1 INTRODUCTION

Stress, formerly defined as “general adaptation syndrome”, refers to the struggle of an organism to adapt to changing situations, including physiological, psychological and social factors (Szabo et al., 2017). Stress can have a significant impact on the entire organism, resulting in organic damage to the central nervous system and a decline in cognitive abilities. These factors, referred to as stressors, can lead to changes in the levels of various hormones and neurotransmitters (de Quervain et al., 2009; Swanson et al., 2005). Stressors not only cause acute changes but also long-term adaptive changes. Stress-induced overstimulation of the hypothalamic–pituitary–adrenal (HPA) axis results in excessive cortisol secretion. This has been linked to chronic inflammatory diseases, metabolic disorders, anxiety and depression (Caraci et al., 2010; de Quervain et al., 2009).

Chronic stress affects the adrenergic system, particularly the prefrontal cortex, amygdala, hippocampus and hypothalamus in the central nervous system. Studies have demonstrated the impact of chronic stress on affective state and affective memory (d'Alessio et al., 2013; de Almeida et al., 2012; de Quervain et al., 2009; Eddin et al., 2021; Zhang et al., 2019). Chronic stress can lead to major depressive and anxiety disorders. Anxiolytic and antidepressant treatments are commonly used to treat these disorders (Sánchez-Vidaña et al., 2017; Swanson et al., 2005). However, treatment for stress-related disorders can be prolonged and may result in side effects such as weight gain, sexual dysfunction and insomnia (Caraci et al., 2010; Qureshi & Al-Bedah, 2013). However, approximately 30% of the patients said they did not benefit from these treatments (Caraci et al., 2010). Aromatherapy has been traditionally used in almost all cultures to manage stress and mood disturbances. Arias and Laca reported that citrus species have had therapeutic properties since ancient times and are still used therapeutically in almost all Mediterranean societies (Arias & Ramón-Laca, 2005).

D-limonene, also known as 4-isopropenyl-1-methylcyclohexene (C10H16), is a monocyclic monoterpene compound that is found in plants of the citrus family. It is commonly used as an additive in cleaning, cosmetics and food products due to its citrus scent (Anandakumar et al., 2021). Recent studies have shown that monoterpenes, including D-limonene, have various pharmacological effects (Anandakumar et al., 2021; da Silva et al., 2021; Eddin et al., 2021). Previous studies show it has beneficial anti-inflammatory, anticancer, gastroprotective and neuroprotective properties (Anandakumar et al., 2021, da Silva et al., 2021, Eddin et al., 2021).

The studies indicate that plants containing D-limonene and limonene have effects on the central nervous system (Boiangiu et al., 2020; de Almeida et al., 2012; Eddin et al., 2021; Fukumoto et al., 2006; Sánchez-Martínez et al., 2021; Tang et al., 2019; Yun, 2014; Zhang et al., 2019). Lorigooini et al. reported that it showed antidepressant-like effects by reducing nitrite levels and inflammation in mice with maternal separation stress (Lorigooini et al., 2021). Studies have shown that D-limonene decreases locomotor activity in methamphetamine-induced rats by affecting 5-HT receptors and dopamine release (Yun, 2014). It has also been reported to have anxiolytic effects (de Almeida et al., 2012).

In this study, we aimed to investigate the effects of D-limonene, which has been suggested to have anti-inflammatory effects, on long-term stress-causing chronic inflammation. Therefore, we evaluated the effects of D-limonene on brain hippocampus tissue and cognitive functions in male rats subjected to restraint stress.

2 MATERIALS AND METHODS

2.1 Animals and experimental procedure

Adult male Sprague Dawley rats were obtained from the Giresun University animal research facility. Rats were housed in polypropylene cages (40 × 25 × 16 cm) with food and water ad libitum. Animals were housed in a cage and maintained on a 12 h light/dark cycle in a temperature (25 ± 1°C) and humidity (50–55%) controlled environment. All experiments were carried out in accordance with the National Guidelines for the Care and Use of Mammals in Animal Research, and experimental protocols were approved by the university animal ethics committee (2020/33).

Animals were divided into five groups (n = 8): i) control (C), ii) D-Limonen (Lim), iii) stress (Str), iv) stress+ D-Limonene (Str + Lim) and v) stress+ Fluoxetine group (Str + Flx). D-limonene of 97% purity a dose of 10 mg/kg was selected (d'Alessio et al., 2013; Song et al., 2021), and a fresh solution of D-limonene was prepared daily by dissolving in 0.25% Tween-80 (Polyoxyethylene 80 sorbitan monooleate) in water (Song et al., 2021). A dose of 10 mg/kg was selected for fluoxetine from previous studies (Misztak et al., 2021). Fluoxetine hydrochloride was dissolved in water and a freshly prepared solution was administered orally via gavage (Yang et al., 2017). All oral administrations were set at a volume of 0.1 ml per 100 g of body weight. The restraint stress protocol has been modified from a previous study (Sántha et al., 2015). The rats were habituated for one week to acclimate to the laboratory conditions. The experiment employed a cylindrical-shaped restrictive apparatus with a 7 cm diameter and a 23 cm length made of plexiglass materials. Animals were restrained for 5 h between 10:00 a.m. and 3:00 p.m. for 21 consecutive days. During the restraint period, animals were also not allowed to eat or drink. Figure 1 presents a timeline of all experimental stages in the study.

3 RESULTS

3.1 Behavioural test results

At the beginning of the behavioural tests, the sucrose preference test was conducted. The results showed a significant difference between the groups in SPT results (F(4, 35) = 33.08, p < 0.001). The sucrose preference in the Str group was almost equal to tap water (58.7 ± 4.3%), while it increased in the Str + Lim group (84.8 ± 1.3). Post-hoc evaluations did not reveal any difference between the C and Lim groups. The group treated with Str showed a significant decrease compared to both the C group (mean diff = −34.7 ± 3.4, p < 0.001) and the Lim group (mean diff = −32.8 ± 3.4, p < 0.001). However, there was a significant difference between the Str group and the Str + Lim group (mean diff = −26.1 ± 3.4 [p < 0.001]) as shown in Figure 2.

The OFT was conducted as the second behavioural test, and the results are presented in Table 1. The table shows significant differences between groups in the number of distances travelled [F(4,35) = 5.70, p = 0.001], rat speed [F(4,35) = 5.91, p = 0.001], hypothetically divided platform crossing line counts [F(4,35) = 4. 14, p = 0.008], mean distance from the walls [F(4.35) = 7.44, p < 0.001] and absolute turn angle of the animal's body [F(4,35) = 3.98, p = 0.009].

TABLE 1. Effect of D-limonene on open field test results in male rats subjected to chronic restraint stress.

	Groups (n = 40)
	Control (n = 8)	Lim (n = 8)	Str (n = 8)	Lim + Str (n = 8)	Str + Flx (n = 8)	p
Traveled distance (m)	10.68 ± 1.36	11.40 ± 1.45	3.56 ± 0.96	8.01 ± 1.71	8.22 ± 0.63	=0.001
Speed (cm/sec)	3 ± 0.4	4 ± 0.5	0.9 ± 0.3	2 ± 0.6	2 ± 0.2	=0.001
Crossing lines (count)	102.0 ± 13.36	95.6 ± 13.16	35.63 ± 8.31	74.38 ± 14.59	82.25 ± 13.55	=0.008
Mean distance from walls (m)	10.28 ± 1.23	10.68 ± 1.34	2.46 ± 0.91	7.78 ± 1.63	8.14 ± 0.64	<0.001
Absolute turn angle (deg)	6464.3 ± 1239.8	7176.7 ± 916.1	2233.2 ± 626.4	5627.7 ± 1045.4	5133.1 ± 812.7	=0.009

• Note: All data are expressed as mean ± SEM. Abbreviations: Lim, D-limonene; Str, stress; Flx, fluoxetine; deg, degree.

Object exploration times and discrimination indexes were recorded and calculated in the training session, which is the first course of the test. As seen in Figure 3a, there was no significant difference in time to explore object 1 and object 2 between the groups in the training session (p > 0.05). Otherwise, when comparing object exploration times between the groups, it was found that the Str group duration was lower than the rest. During the testing session (Figure 3b), all groups increased the time to recognize the novel object except the Str group. Comparing the discrimination indexes between the groups, there was no significant difference in the training session (p > 0.05) (Figure 3c), However, statistical differences were observed between the groups in the test session [F(4,35) = 3.81, p = 0.011] (Figure 3d). The post-hoc test results showed that the Str group (48.6 ± 5.5) was lower than the C (62.8 ± 9.8) and the Lim group (60.5 ± 3.8) respectively, p = 0.008 and p = 0.033. Although Str + Lim (58.2 ± 11.3) and Str + Flx group (58.6 ± 5.2) had higher discrimination index values than Str group, the difference was not statistically significant (p > 0.05).

The final behavioural test was the FST. Movement scores, which express the subject's struggling behaviour on the water surface, and the number of freezing episodes, which are used in the depression scale as a sign of self-despair, were recorded in the FST. Despite the lower movement score in the Str group (116.8 ± 8.3) compared to the C (133.0 ± 6.3) and Lim (128.7 ± 4.6) groups, there was no statistically significant increase in the number of freezing episodes (p = 0.078).

3.2 ELISA results

Following completion of all behavioural tests, the animals were sacrificed and the levels of BDNF, IL-1β, caspase-1 and IL-6 in hippocampal tissues were measured using the ELISA method. There were significant differences in BDNF levels between the groups [F(4,35) = 10.05, p < 0.001]. As seen in Figure 4a, we found that the BDNF level was significantly decreased in the Str group compared to the C. However, the Lim group had increased BDNF levels compared to the Str group (p < 0.05). In addition, the BDNF level, which is decreased in the Str group was, elevated with D-limonene administration in the Str + Lim group (Figure 4a).

There was a significant difference in IL-1β levels between the groups [F(4,35) = 10.95, p < 0.001]. Post-hoc test results revealed that in the Str group IL-1β levels (5.33 ± 0.42) were higher than the C group (3.16 ± 0.41) and Lim group (2.85 ± 0.24). On the other hand, in the Str + Lim group, IL-1β levels (4.07 ± 0.1) were lower than in the Str group (5.33 ± 0.42). These results indicate that D-limonene administration reduces IL-1β production in the hippocampus tissue (Figure 4b). These results were confirmed by the caspase-1 levels in hippocampus tissue. As seen in Figure 4c, we found that caspase-1 levels in the Str group increased compared to the C group. The increase in Str results was reversed in the Str + Lim group levels of caspase-1 in the Str + Lim group (0.55 ± 0.06) were significantly higher (p = 0.009) than those in the Lim group (0.32 ± 0.04). However, there were no significant differences between the groups when considering IL-6 levels in hippocampus tissue (p > 0.05).

4 DISCUSSION

D-limonene is a monoterpene compound found in the citrus family and some studies have shown it to have neuromodulatory, antidepressant, anxiolytic and antinociceptive effects (d'Alessio et al., 2013; Fukumoto et al., 2006; Gu et al., 2019; Yun, 2014; Zhang et al., 2019). In this study, we aimed to evaluate the effects of D-limonene administration on depressive-like behaviour and learning memory under restraint stress in rats. Our findings were confirmed by the administration of fluoxetine, which washed out the effects of the restraint stress model in both behavioural and biochemical tests.

Anhedonic behaviour is the inability to experience pleasure from reward and is a core feature of depression (Liu et al., 2018). The sucrose preference test was used to measure anhedonic behaviour in this study. Our results showed that the reduced sucrose consumption in the Str group was reversed by D-limonene administration (Figure 2). Similar to the findings of Zhang et al., our results also demonstrated that the administration of citrus sinensis, the main constituent of which is D-limonene, increased sucrose consumption. The study concluded that D-limonene has antidepressant properties in mice subjected to chronic unpredictable mild stress. The study shows that D-limonene had a beneficial effect in reversing the reduction of corticosterone and corticosterone-releasing factor in the hippocampus and reversing the reduction of BDNF in the hippocampus (Zhang et al., 2019). Similarly, our results demonstrate that chronic restraint stress-induced decreases in hippocampal BDNF levels were reversed in the Str + Lim group (Figure 4a). In addition, BDNF levels in the Str + Lim group were similar to those in the control and Str + Flx groups. These findings indicate that D-limonene may have a protective effect against stress-induced neurodegeneration. BDNF plays a pivotal role in synaptic plasticity development and neuronal survival. It is known to be involved in the pathophysiology of acute and chronic stress and the associated disorders. It has been demonstrated that BDNF levels decrease in serum and the prefrontal cortex and hippocampus in stress disorders (Tripp et al., 2012). Brivio et al. reported that chronic restraint stress decreased BDNF levels and even chronic restraint stress was effective on acute stress induced as a second hit. It is understood that the lasting effects of restraint stress are mediated through BDNF by affecting the intracellular cascade of Tropomyosin receptor kinase B (TRKB), PI3K-AKT and the MEK-MAPK/ERK pathways (Brivio et al., 2020).

Several studies have demonstrated the effects of the citrus family on other neurotransmitters, with a particular focus on monoamines. Citrus essential oil had effects on monoamine release in the striatum and cortex of the rat brain (Fukumoto et al., 2006). The striatum is known as an important region for dopaminergic neurons. Dopamine mediates the effects of stimuli related to reward in the ventral striatum (Robbins & Everitt, 1992). Both animal and human studies have shown that striatal dopamine levels play a crucial role in emotional processing. It is well-established that adenosine A2A receptors regulate the circadian oscillation of corticosterone and restore stress-related decreased levels in the hippocampus (Batalha et al., 2013). D-limonene reduces anxiety-related behaviour by regulating dopaminergic and GABAergic neuronal activity in the striatum through adenosine A2A receptors (Song et al., 2021). Yun demonstrated that D-limonene alters the activity of dopamine and serotonin 5-HT receptors, inhibiting the behavioural changes induced by methamphetamine-like stimulants (Yun, 2014). Antioxidants can prevent methamphetamine-induced neurotoxicity that is associated with the activation of 5-HT receptors. D-limonene may affect hydroxytryptophan receptors and dopaminergic activity due to its antioxidant properties (Anandakumar et al., 2021; Boiangiu et al., 2020; Eddin et al., 2021). As previously stated, D-limonene's antioxidant properties may help in the treatment of various mood disorders.

In the OFT, distance travelled, average speed and number of passes through virtually separated sections decreased in the Str group compared to the C group (Table 1). The OFT is commonly used to assess exploratory behaviour and general activity in rodents (Crusio, 2001). The results of the OFT showed a decrease in absolute turning angles and mean distance from the platform wall in the Str group. Time spent in the central area of the platform is associated with exploratory behaviour in the OFT, whereas time spent near the sides and corners of the platform is considered to indicate defensive behaviour. Rodents prefer to stay close to the platform wall as an indicator of defensive behaviour, and this effect is called thigmotaxis behaviour, but they also need to explore the new environment (Seibenhener & Wooten, 2015; Simon et al., 1994). Another measure of exploratory behaviour is the angle of rotation of the rodent during its orientation attempts in the environment (Seibenhener & Wooten, 2015). Our results showed that the total angle of rotation and the mean distance from the platform wall decreased in the Str group. D-limonene administration increased both of these measures in the Str + Lim group. According to the OFT results, D-limonene decreased defensive behaviour and increased exploratory behaviour on the platform. Our results suggest that the administration of D-limonene reduces the defensive behaviour induced by chronic restraint stress.

In aromatherapy, essential oils are often used to alleviate anxiety symptoms caused by stress (Anandakumar et al., 2021, Eddin et al., 2021). Almeida et al. reported that the administration of D-limonene epoxide has an anxiolytic-like effect. These results suggest that D-limonene epoxide may have potential as an anxiolytic agent. The study found a reduction in stereotypical movement and the number of crossings in the OFT, as well as an increase in the time spent with open arms in the elevated plus maze test. However, flumazenil, a benzodiazepine receptor antagonist, reversed these effects (de Almeida et al., 2012). It is understood that benzodiazepine-type receptors mediate the anxiolytic-like effects of D-limonene. Another study by Almeida et al. revealed that the anxiolytic effect was reversed by flumazenil, similar to diazepam. This suggests that D-limonene epoxide and diazepam may have identical effects. The study used an anxiogenic procedure and also evaluated the antioxidant capacity in the hippocampus tissue of mice. The administration of D-limonene epoxide led to a 50% reduction in nitrite ions, hydroxyl radicals, lipid peroxidation and the production of thiobarbituric acid-reactive compounds. Moreover, in the hippocampi of mice, D-limonene epoxide increased the activity of antioxidant enzymes such as catalase and superoxide dismutase (de Almeida et al., 2014).

The NORT is a memory test that utilizes the exploratory behaviour of the subjects. Rodents tend to recognize novel objects rather than familiar ones, during the testing session. Our findings showed that the Str group was less interested in the objects than the control and Lim groups during the training session. Also, the test showed that the Str group has less interest in exploring novel objects compared to the Str + Lim group in the testing session (Figure 3b). The study found that restraint stress caused cognitive impairment in rats, affecting their short-term working memory, characteristic exploratory features, spatial learning and memory performance. However, the administration of D-limonene reversed this impairment and improved spatial memory. These results suggest that D-limonene may have the potential as a treatment for stress-induced cognitive decline. Bigdeli et al. demonstrated that administering D-limonene improved spatial memory in rats undergoing immobilization stress, as demonstrated by the Morris water maze test. Additionally, the authors observed a reduction in stress-induced neuronal loss in hippocampal CA1 neurons with D-limonene treatment (Bigdeli et al., 2019). These results can be explained by processes that affect the hippocampal neurotransmitter or the neuroprotective mechanisms of D-limonene. The cholinergic system, which mediates hippocampus-dependent learning, is responsible for the formation of both episodic and semantic memory (Haam & Yakel, 2017). Yadav et al. demonstrate that citrus lemon juice has neuroprotective potential against scopolamine-induced amnesia in rats. This neuroprotective effect alters hippocampal activity by affecting cholinergic neurotransmission (Yadav et al., 2022).

Proinflammatory cytokines such as TNF-α, IL-1 and IL-6 play a crucial role in the behavioural and cognitive effects of stress responses (Cunningham et al., 1996; Frank et al., 2020; Goshen & Yirmiya, 2009). IL-1 plays a crucial role in the behavioural effects of stress and acts as a ‘gatekeeper’ of neuroinflammation (Basu et al., 2004). In this study, we showed that IL-1β and caspase-1 levels were elevated in the Str group, while BDNF levels decreased in hippocampal tissue. “pyroptosis”, a form of inflammation-induced cell death, is caused by cytokine release and involves the activation of caspase-1, maturation of Il-1 and release of IL-1 into the extracellular space (Frank et al., 2020). Impaired spatial/contextual memory is caused by increases in IL-1 and caspase-1 levels in the hippocampus, which are driven by immunological challenges under conditions of stress induction (Frank et al., 2020, Goshen & Yirmiya, 2009). Previous studies have shown that IL-1β and IL-18 reduce long-term potentiation in the dentate gyrus (Curran et al., 2003; Curran & O'Connor, 2001). IL-1ra blocked its ability to reduce NMDA receptor-mediated potentials. Caspase-1, which is induced by pyroptosis, initiates both IL-18 and IL-1β (Curran et al., 2003; Curran & O'Connor, 2001; Frank et al., 2020; Goshen & Yirmiya, 2009). In our study, we found that D-limonene decreased IL-1β and caspase-1 levels in the Str + Lim group compared to the Str group in the hippocampus tissue, as shown in Figure 4b. It is suggested that D-limonene reduces neuroinflammation at a dose of 10 mg/kg.

Clinical and animal studies have demonstrated that stress can lead to an increase in IL-6 levels. Elevated IL-6 may result in dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis, alterations in synaptic neurotransmission and a decrease in neurotrophic factors (Rohleder et al., 2012) (Ting et al., 2020). Although some clinically effective antidepressants have been shown to reduce IL-6 levels, tricyclic antidepressants have been found to have little effect on serum IL-6 levels in individuals with major depressive disorder. Studies investigating the genetic relationship between IL-6 and major depressive disorder have produced contradictory results (Ting et al., 2020). Similarly, in our study, although an elevated level of IL-6 was observed in the stress group, no statistically significant difference was found between the limonene groups and the control group (Figure 4d).

In our study, we found that chronic restraint stress led to increased levels of IL-1β and Caspase-1, as well as decreased levels of BDNF. However, these effects were reversed with D-limonene administration in the Str + Lim group, similar to the Str + Flx group as shown in Figure 4b. It is also thought that D-limonene at a dose of 10 mg/kg reduces neuroinflammation and may contribute to the stress-induced memory impairment that is prevented by the administration of D-limonene as shown in the NORT study.

Lorigooini et al. found that administration of 20 mg/kg D-limonene reduced neuroinflammation, while no significant effect was observed at a dose of 10 mg/kg. They reported that D-limonene decreased the expression of IL-1β, TNF and NO levels. These results suggest that D-limonene has an antidepressant-like effect by reducing neuroinflammation during maternal separation stress in mice (Lorigooini et al., 2021). However, our results contradict the previous study where D-limonene was co-administered with a non-selective nitric oxide synthase (NOS) inhibitor L-NAME, at a dose of 10 mg/kg, which was defined as sub-effective, and it was stated that L-NAME potentiated the effects of D-limonene. In our study, we found that a 10 mg/kg dose of D-limonene was effective even without the co-administration of L-NAME. Also, the use of L-NAME to investigate whether D-limonene's effect on the stress response is mediated via the nitric oxide pathway makes it difficult to understand the effects of D-limonene on the nitric oxide pathway. A number of studies have shown that different NOS inhibitors exert their effects on the stress response through many different pathways (Coelho et al., 2022; Gilhotra et al., 2010; Joung et al., 2012). Joung et al. explained that nitric oxide synthase inhibitors (NOS) have diverse effects on stress responses and that NO inhibition may be responsible for the differential beneficial effects on stress regulation. Pretreatment with L-NAME or 7-nitro-indazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase, significantly reduced the stress-induced anxiety response. L-NAME also reversed the stress-induced increase in plasma corticosterone and the NO metabolites. However, administration of 7-NI, but not L-NAME, reversed stress-induced NO in the paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus, accompanied by a decrease in NADPH-d reactivity in the PVN and lateral dorsal tegmental nucleus (Joung et al., 2012). In addition, iNOS decreased the anxiogenic effect of restraining stress by acting on endocannabinoid signalling in the prefrontal cortex (Coelho et al., 2022).

5 CONCLUSION

Administration of D-limonene reduced the defensive and anhedonic behaviours induced by restraint stress, as observed in behavioural tests. D-limonene administration also improved the results of learning and memory tests in the male rats. In addition, it was found that D-limonene reduced caspase-1 and IL-1β levels while increasing BDNF levels. The results indicate that D-limonene could enhance neurogenesis by reducing pyroptosis and neuroinflammation while increasing neurotrophic factors, including BDNF, in brain tissue. Our results demonstrate that D-limonene displays antidepressant properties and improves learning and memory.

AUTHOR CONTRIBUTIONS

Mehmet Alkanat: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Writing original draft, Writing - review & editing. Hafize Özdemir Alkanat: Investigation, Methodology, Project administration, Validation, Writing - review & editing.

DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA SHARING STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. The data that support the findings of this study are available on request from the corresponding author. Some data are licensed from the software company (e.g., ANY-maze Dublin/Ireland), and third-party permission is required. The datasets produced by this software are subject to licensing restrictions.