This study investigated the antidepressant-like effects of CSL in an LPS-induced depression model. The results from the SPT, TST, and FST indicated that CSL significantly reduced depressive-like behaviors. Furthermore, CSL notably modulated mast cell (MC) activation in the smLNs, dcLNs, and dura mater. Hematological analysis demonstrated that CSL effectively lowered the neutrophil-to-lymphocyte ratio (NLR), and analysis of the PFC revealed a significantly inhibited expression of pro-inflammatory cytokines like TNF-α and IL-1β.

Behavioral tests such as the SPT, TST, and FST are well-recognized tools for evaluating depression-like behaviors in preclinical models of depression. Anhedonia, characterized by a diminished capacity to experience pleasure, is a core symptom of depression and can be effectively modeled using the SPT, where a reduction in sucrose preference is indicative of anhedonia [26]. Conversely, the TST and FST are designed to assess behavioral despair, with increased immobility interpreted as a lack of escape-driven behavior, a feature commonly associated with depression [27,28]. LPS-induced depression models are widely used to replicate inflammation-related depressive conditions, as LPS administration elicits sickness behaviors, including decreased sucrose preference and increased immobility, by activating peripheral and central inflammatory responses. [29]. In our study, LPS administration led to decreased sucrose preference in the SPT and heightened immobility in both the TST and FST, aligning with its established depressive-like effects [18,30]. Treatment with CSL reversed these effects in a dose-dependent manner, restoring sucrose preference and reducing immobility, which suggests an antidepressant-like action. These results are consistent with previous research showing the efficacy of cannabinoids in mitigating inflammation-induced behavioral changes, likely through the modulation of neuroimmune pathways [14,18,31]. However, it is important to note that while a clear dose-dependent effect of CSL was observed in the FST and SPT, the TST did not demonstrate the same level of dose-dependency at the 30 mg/kg dose. This discrepancy may be attributed to the TST’s lower sensitivity to cannabinoid effects compared to the FST and SPT, which may engage distinct neurobiological pathways [17]. Additionally, pharmacokinetic factors, such as the distribution and metabolism of cannabinoids, could influence receptor activation differently across these tests [32]. Further investigations are warranted to explore these differences, with future studies incorporating additional dosing regimens and behavioral assays to more comprehensively assess the therapeutic potential of CSL.

The NLR is gaining recognition as a reliable indicator of systemic inflammation and is widely used in clinical and preclinical models to assess immune responses during stress conditions [33]. An elevated NLR reflects a disruption in immune regulation, primarily resulting from increased neutrophil activity and decreased lymphocyte levels, both indicative of heightened inflammatory responses [34]. Neutrophils function as the primary line of defense in the host immune response against pathogens, utilizing mechanisms such as chemotaxis, phagocytosis, and the release of reactive oxygen species (ROS), granular proteins, and cytokines [35]. As essential regulators of innate immunity, neutrophils recruit, activate, and modulate the functions of other immune cells through the secretion of pro-inflammatory and immunomodulatory cytokines and chemokines. This activity promotes the recruitment and activation of immune cells such as dendritic cells, natural killer (NK) cells, and CD4 and CD8 T cells [36]. In animal models, the administration of LPS is known to cause systemic inflammation, as evidenced by elevated NLR, indicating a peripheral inflammatory burden [37]. LPS activates various immune cells, including MCs and neutrophils [38,39]. Specifically, LPS binds to receptors on leukocytes, inducing the release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β [40]. NLR has been shown to change in MDD and has been associated with various factors, including chronic stress and impulsivity, both of which have been previously linked to suicidal behavior [41]. In our study, LPS administration significantly elevated NLR, indicating increased systemic inflammation. However, CSL treatment dose-dependently reduced NLR, suggesting a potential immunomodulatory effect. This reduction in NLR implies that CSL may help mitigate systemic inflammation, which plays a crucial role in inflammation-driven psychiatric disorders such as depression. Given that the endocannabinoid system regulates immune cell activity through cannabinoid receptors, it is plausible that CSL has an effect by inhibiting neutrophil activation and reducing cytokine production. While further research is needed, these findings suggest that CSL could be a promising therapeutic approach for managing inflammation-induced depressive disorders [42].

MCs are prevalent in nearly all vascularized tissues. Their close anatomical relationship with lymphatic vessels, combined with their capacity to synthesize, store, and release a wide range of inflammatory and vasoactive mediators, highlights their crucial role in regulating lymphatic vascular functions [43]. Recent studies suggest that MCs function as immune sentinels by presenting antigens through the expression of major histocompatibility complex II (MHC II) molecules. Additionally, they regulate the activity of various immune cells, including macrophages, dendritic cells (DCs), eosinophils, fibroblasts, and both T and B lymphocytes [44,45,46,47]. MC-derived histamine is essential for the LPS-induced phosphorylation of NF-κB [48]. LPS stimulates TLR4, initiating the histamine- and NF-κB-dependent production and secretion of various cytokines by MCs and surrounding tissue [48]. Upon encountering Gram-negative bacteria, MCs release inflammatory mediators, including TNFα, IL-6, and IL-1β. This response facilitates the chemotactic migration of NK cells, eosinophils, and neutrophils, while also enhancing the expression of adhesion molecules and chemokine ligands in lymphatic and blood vessels [49]. Additionally, MC-derived mediators affect lymphatic pumping by modulating the contractility of lymphatic muscle cells, which can either enhance or delay the transport of pathogens and immune cells to draining LNs [48,50]. In our study, the administration of CSL significantly inhibited excessive MC degranulation in both the smLNs and dcLNs, which are draining LNs connected to the meningeal lymphatic system [51,52]. Particularly, dcLNs are more directly connected to the meningeal lymphatic vessels and function as drainage LNs for macromolecules, cellular waste, toxic substances, and immune cells [52]. Aging is acknowledged as a significant risk factor for neurodegenerative disorders, including Alzheimer’s disease [53]. In aged mice, lymphatic drainage becomes compromised, resulting in a reduction diameter and coverage of meningeal lymphatic vessels, as well as diminished transport of CSF macromolecules to dcLNs [54]. Additionally, MC numbers in cervical lymph nodes are lower in young rats (2 months old) compared to other lymph nodes, but a more pronounced increase in MC density is observed in older rats (12 months old) [55]. Aging also leads to the basal activation of peri-lymphatic MCs, hindering the recruitment and activation of immune cells and potentially exacerbating inflammatory responses in these areas [56,57]. While this study was conducted on young healthy animals, the findings provide a baseline understanding of how CSL affects MC degranulation and neuroinflammation. The potential anti-inflammatory and neuroprotective effects observed in this context suggest that CSL may also have therapeutic relevance for age-related increases in MC activation and inflammation. Future studies should investigate whether these effects are preserved or enhanced in aging models, where chronic inflammation and impaired lymphatic function are more pronounced. MCs express both CB1 and CB2 cannabinoid receptors, and they exhibit diverse responses to cannabinoid exposure [58,59,60]. MCs also produce endocannabinoids, including an andamide, palmitoylethanolamide (PEA), suggesting the presence of a potential autocrine regulatory mechanism. Notably, CB1 possesses a unique ability to suppress FcεRI-induced secretory responses in MCs [61]. The inhibition of degranulation in dcLNs is consistent with our previous study on another species of Cannabis sativa L. [62], suggesting that CSL regulates MC activity in drainage LNs. Further research is needed to determine whether this regulation is associated with CSF drainage functions in the meningeal lymphatic system.

The vascularized meninges of the central nervous system (CNS) are essential for the protection, development, and maintenance of neural parenchyma [63]. The meninges are normal residents of innate immune cells, including macrophages, MCs, and innate lymphoid cells (ILCs). Additionally, circulating immune cells like neutrophils and T cells pass through the meninges as part of routine immunosurveillance [64,65,66]. Within the dura mater, the meningeal layer closest to the skull, MC distribution varies notably, with the highest density found in the interparietal region and the most pronounced activation occurring in the transverse region [67]. In humans, MCs in the convexity of the intracranial dura mater are situated near arterial and venous vessels, with a greater concentration observed around venous vessels within the periosteal layers and near the SSS [68]. In our study, MCs were more densely distributed in the parietal region compared to the SSS region, which is consistent with previous research [68]. No significant changes in MC number were observed following LPS or CSL administration. However, a notable increase in the degranulation ratio was observed in both regions, with the SSS region exhibiting a higher degranulation ratio after LPS administration. CSL treatment showed a dose-dependent suppression of degranulation in the SSS region. This suggests that the regulation of MC activation in the SSS region is functionally important, as this area is not only a venous site, but is also a critical region for the meningeal lymphatics. Since the discovery of meningeal lymphatics in 2015, an increasing body of evidence has highlighted the essential role of the meningeal lymphatics in regulating immune responses and inflammation within the central nervous system [52,54,69,70,71,72]. Moreover, recent studies suggest that the drainage of meningeal lymphatics, driven by VEGF-C, plays a crucial role in regulating depression-like behavior and enhancing the immune response against brain tumors [73,74,75]. MCs in the meninges may play a critical regulatory role in controlling brain inflammation and the progression of injury following a stroke [76]. Also, aging triggers the basal activation of perilymphatic MCs, which subsequently limits the recruitment and activation of various immune cell types within perilymphatic tissues [77]. CSL effectively suppressed the degranulation of MCs in the dura mater, particularly in the SSS region, suggesting that it plays a critical role in modulating mast cell activation in this area. These findings imply a complex interaction between mast cell activity and meningeal lymphatic drainage, which may serve as a crucial mechanism for regulating immune responses and inflammation in the central nervous system. Future studies should focus on elucidating the mechanisms by which CSL regulates meningeal lymphatic pathways through mast cell modulation, with particular emphasis on the VEGF-C-mediated pathway and its potential connections to neuroinflammatory or neurodegenerative diseases. Furthermore, it will be important to investigate how the basal activation of dural MCs during aging progresses and how CSL affects this activation. These studies could provide vital insights into developing preventive or therapeutic strategies for age-related neurological disorders and inflammatory diseases.

Pro-inflammatory cytokines such as TNF-α and IL-1β are pivotal in the neuroinflammatory response observed in LPS models, playing critical roles in the pathogenesis of neurodegenerative and psychiatric disorders, including depression and cognitive impairments. Our study demonstrates a significant increase in TNF-α and IL-1β expression in the PFC following LPS administration, consistent with previous reports in LPS models [18,29,30]. The administration of CSL markedly reduced these pro-inflammatory cytokines in a dose-dependent manner, particularly at the 30 mg/kg dose. This aligns with studies showing that cannabinoids like CBD and Δ9-THC, along with terpenes such as β-caryophyllene, exert potent anti-inflammatory and antidepressant effects in LPS-induced models by inhibiting pro-inflammatory cytokines through modulation of the NF-κB pathway and interactions with CB1 and CB2 receptors [78,79]. Further studies should focus on the precise mechanisms by which CSL exerts its anti-inflammatory and antidepressant effects. Investigating how CSL modulates the endocannabinoid system, including enzymes like FAAH and receptors such as GPR55, would provide key insights [80]. Additionally, exploring the roles of PPAR-γ, TRPV1, and the inhibition of NF-κB signaling in reducing cytokine expression and oxidative stress is needed [78,79,80].

Studies on the therapeutic potential of cannabis have predominantly focused on isolated cannabinoids, such as CBD and Δ9-THC, to explore their pharmacological properties. However, an increasing amount of evidence shows that treatments using the whole cannabis plant tend to produce more effective therapeutic outcomes compared to those relying solely on purified cannabinoids [81,82,83,84,85]. This superior efficacy is attributed to the “entourage effect,” a synergistic interaction between multiple bioactive compounds, including cannabinoids, terpenes, and flavonoids. In our study, we identified a variety of bioactive components in CSL, such as CBDA, CBCA, myrcene, and α-Pinene, which are likely to work together to produce the antidepressant-like effects observed. We propose that this synergistic interaction amplifies the therapeutic benefits, supporting the idea that whole plant formulations may offer superior efficacy for managing depression-like symptoms.

The limitations of our study include several important factors. First, we relied on the LPS-induced neuroinflammation model to assess the anti-inflammatory and antidepressant effects of CSL, which may not fully capture the complexity of depressive disorders in humans. Second, while CSL demonstrated significant effects on cytokine levels and behavior, the exact molecular pathways, particularly the interactions between cannabinoids and signaling mechanisms such as NF-κB and PPAR-γ, remain unclear. Furthermore, we did not assess the long-term effects or potential side effects of CSL, nor its efficacy in chronic depression models. Future studies should incorporate additional depression models and explore these mechanisms in greater depth to improve the translational relevance of our findings.