Exploring the Anxiolytic, Antidepressant, and Immunomodulatory Effects of Cannabidiol in Acute Stress Rat Models

Abstract

Cannabidiol (CBD), a non-psychoactive compound derived from Cannabis sativa, is believed to have anxiety-reducing and antidepressant effects. However, existing data are inconsistent, likely due to variations in experimental designs, dosages, and stress models. This study sought to assess the impact of CBD on anxiety and depression-like behaviors in Wistar rats exposed to acute cold stress, as well as its impact on pro- and anti-inflammatory cytokines. Male rats were treated with CBD (2.5, 5, or 10 mg/kg) or vehicle for 14 days and subjected to behavioral tests, including the elevated plus maze, social interaction, and forced swim tests. Serum levels of cytokines (IL-6, TNF-α, IL-1β, and IL-10) were analyzed post-experiment using ELISA. Results demonstrated a dose-dependent anxiolytic effect of CBD, with significant improvements in social interaction and reductions in anxiety-like behaviors at 5 and 10 mg/kg. All doses of CBD decreased immobility in the forced swim test, suggesting antidepressant effects. Furthermore, CBD selectively lowered IL-6 levels, a key cytokine in acute stress and depression pathogenesis. These findings indicate that CBD has anxiety-reducing and antidepressant properties, partially mediated by modulation of inflammatory processes, particularly IL-6.

1. Introduction

The complex interplay between stress, depression, and systemic inflammation has emerged as a key focus in behavioral and biomedical research due to its profound implications for mental health and overall physiological well-being. Acute and chronic stress can disrupt the hypothalamic–pituitary–adrenal (HPA) axis and immune homeostasis, leading to altered cytokine profiles and neuroinflammatory responses that contribute to the development of depressive disorders. Understanding these interconnected pathways is essential for identifying effective therapeutic interventions.

Cannabidiol (CBD), a non-intoxicating phytocannabinoid sourced from Cannabis sativa, has emerged as a promising neuromodulatory and anti-inflammatory agent in this context [1,2]. Unlike its psychoactive counterpart Δ-9-tetrahydrocannabinol (THC), CBD does not produce psychotropic effects but exerts its therapeutic actions through diverse mechanisms. These include modulation of the endocannabinoid system via cannabinoid receptors (CB1 and CB2), activation of transient receptor potential (TRPV) channels, interaction with serotonin (5-HT1A) receptors, and inhibition of enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) [3,4]. These mechanisms underpin its anxiolytic, antidepressant, and immunomodulatory effects, as demonstrated in both preclinical and clinical studies.

One key aspect of CBD’s therapeutic potential lies in its ability to attenuate systemic inflammation, a hallmark of stress-related disorders [5,6]. Elevated levels of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), have been associated with the pathophysiology of depression and cognitive dysfunction. Stress-induced immune activation exacerbates this inflammatory cascade, perpetuating a vicious cycle of neuroinflammation and depressive symptoms [7]. CBD has been shown to counteract these processes by modulating cytokine production, enhancing adenosine signaling, and reducing oxidative stress [2]. Moreover, the anxiolytic and neuroprotective properties of CBD might normalize the dysfunction of the HPA axis, one of the hallmarks of stress-related disorders [3].

Despite promising preclinical findings, the clinical application of CBD in stress-related disorders remains limited due to challenges such as variability in product formulations, inconsistent dosing protocols, and a scarcity of comprehensive studies. Few investigations have integrated behavioral, neuroendocrine, and immunological outcomes to offer a comprehensive understanding of CBD’s therapeutic potential [1,4].

This study aims to address these gaps by systematically evaluating the impact of CBD oil on behavioral responses to acute stress, depressive symptoms, and serum cytokine profiles in a rat model. By combining behavioral assessments with biomolecular analyses, this work seeks to clarify the mechanisms through which CBD modulates systemic inflammation and stress-related pathophysiology, contributing to the development of novel therapeutic approaches.

Our findings reveal that CBD significantly enhances social interaction, alleviates depressive-like behaviors, and selectively reduces pro-inflammatory cytokine IL-6, emphasizing its potential as a targeted therapeutic agent in stress-related disorders.

2. Materials and Methods

2.1. Chemicals and Reagents

Cannabidiol (organic hemp oil, containing 500 mg CBD in 30 mL), batch number 20116459, Dr. Biomaster Ltd., Sofia, Bulgaria; Rat TNF-α ELISA kit Cat. No. 670.000.096; Rat IL-6 ELISA kit Cat. No. 670.010.096; Rat IL-10 ELISA kit Cat. No. 670.070.096; Rat IL-1β ELISA kit Cat. No. 670.040.096 (Diaclone, Medix Biochemica, Finland).

2.2. Acute Stress Model

The anxiolytic and antidepressant effects of CBD were investigated using six-week-old male Wistar rats (approx. 200 g). The rats were housed in groups under standard laboratory conditions (12-h light/dark cycle, temperature 22 ± 2 °C, and access to food and water ad libitum). The study design included five groups (n = 8):

• Control group administered olive oil (vehicle for CBD delivery).
• Control group exposed to acute cold stress, with olive oil treatment.
• Experimental groups treated with CBD at doses of 2.5, 5, and 10 mg/kg body weight.

CBD or olive oil was administered orally once daily for 14 consecutive days. On the 15th day, 60 min after the final treatment, the animals in the stress groups were subjected to acute cold stress by being placed in a refrigerator at 4 °C for 60 min.

Behavioral Assessments

To assess the anxiety-reducing effects of CBD, the social interaction test was employed immediately after stress induction. The social interaction test measured the duration of active interaction with a conspecific, a marker of social behavior and anxiety reduction.

To evaluate antidepressant-like activity, the forced swim test (FST) was performed. Each rat was positioned in a clear cylinder (30 cm tall, 20 cm wide) filled with water at 25 ± 1 °C to a depth of 20 cm. Behavior was recorded for 5 min. Immobility (minimal movement to maintain floating) was taken as an indicator of depressive-like behavior, while reduced immobility time indicated antidepressant-like effects of CBD.

2.3. Evaluation of Cannabidiol’s Effects on Serum Cytokine Levels in an Acute Stress Model

Following behavioral testing, blood samples were obtained from the retro-orbital sinus of each rat under light isoflurane anesthesia. Blood samples were collected using pyrogen- and endotoxin-free tubes to ensure sample integrity. After allowing the blood to clot, samples were centrifuged at 3000 rpm for 10 min to separate the serum. The serum was carefully aliquoted and stored at −70 °C for subsequent analysis.

Cytokine Quantification Using ELISA

Serum levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and the anti-inflammatory cytokine (IL-10) were quantified using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instructions. The specific steps included the following:

• Diluting serum samples (1:2 for IL-10, IL-6, and IL-1β; 1:1 for TNF-α).
• Adding diluted samples, standards, and internal controls to microplate wells pre-coated with monoclonal antibodies specific to each cytokine.
• Incubating the plate at room temperature for the recommended period.
• Washing the wells thoroughly to remove unbound materials.
• Adding peroxidase-conjugated secondary antibodies to form cytokine-antibody complexes.
• Performing another wash step to eliminate unbound conjugates.
• Adding a chromogenic substrate to initiate the enzymatic color reaction.

Absorbance was recorded at 450 nm using a TECAN ELISA reader. Cytokine concentrations (pg/mL) were calculated by constructing standard curves from known concentrations of each cytokine.

2.4. Statistical Analysis

Statistical analysis was conducted using IBM SPSS Statistics version 19.0. Group differences were assessed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for pairwise comparisons. The results are expressed as the mean ± standard error of the mean (SEM), with a p-value of <0.05 considered statistically significant.

3. Results

3.1. Social Interaction Test

In the social interaction test, each rat was paired with an unfamiliar conspecific, and their behavior was monitored for 5 min. The duration of social interactions, including activities like sniffing, following, and other affiliative behaviors, was recorded. Reduced interaction time indicated anxiety-like behavior, whereas increased interaction time suggested an anxiolytic effect.

Rats in the cold-stressed control group showed a significant reduction in social interaction time compared to the non-stressed control group, indicating increased anxiety due to stress (p < 0.05). Treatment with CBD at doses of 5 mg/kg and 10 mg/kg body weight significantly enhanced social interaction times compared to the stressed control group (p < 0.05) (Figure 1). These results suggest that CBD effectively alleviates stress-induced anxiety, with more noticeable anxiolytic effects at higher doses.

3.2. Forced Swim Test

FST was utilized to evaluate antidepressant-like activity. Each rat was placed in a water-filled cylinder, and behavior was monitored for 5 min. Immobility, defined as the absence of active movements other than those required for balance, was interpreted as depressive-like behavior. Reduction in immobility time indicated an antidepressant effect.

CBD treatment at all administered doses (2.5, 5, and 10 mg/kg body weight) significantly reduced immobility time compared to the control group (p < 0.05, p < 0.01, and p < 0.001, respectively). Moreover, the 10 mg/kg dose further decreased immobility time compared to the lowest dose of 2.5 mg/kg (p < 0.05). These findings suggest a dose-dependent enhancement of antidepressant activity at higher doses (Figure 2).

Although all doses demonstrated significant antidepressant effects, the findings indicate that CBD’s efficacy may not be strictly dose-dependent across the tested range. However, the substantial reduction in immobility at higher doses reinforces the therapeutic potential of CBD in alleviating depressive-like behaviors.

3.3. Cytokine Analysis

Given the established role of inflammation in stress pathogenesis and the known immunomodulatory properties of CBD, we investigated the impact of CBD on serum levels of pro- and anti-inflammatory cytokines in animals exposed to acute stress.

Cold-stressed control animals showed significantly higher serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (p < 0.05, p < 0.01, and p < 0.05, respectively), along with a notable decrease in the anti-inflammatory cytokine IL-10 (p < 0.05) compared to the non-stressed control group (Figure 3). These findings align with the inflammatory response commonly observed in acute stress.

3.3.1. Changes in Serum TNF-α Levels

In animals treated with the two lower doses of CBD, a slight but statistically insignificant reduction in serum TNF-α levels was observed compared to the cold-stressed control group. No significant difference was noted when compared to the non-stressed control group. However, in animals treated with CBD at 10 mg/kg body weight, serum TNF-α levels were significantly higher compared to the non-stressed control group (p < 0.05), with no significant difference observed when compared to the stressed control group (Figure 3A).

3.3.2. Changes in Serum IL-6 Levels

CBD treatment led to a significant reduction in serum IL-6 levels in stressed animals. The most substantial reductions were seen at doses of 2.5 mg/kg and 5 mg/kg body weight (p < 0.01 compared to the cold-stressed control group), while the 10 mg/kg dose produced a smaller but still significant reduction (p < 0.05). No significant differences in IL-6 levels were observed between the vehicle control and CBD-treated groups (Figure 3B).

3.3.3. Changes in Serum IL-10 Levels

Serum IL-10 levels in CBD-treated animals were significantly lower than those in the non-stressed control group (p < 0.05) and were similar to the levels observed in the stress-exposed control animals (Figure 3C).

3.3.4. Changes in Serum IL-1β Levels

CBD treatment had no significant impact on serum IL-1β levels. In all CBD-treated groups, IL-1β concentrations were comparable to those in the stressed control animals but remained significantly higher than those in the non-stressed control group (p < 0.01 for the 2.5 mg/kg and 5 mg/kg doses, and p < 0.05 for the 10 mg/kg dose). The 10 mg/kg dose showed a slight, though non-significant, reduction in IL-1β levels compared to the stressed control group (Figure 3D).

4. Discussion

The findings of this study provide strong evidence for the anxiety-reducing and antidepressant effects of CBD in rat models of acute stress. The observed increase in social interaction and reduction in depressive-like behaviors align with a growing body of research on CBD’s therapeutic potential, particularly its ability to modulate stress responses and emotional regulation. These findings are consistent with prior studies reporting that CBD alleviates anxiety and depression-like symptoms across various preclinical models [8,9,10]. For instance, Blessing et al. highlighted CBD’s anxiolytic effects in a wide range of stress paradigms [11], while Linge et al. demonstrated CBD’s antidepressant-like properties in mice [12]. The anxiolytic effects observed in this study, as indicated by increased social interaction, are particularly noteworthy. This finding aligns with studies such as De Gregorio et al., which reported that CBD modulates stress responses through its interaction with serotonin (5-HT1A) receptors [13]. Similarly, Zieba et al. demonstrated that CBD reduces anxiety in rodents in various behavioral tasks [14]. However, discrepancies in the literature remain, with Liu et al. noting variability in CBD’s anxiolytic effects depending on factors such as dose, stress paradigm, and animal strain [9]. These inconsistencies may be explained by the inverted U-shaped dose-response relationship of CBD, where intermediate doses are most effective, while lower and higher doses may fail to produce significant effects. The doses of 5 and 10 mg/kg used in this study appear to fall within the optimal range, producing robust anxiolytic and antidepressant outcomes. These effects are likely driven by multiple mechanisms, including the activation of serotonin 5-HT1A and CB1 receptors, as well as interactions with vanilloid TRPV1 receptors. The endocannabinoid system plays a vital role in regulating stress responses. CBD increases endocannabinoid levels, indirectly activating cannabinoid receptors. Anandamide, a major endocannabinoid, facilitates neuroadaptive changes that promote resilience to stress [10]. Additionally, local endocannabinoid levels in the ventromedial prefrontal cortex have been shown to modulate serotonergic activity, which may contribute to the antidepressant effects of CBD [15]. Notably, blocking 5-HT1A receptors has been demonstrated to prevent the antidepressant-like effects of CBD and anandamide, while blocking CB1 receptors specifically inhibits CBD’s effects [16]. The GABAergic system, another critical pathway in emotional regulation, is also implicated in CBD’s effects. Reduced gamma-aminobutyric acid (GABA) levels have been observed in patients with major depressive disorder and are normalized following chronic antidepressant treatment [17,18]. Interactions between cannabinoid receptors and GABA signaling have been documented, with changes in GABA subunits noted in CB1 knockout mice and CB2 overexpressing mice. Pharmacological studies using CB1 and CB2 receptor-targeting drugs have demonstrated altered responses to stress, anxiety, and behavioral despair, along with changes in the gene expression of GABA(A) subunits, including α2 and γ2 [19,20].

The antidepressant effects of CBD, as evidenced by reduced depressive-like behaviors, are consistent with studies showing that CBD enhances serotonergic signaling and promotes neuroadaptive changes. In FST, a single injection of CBD (30 mg/kg, ip) produced antidepressant-like effects comparable to those observed with conventional treatments such as imipramine [21]. Additionally, in the olfactory bulbectomy (OBX) model of depression, both acute and chronic administration of CBD reduced depressive-like behaviors while also reversing associated neurochemical and anatomical abnormalities [12]. Furthermore, CBD demonstrated beneficial effects in genetic models of depression, including Wistar Kyoto and Flinders Sensitive Line rats, which exhibit behavioral and physiological characteristics resembling major depressive disorder [22].

Inflammation is increasingly identified as a key factor in stress-related disorders, and the observed reduction in serum IL-6 levels in CBD-treated animals in this study underscores its immunomodulatory potential. Acute stress activates the immune system, promoting the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, while suppressing anti-inflammatory cytokines like IL-10. This imbalance significantly contributes to the onset and progression of stress-related conditions, including anxiety and depression. Stress-induced inflammation, characterized by a dysregulated immune response, has gained attention due to its profound implications for mental and physical health. TNF-α, a pro-inflammatory cytokine, is particularly pivotal in stress responses. It regulates cellular processes such as proliferation, differentiation, apoptosis, and necrosis, playing a central role in stress-induced inflammatory changes [23]. Similarly, IL-6 not only participates in immune responses but also influences metabolic, regenerative, and neuronal functions [24]. In animal models, acute stressors like cold exposure, immobilization, or forced swimming increase levels of IL-6 and TNF-α, with peak concentrations typically occurring within 30 min of stress exposure [25]. These models, which replicate depressive symptoms, demonstrate a correlation between elevated IL-6 levels and behavioral changes, such as increased immobility in the forced swim test and decreased sucrose consumption, both of which are markers of depression [26,27]. Clinical studies further confirm the involvement of pro-inflammatory cytokines in depression. A meta-analysis found significantly higher levels of IL-2, IL-6, TNF-α, and IFN-γ in patients with depressive disorders compared to healthy controls [28]. This inflammatory profile is believed to contribute to the pathophysiology of depression, emphasizing the role of inflammation in mood disorders. In contrast, the anti-inflammatory cytokine IL-10 helps regulate immune function and maintain balance by inhibiting pro-inflammatory Th1 cells and promoting autoreactive B cell differentiation [29]. Elevated IL-10 levels in acute and chronic stress models, such as forced swimming and immobilization, suggest that stress affects both pro- and anti-inflammatory responses [30]. CBD’s anti-inflammatory and immunomodulatory properties are well-documented and offer promising therapeutic potential for stress-related disorders. CBD interacts with multiple biological targets, including CB1 and CB2 cannabinoid receptors, peroxisome proliferator-activated receptors (PPARs), and GPR receptors, to modulate immune responses. Specifically, CBD negatively modulates CB1 receptors, leading to a reduction in the production of pro-inflammatory cytokines like TNF-α and reactive oxygen species (ROS). It also activates PPAR-γ, which downregulates gene expression of pro-inflammatory mediators like cyclooxygenase-2, TNF-α, IL-1β, and IL-6 [31]. Furthermore, CBD’s interaction with adenosine A2A receptors enhances its anti-inflammatory effects, illustrating its multifaceted approach to immune modulation. In this study, CBD significantly reduced serum IL-6 levels in animals exposed to acute cold stress, aligning with in vitro findings that CBD inhibits toll-like receptor (TLR)-stimulated IL-6 production [32]. Given the established link between elevated IL-6 levels, acute stress, and depression [33,34], this reduction is a key indicator of CBD’s therapeutic potential. However, CBD did not have a significant impact on serum levels of TNF-α or IL-10, suggesting a selective modulation of the inflammatory response. This specificity indicates that CBD may target aspects of inflammation, potentially providing therapeutic benefits without broadly disrupting immune function.

While the results of this study provide valuable insights, several questions remain unanswered. For instance, the mechanisms underlying CBD’s selective reduction of IL-6 but not TNF-α or IL-10 require further investigation. Additionally, the long-term effects of CBD on inflammatory and neurochemical pathways, particularly in chronic stress models, are not yet fully understood. Variability in CBD’s effects across studies underscores the importance of dose optimization and standardized experimental protocols.

Future research should focus on combining behavioral, neuroendocrine, and immunological analyses to gain a more thorough understanding of CBD’s therapeutic potential. Expanding studies to include diverse stress paradigms and clinical populations will be essential for translating these findings into practical applications. The selective modulation of inflammation and neurochemical pathways observed in this study underscores CBD’s potential as a targeted treatment for stress-related disorders, offering hope for better outcomes in conditions like anxiety and depression.

5. Conclusions

This study aimed to explore the anxiolytic, antidepressant, and immunomodulatory effects of CBD in rat models of acute stress. The results demonstrated that CBD significantly increased social interaction, reduced depressive-like behaviors, and decreased serum levels of the pro-inflammatory cytokine IL-6. These findings underscore CBD’s therapeutic potential in stress-related disorders, with its effects likely mediated by interactions with the endocannabinoid, serotonergic, and immune systems. Importantly, CBD’s selective modulation of inflammatory markers, such as IL-6, without altering TNF-α or IL-10, highlights its potential as a targeted therapeutic agent.

Future research should concentrate on optimizing dosing regimens and investigating the long-term effects of CBD in chronic stress models, and integrating behavioral, neurochemical, and immunological outcomes in both preclinical and clinical settings. These efforts will help clarify CBD’s mechanisms of action and therapeutic applications, paving the way for its development as a safe and effective treatment for stress-induced psychiatric and inflammatory disorders.