locomotor and e exploratory activity in the OFT. f AST and g ALT enzymatic activity in plasma and h plasma levels of urea. Values were expressed as mean ± standard error of the mean (S.E.M.) (n = 6 animals/group). Data were analyzed by unpaired t test used to compare columns from each experiment individually. (**) p < 0.001 compared to the control group. Abbreviations: MeSeI, 1-(phenylselanyl)-2-(p-tolyl)indolizine; BW, body weight; AST, aspartate aminotransferase; ALT, alanine aminotransferase; OFT, open field test

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Discussion

In the present study, it was demonstrated that the selenoindolizine MeSeI has a significant antidepressant-like effect in male mice. This activity is dependent on the interaction with the serotonergic system through an involvement of 5-HT2A/2C and 5-HT4 receptors. In addition, the treatment did not cause changes in the locomotor and exploratory activities of the animals. The results also revealed that MeSeI at a high dose did not cause systemic toxicity in female mice.

The antidepressant-like effect of MeSeI was shown through the reduction of the total immobility time (1–50 mg/kg, i.g.) and increase of the latency time to the first episode of immobility (50 mg/kg, i.g.) in the FST and TST in mice. Both tests assess the animal’s despair when subjected to unavoidable stress in an unpleasant environment. During the beginning of the tests, the mice exhibit active behaviors to escape until they manifest an immobile posture. The search for new drugs for the treatment of depression seeks to effectively reverse this state of immobility, since an antidepressant-like effect in rodents refers to a decrease in immobility time (Hao et al. 2019; Wang et al. 2017). The FST and TST have been largely used as preclinical screening tests to identify the efficacy of new compounds with antidepressant-like properties (Domingues et al. 2022; Wang et al. 2017), as they have strong predictive validity and are fast and inexpensive methods with wide applicability to different compounds (Hao et al. 2019). FST has a broad spectrum for testing the efficiency of antidepressants and TST has been considered a supplementary method for FST. Usually, the effects of antidepressants are more prominent in the TST than in the FST (Hao et al. 2019). This was confirmed by the results of this study, because at the dose of 50 mg/kg, MeSeI reduced immobility time by around 35% in the FST and 55% in the TST. Considering that (i) an excessive pharmacodynamic effect of antidepressants is usually identified in the TST, (ii) FST has high predictive validity, and (iii) the results obtained in this study were more homogeneous in FST, we chose FST to investigate the involvement of the serotonergic system in the antidepressant-like effect of MeSeI.

The antidepressant-like action of MeSeI may be associated with the presence of selenium in its chemical structure, as different studies have demonstrated the antidepressant-like property of organic selenium compounds in animal models (Besckow et al. 2020; Gall et al. 2020; Rech et al. 2021). Selenium is a vital chemical element for the proper functioning of several selenoproteins, protecting the brain against damage from oxidative stress. Currently, it is known that there is an association between low selenium levels and depression because it has an important neuromodulatory role in the CNS through the maintenance of metabolism and due to its antioxidant function (Wang et al. 2018; Nogueira et al. 2021).

Indolizine-based compounds have pharmacological properties that could contribute to the treatment of several diseases, including neuropsychiatric ones, such as Alzheimer's disease and schizophrenia (Xue et al. 2016; Baussanne et al. 2021). Thus, the presence of the indolizine nucleus in the MeSeI may contribute to its antidepressant-like effect in animals. Indolizine derivatives began to be synthesized in the 1960s, when James M. Price suggested replacing the biologically active indole ring with an indolizine one, and this replacement allowed the synthesis of β‑(1‑indolizyl) alanine, a tryptophan analogue (Venugopala et al. 2021). Tryptophan is the precursor of serotonin, a neurotransmitter involved in depressive disorder, as its role at the CNS level involves modulation of mood, cognition, and reward (Maffei 2020). In addition, the indolizine nucleus has antioxidant properties (Baussanne et al. 2021) and may have beneficial effects in reducing or delaying neural death, since the production of reactive oxygen species (ROS) is also a contributing factor for the development of depression (Bhatt et al. 2020). Accordingly, a recent study demonstrated that a selenoindolizine structurally similar to MeSeI showed scavenger activity of the 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical at low concentrations, indicating the efficacy of chalcogenylindolizines as antioxidants (Penteado et al. 2019).

The MeSeI is a hybrid molecule that includes an indolizine nucleus, selenium, and a methyl substituent. The hybridization process aims to unite the pharmacological actions of each component and can also introduce complementary physicochemical properties, producing drugs with greater binding affinity to their target site, and intensifying their pharmacological action (Alkhzem et al. 2022). The MeSeI also presents the methyl substituent in its structure, and this component can also modulate the physicochemical, pharmacodynamic, and pharmacokinetic properties and improve the metabolic stability of the compound (Lian et al. 2013; Sun and Fu 2018). The results obtained in this study demonstrated that MeSeI presents an antidepressant-like effect in both FST and TST in mice from the low dose of 1 mg/kg i.g.

There are different neural pathways that are associated with the neurophysiology of depression, among them, the hypofunction of the serotonergic system (Malhi and Mann 2018). Serotonin is a crucial factor in the pathogenesis of depression and a critical mediator in regulating the efficacy of antidepressant agents. In general, antidepressants that act through the serotonergic system seek mainly to increase the extracellular concentrations of serotonin, acting on the different subtypes of serotonergic receptors, via the neurotransmitter reuptake in the synaptic cleft or its degradation (Köhler et al. 2016).

In addition, to explore the influence of the serotonergic system on the antidepressant-like activity of MeSeI, a simple protocol was adopted by decreasing the serotonin availability in the brain by inhibiting its synthesis with p-CPA. The enzyme tryptophan hydroxylase is the key enzyme in the synthesis of this neurotransmitter in the CNS, as it converts tryptophan into 5-hydroxytryptophan, an immediate precursor of serotonin. The administration of p-CPA inhibits tryptophan hydroxylase and reduces the levels of this monoamine in rodent brains (Koe and Weissman 1966; Redrobe et al. 1998).

In the present study, the involvement of the serotonergic pathway in the antidepressant-like effect on MeSeI was demonstrated since p-CPA-induced serotonin depletion blocked the decrease in the total immobility time induced by MeSeI on the FST. On the other hand, p-CPA did not block the increase in the latency for the first episode of immobility of mice in the FST induced by MeSeI. However, the total immobility time is the main parameter in the FST test (Nasehi et al. 2019; Qu et al. 2021; Szopa et al. 2022). Importantly, the animals that received p-CPA associated with canola oil showed no statistical difference compared to the control group in both parameters, demonstrating that p-CPA did not induce depressive-like behavior per se at this dose.

The present study showed that the antidepressant-like action of MeSeI is mediated by 5-HT2A/2C and 5-HT4 receptors, since the antagonists of these receptors, ketanserin, and GR113808, respectively, prevented the anti-immobility effect of MeSeI in the FST. The 5-HT2A receptors, widely distributed in the CNS, are considered one of the most important therapeutic targets in the treatment of depression, as they act as modulators of the cognitive process by interacting with other receptors, such as the 5-HT1A receptor (Borroto-Escuela et al. 2021; Zięba et al. 2021). In addition, dysfunction of the 5-HT2C receptor was demonstrated to be implicated in depressive conditions (Chagraoui et al. 2016), providing a valuable pharmacological target for the screening of new antidepressant agents. However, preclinical tests have presented contradictory effects, as they demonstrate the capacity of 5-HT2C agonists and antagonists to attenuate depressive-like behavior in animal models (Rosenzweig-Lipson et al. 2007; Dekeyne et al. 2008). Rosenzweig-Lipson et al. (2007) showed that the compound WAY-163909, with agonist properties for the 5-HT2C receptor, presented effects comparable to Selective Serotonin Reuptake Inhibitor (SSRI) drugs, decreasing the immobility time in the FST in animals. Furthermore, different 5-HT2C receptor agonists have been shown to be active with antidepressant activity in different preclinical models (Martin et al. 1998; Berg et al. 2008). Curiously, the pharmacological treatment for depression may be mediated by 5-HT2C receptor antagonists (Villas-Boas et al. 2021), and in preclinical models, it can be observed that the use of antagonists RS102221 and SB242084 reduces the immobility time in the FST in acute and subchronic treatment for depression (Opal et al. 2014). Further studies focused on the 5-HT2C receptor are necessary to elucidate this contradiction.

The 5-HT4 receptors are good targets for new antidepressant candidates, as agonists of these receptors produce behavioral and biochemical responses in the hippocampus (increased expression of neuroplasticity-related proteins, CREB phosphorylation, and neurogenesis, for example) compatible with responses to treatment with SSRIs (Rebholz et al. 2018; Murphy et al. 2021). Preclinical studies corroborate that 5-HT4 receptors play a critical role in depression. There are indications that antidepressants like fluoxetine seem to depend on this receptor to obtain a satisfactory action (Amigo et al. 2016). Activation of 5-HT4 receptors promotes several intracellular changes, such as increased levels of cyclic adenosine monophosphate (cAMP), activation of protein kinase A and transcription of brain-derived neurotrophic factor (BDNF) that are associated with the antidepressant response (Samuels et al. 2016).

By integrating in vivo tests with in silico tools, it was possible to investigate if MeSeI could interact with 5-HT2A, 5-HT2C, and 5-HT4 receptors. According to molecular docking, MeSeI showed a lower affinity with the 5-HT2A receptor compared with the 5-HT2C and 5-HT4 receptors. In the 5-HT2C receptor docking study, MeSeI makes Pi-alkyl hydrophobic interactions with Val215, Leu209, Ala222, Ile142 e Phe223. The hydrophobic contact with Val215, Leu209 and Phe223 was already described and could be relevant to the action of a 5-HT2C agonist (Zuo et al. 2007). These ring-stacking interactions are further supplemented by contacts with a cluster of other nonpolar residues, including Leu209, Ala222, and Ile142 (Yuen et al. 2020).

The structural data related to the 5-HT4 receptor are scarce. However, through homology modeling, MeSeI demonstrated strong affinity with the 5-HT4 receptor, with mostly hydrophobic interactions. MeSeI phenolic rings make Pi-Pi-Stacked interactions with Tyr57 and Phe64 residues and Pi-Sigma interactions with Val44 residue. Based on these observations, it is plausible to suggest that MeSeI phenolic rings are significant to the interaction with the receptor. Furthermore, based on the results, we propose that a hydrophobic pocket may exist within the region demonstrated in Fig. 8a, which may be relevant to the MeSeI affinity towards the 5-HT4 receptor.

The modulation of the serotonergic system in the antidepressant-like effect of MeSeI may be associated with the presence of selenium in its structure, since this micronutrient has a modulatory role in several neurotransmitter systems, including the serotonergic pathway (Wang et al. 2018). Different studies show that organoselenium compounds have antidepressant-like properties through the modulation of the serotonergic system, especially the 5-HT1A, 5-HT2A/2C, and 5-HT3 receptors or by MAO inhibition (Pinto Brod et al. 2016; Fronza et al. 2017; Besckow et al. 2020). In addition, indolizine derivatives have demonstrated pharmacological properties through the modulation of different receptors, such as serotonergic, cannabinoid, cholinergic, or neurokinin A and B (Bermudez et al. 1990; Millet et al. 2002; Xue et al. 2016; Belal 2018).

In the present study, the evaluation of the acute oral toxicity of MeSeI (300 mg/kg, i.g.) in female mice showed no mortality of the animals, signs of general toxicity and alteration in the analyzed biochemical parameters. MeSeI did not cause acute liver or kidney toxicity because it did not alter AST and ALT enzymatic activities nor elevate plasma levels of urea. Furthermore, the compound did not change the water and food intake and the locomotor activity of the mice. The acute toxicity experimental protocol was performed by other researchers, elucidating those different organoselenium compounds that are promising therapeutic strategies for not triggering toxicity in animals at the doses recommended by the OECD guidelines (Jesse et al. 2010; Gall et al. 2020; Carraro Junior et al. 2021).

The mice that received oral administration of MeSeI (300 mg/kg, i.g.) did not lose weight, although they gain less weight than the control group. However, these alterations can also be observed for other drugs. For example, the treatment with antidepressants that modulate the serotonergic system can produce weight loss or gain, such as drugs that act by inhibiting serotonin reuptake (Gill et al. 2020). Fluoxetine, a commercially established SSRI, can induce weight loss during an acute period of treatment (Serretti and Mandelli 2010) or long-term weight gain, thus requiring weight monitoring throughout drug administration (Arterburn et al. 2016; Gill et al. 2020). Furthermore, another parameter altered in the investigation of the acute oral toxicity of MeSeI (300 mg/kg, i.g.) was the exploratory activity of the treated group in relation to the control one. It is noteworthy that the OFT is a behavioral test also used to research new drugs with anxiolytic properties (Nie et al. 2017; Bao et al. 2021) and, under these circumstances, more studies are needed to better understand this effect.

This study presents some limitations. It was demonstrated through behavioral tests that 5-HT2A/2C and 5-HT4 receptors are involved in the antidepressant-like effect of MeSeI, and the docking analysis suggested that MeSeI could interact with these receptors. However, the interaction of MeSeI with these receptors remains to be confirmed through binding studies. In addition, other possible effects of MeSeI on serotonergic systems, such as modulation on serotonin transporter or 5-HT release need to be investigated. This study pointed out the acute antidepressant-like effect of MeSeI and does not provide the effects of chronic treatments with MeSeI and does not bring results to solve one of the main problems of current antidepressants that is the long time to achieve effects in humans, although the translation of the effects from mice to humans is difficult. Although many studies screened the antidepressant-like effects of new compounds only in male mice, this is also a limitation, and the antidepressant-like effect of MeSeI should also be demonstrated in female mice in future investigations.

Conclusion

In summary, the results of this study show the antidepressant-like effect of oral administration of MeSeI in male mice and the involvement of the serotonergic system, more specifically, the 5-HT2A/2C and 5-HT4 receptors in this effect. MeSeI also presented a low potential to cause adverse effects. Furthermore, more studies are needed to investigate other mechanisms involved in the antidepressant-like effect of this selenoindolizine.