A crucial factor in various neurological conditions, including neurodegenerative disorders like Alzheimer’s and Parkinson’s diseases, as well as chronic pain, is neuroinflammation. This process is marked by the release of pro-inflammatory cytokines, which significantly contribute to the development and progression of these disorders. ¹

Central sensitization, a key mechanism in pain modulation, is significantly influenced by proinflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α) and interleukin-6 (IL-6). TNF-α markedly enhances excitatory neurotransmission in the spinal cord. Concurrently, IL-1β and IL-6 contribute to the dysregulation of neurotransmitters in the pain-related neural pathways. This alteration in neurotransmitter balance promotes central sensitization and amplifies pain sensitivity, establishing these cytokines as crucial components in the development of pathological pain. ^{2, 3} Microglia, the primary immune cells of the central nervous system (CNS), play a crucial role in the development of neuropathic pain. In recent decades, substantial evidence has highlighted the capacity of microglia to induce dysfunction in the nervous system following nerve injury, thus contributing to pain regulation. ⁴ These cells exert their influence by releasing cytokines such as TNF-a, ⁵ and IL-6, ^{6, 7} which drive the process of central sensitization, a state in which CNS neurons become hyper-responsive to stimuli. Therefore, neuroinflammation is a critical factor in the transition from acute to chronic pain. ^{2, 4} Cannabinoid receptor type 2 (CB2R) expressed on microglia has emerged as a promising target for therapies addressing pain related to neuroinflammatory conditions, such as chronic pain, owing to its role in mediating analgesia with minimal psychoactive effects. Modulation of CB2R has been shown to affect spinal microglial activity and offers potential for developing novel treatment strategies for neuroinflammation. ⁸ Additionally, microglia, as key regulators of both the central nervous and immune systems, play a crucial role in orchestrating neuroinflammatory responses. Investigating these responses under well-controlled in vitro conditions is essential for elucidating their underlying mechanisms and exploring potential therapeutic strategies.

The endocannabinoid system (ECS) modulates neuroinflammation and may offer potential therapeutic options for the treatment of chronic pain. ⁹ Two types of cannabinoid receptors have been characterized, CB1 and CB2. CB1 is expressed in neural cells and mediates the psychoactive activity of cannabinoids, whereas CB2 is abundant in the immune system and is involved in immunomodulation. ¹⁰ Cannabinoid derivatives interact with the ECS and have demonstrated immunosuppressive, ¹¹ anti-inflammatory, ¹² pro-apoptotic, ¹³ neuroprotective ¹⁴ and antitumor properties. ^{13, 15} These properties make them promising candidates for the treatment. ¹⁶ Approximately 120 different cannabinoids have been identified in preparations from the Cannabis plant, including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC is the main psychoactive constituent of cannabis, whereas CBD has no known psychoactive effects. ¹⁷

Both THC and CBD are widely utilized for therapeutic applications, ¹⁸ as they reduce cytokine production in human immune cells while inhibiting both T cell proliferation and effector functions. ¹⁹ When exposed to proinflammatory stimuli, microglia treated with THC or CBD exhibit decreased secretion of cytokines, chemokines, and neurotrophic factors. ²⁰ Nevertheless, the precise molecular pathways mediating these cannabinoid effects remain incompletely understood. ²¹ The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) is a key intracellular target that mediates cannabinoid-associated immunosuppression unrelated to the cannabinoid CB1 and CB2 receptors. ²² Other targets have also been suggested, including G protein-coupled receptors (GPR)55 and GPR18 as well as transient potential (TRP) channels. ²³

The use of in vitro microglial models, including BV2 cells, facilitates systematic study of neuroinflammation in controlled and reproducible environments. These models enable the manipulation of experimental conditions, permitting the meticulous examination of cellular responses to diverse stimuli. However, accurate quantification of subtle shifts in cytokine secretion and other inflammatory markers within microglial models has historically presented challenges. Conventional techniques are often intricate and time intensive, potentially interrupting the temporal intricacies of the inflammatory process. ^{24, 25} In the context of understanding neuroinflammation and its modulation, microglia exhibit a functional dichotomy, with their phenotype having the potential to either support neuronal maintenance or contribute to neuronal harm through low-grade inflammation and reduced neurotrophic support, ²⁶ the application of cannabinoids adds a compelling dimension. ^{8, 27, 28} Active compounds from cannabis have demonstrated anti-inflammatory potential, stimulating interest in their effects on microglial responses. ^{29– 31}

This study aimed to evaluate the changes in the production of neuroinflammatory mediators in an in vitro model of microglial cells exposed to different concentrations of natural cannabinoids. A Surface Plasmon Resonance imaging (SPRi) nanobiosensor was used for the real-time evaluation of neuroinflammation biomarkers, while immunocytochemistry (ICC) was employed to assess the expression of proteins related to neuroinflammation. This article highlights the role of these techniques in advancing basic research and in developing potential therapeutic approaches for neuroinflammation-associated diseases.

In our study, none of the tested treatments significantly affected cell viability. Treatment with Dex-SP (4 μM) significantly reduced LPS-induced release of TNF-α (ICC), IL-6, and Bcl-2. In contrast, CBD significantly decreased LPS-induced release of TNF-α (5 and 10 μM), IL-6 (10 μM), Bcl-2 (5 and 10 μM), IFN-γ (5 and 10 μM), and iNOS (10 μM). Additionally, FSE-THC 70% significantly reduced Bcl-2 (5 μM), Bax (10 μM), TNF-α (ICC) (5 and 10 μM), IFN-γ (10 μM), NF-κB (5 μM), and iNOS (5 μM). Finally, FSE-THC:CBD significantly decreased TNF-α (5 and 10 μM), Bax (10 μM), NF-κB (10 μM), and iNOS (10 μM).

We stimulated BV-2 microglial cells using LPS, leading to a notable release of Bcl-2, TNF-α and NF-κB. These compounds are widely acknowledged as critical mediators in inflammatory processes. ⁵⁴ CBD (5 and 10 μM) and FSE-THC 70% (5 and 10 μM) exhibited the greatest ability to modulate inflammatory and apoptotic mediators, significantly reducing TNF-α, IL-6, IFN-γ, NF-κB, iNOS, and Bax, whereas FSE-THC:CBD (5 and 10 μM) showed an intermediate response. In comparison, Dex-SP (4 μM) effectively reduced TNF-α and IL-6 but had no significant impact on IFN-γ, NF-κB, or iNOS, suggesting that phytocannabinoids, particularly CBD and FSE-THC 70%, may represent promising strategies for neuroinflammation modulation.

Previous studies have demonstrated that the endogenous cannabinoid system, which comprises compounds such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), plays a crucial role in protecting neurons from damage. This protection was achieved by reducing cellular apoptosis and enhancing cell viability. ⁵⁵ Another study highlighted the antioxidant and neuroprotective properties of CBD and Cannabigerol (CBG) in rat astrocytes and cortices, demonstrating their effectiveness in reducing oxidative stress and preventing apoptosis. CBD outperformed CBG, with both impacting the neurokinin 3 receptor, indicating a potential multi-target mechanism. ⁵⁶ To the best of our knowledge, this is the first study to compare the effects of Dex-SP, CBD, and FSE-THC 70%, both individually and in combination, on the viability of BV-2 microglial cells.

The inhibitory effects of CBD 5 and 10 μM on the release of TNF-α were statistically superior to Dex-SP 4 μM. On the other hand, IL-6 release was more effectively inhibited by both Dex-SP 4 μM and CBD 10 μM. THC and CBD have been reported to exert distinct pharmacological actions on known cannabinoid receptors, displaying differential effects on exposed cells. THC functions as a partial agonist for both CB1 and CB2 receptors, while CBD shows a markedly low affinity for these receptors. ⁵⁷ CB2 receptors, found on a range of immune cells, including primary microglia and the BV-2 microglial cell line, are considered key mediators in cannabinoid-driven immunomodulation. ⁵⁸ The immunosuppressive effects of THC have been linked to CB2 receptors, as demonstrated through knockout system studies. ⁵⁹ Consequently, studies have demonstrated that CB2 agonists exhibit immunosuppressive effects in rat primary microglial cultures. ⁶⁰ It has been demonstrated that rat primary microglial cells express CB1 receptors, and the activation of these receptors induces the production of NO. ⁶¹ To assess whether various compositions of natural cannabinoids mediate immunosuppression in our system, we applied them prior to exposure to LPS and compared this response with that obtained using Dex-SP.

We observed that CBD (5 μM) and FSE-TSH 70% (5 and 10 μM) reduce TNF-α expression at the cytoplasmic level to a similar extent as Dex-SP. The precise mechanisms underlying these findings remain unclear. One potential explanation is that THC acts as a weak agonist for the CB2 receptor ⁶² because that the CB1 is almost absent in BV-2 cells. ⁶³ Additionally, several studies indicate that certain terpenoids may interact with components of the cannabinoid signaling system. ⁶⁴

Immunocytochemistry (ICC) analysis revealed that CBD (5 and 10 μM), FSE-THC 70% (5 and 10 μM), and FSE-THC:CBD (5 and 10 μM) exerted an anti-inflammatory effect comparable to Dex-SP (4 μM). Similarly, CBD (5 and 10 μM) and FSE-THC 70% (5 μM) demonstrated a similar ability to Dex-SP (4 μM) in regulating cell survival by suppressing LPS-induced Bcl-2 expression. This reduction in Bcl-2 may reflect a shift in cell fate, promoting a less inflammatory environment and facilitating the clearance of hyperactivated or dysfunctional microglial cells, thereby contributing to inflammation resolution. Likewise, FSE-THC 70% (10 μM) and FSE-THC:CBD (10 μM) exhibited a statistically significant effect in reducing LPS-induced Bax expression.

In our model, ICC analysis of IFN-γ and iNOS showed no detectable changes, nor were any alterations observed in NO production. This may be due to the 4-hour LPS exposure, which might have been insufficient to induce a measurable response. Studies suggest that a 6-hour time point is more appropriate for capturing early microglial activation in neuroinflammatory models. ⁶⁵ Similarly, early production of pro-inflammatory cytokines, such as IL-6 and TNF-α, has been reported shortly after intracerebral hemorrhage, highlighting the rapid onset of neuroinflammatory responses in the central nervous system. ⁶⁶

Proinflammatory cytokines are key mediators of the immune response, often peaking within the first six hours of an inflammatory stimulus. This rapid release is a hallmark of acute inflammation, which occurs in response to infections, injuries, or other inflammatory triggers. For instance, IL-1β and TNF-α are among the earliest cytokines released upon exposure to stimuli such as LPS or ionizing radiation, reflecting the swift activation of the inflammatory cascade. ⁶⁷ Their early secretion is crucial for initiating immune responses by recruiting immune cells to the site of injury and amplifying cytokine production. ⁶⁸ Accurately modeling acute neuroinflammatory responses and assessing pharmacological interventions before secondary changes occur are essential for developing effective therapeutic strategies. LPS-induced models combined with pharmacological agents targeting specific neuroinflammatory pathways provide a valuable framework for studying the acute phase of neuroinflammation. ⁶⁹

Dex-SP, a widely used anti-inflammatory drug, has also been employed clinically to manage various neuroinflammatory processes. ⁷⁰ Dex-SP inhibits inflammatory responses by regulating the activity of transcription factors such as nuclear factor kappa B (NF-κB), ⁷¹ which was observed in our study. In neuroinflammatory processes, NF-κB plays a crucial role in neurodegeneration mediated by neuroinflammation. ⁷² The upregulation of NF-κB is associated with some neuroinflammatory disease, and targeting its inhibition could potentially serve as an effective therapeutic strategy for treating Alzheimer’s disease, ⁷³ Parkinson’s disease, ⁷⁴ and chronic pain. ⁷⁵ Evidence suggests that proinflammatory cytokines, including TNF-α and IL-6 significantly contribute to the NF-κB signaling pathway. ⁷⁶ NF-κB is known to activate these cytokines in cells, thereby triggering an inflammatory response. ⁷⁶ Prior research has shown that in LPS-stimulated BV2 cells, the levels of proinflammatory cytokines TNF-α and IL-6 are elevated. ⁷⁷ Elevated levels of pro-inflammatory cytokines in BV2 cells are believed to play a direct role in the development of neuroinflammation. ⁷⁸ In the present study, Dex-SP (4 μM) significantly decreased the expression of Bcl-2 and TNF-α in LPS-stimulated BV2 cells.

IL-1β is an important pro-inflammatory cytokine in microglial activation; however, in this study, we prioritized the quantification of IL-6 and TNF-α, which are rapidly induced and play central roles in the inflammatory cascade. TNF-α is a key upstream regulator of the inflammatory response, capable of inducing both IL-1β and IL-6 expression, ^{52, 53} while IL-6 serves as a crucial modulator of neuroinflammation and is strongly associated with microglial activation. Given their well-documented roles in neuroinflammatory processes, the measurement of IL-6 and TNF-α provided a comprehensive assessment of the inflammatory response in our model. Future studies could complement these findings by including IL-1β measurements to further characterize the cytokine profile over time

On the other hand, cannabinoid derivatives, particularly CBD, have been shown to modulate inflammation, yet there are still gaps in understanding their mechanisms of action or biological responses. It has been reported that the combination of THC and CBD can enhance these properties. ⁷⁹ To date, there are no published studies comparing the anti-inflammatory properties of cannabinoids with Dex-SP in a neuroinflammation model using microglial cells. However, there are studies in other cell types showing that extracts are more potent than CBD alone, ⁸⁰ which aligns with our findings.

The expression of Bax, IFN-γ, NF-κB, and iNOS was significantly reduced in microglial cells exposed to cannabinoids compared to that in cells treated with Dex-SP (4 μM). These differences may be attributed to the distinct modulatory mechanisms of cannabinoids, in contrast to the effects of corticosteroids, such as Dex-SP, on BV-2 microglial cell activity. Previous research has also shown that CBD can exert anti-inflammatory effects on LPS-stimulated microglial cells. ⁸¹ The specific molecular mechanisms underlying CBD’s actions have yet to be fully elucidated, and more in-depth studies directly comparing the effectiveness and therapeutic potential of CBD, other cannabinoids, and their mixtures are needed. Our study demonstrated a greater anti-inflammatory effect with the presence of EFS-THC, in line with findings reported in other studies. ⁷⁹

The limitation of our study was that no RNA level tests were conducted; hence, the effect of the observed changes on genetic expression was not determined. The exposure duration to LPS was brief, so we were unable to detect changes in the response after the 6-hour period. Moreover, a broader range of inflammatory proteins, such as TLR4 and MyD88, and modulators, including IL-1β, IL-4, IL-10, and Nerve Growth Factor (NGF), are required to further validate the roles of Dex-SP and cannabinoids in the regulation of neuroinflammation in LPS-stimulated BV2 cells.

Our findings indicated that Dex-SP has an anti-inflammatory effect on LPS-stimulated BV2 cells. Furthermore, cannabinoids, particularly at certain concentrations and combinations, demonstrate efficacy comparable to or even superior to that of Dex-SP in reducing certain inflammatory mediators. This includes a reduction in the expression of anti-apoptoic protein as Bcl-2, pro-apoptotic proteins such as and Bax, as well as proinflammatory cytokines such as TNF-α, IFN-γ, NF- κB and iNOS This study will aid in elucidating the therapeutic effects of cannabinoids on processes associated with neuroinflammation. Further research is needed to understand the mechanisms and determine the combinations of cannabinoids that significantly affect inflammation modulation.

Ethical approval and consent were not required.

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Conceptualization J.-M.Q., R.-H. B., L.E.D., and M.X.L.; methodology J.-M.Q., R.-H. B. and L.E.D.; writing—original draft preparation, J.-M.Q., R.-H. B. and L.E.D; writing—review and editing, J.-M.Q., R.-H. B. and L.E.D; supervision, J.-M.Q., R.-H. B. and L.E.D; project administration, R.-H.B.; funding acquisition, R.-H. B., L.E.D., and M.X.L. All authors have read and agreed to the published version of the manuscript.