The Protective Effects of Dendrobine on LPS-Induced Neuroinflammation and Related Mechanisms Based on Microglial M1/M2 Polarization

TL;DR

Dendrobine reduces neuroinflammation by shifting microglial cells from a harmful to a protective state.

Contribution

This study reveals DDB's novel ability to modulate microglial M1/M2 polarization in LPS-induced neuroinflammation.

Findings

Dendrobine significantly reduced pro-inflammatory mediators like TNF-α, IL-6, and IL-1β in LPS-induced BV2 cells.

DDB increased anti-inflammatory markers IL-10 and Arg-1, indicating a shift toward an anti-inflammatory microglial profile.

DDB decreased M1 markers (iNOS, CD16/32) and increased M2 markers (CD206, Arg-1), suggesting polarization toward M2.

Abstract

Objectives: Dendrobine (DDB) is one of the active ingredients in Dendrobium and has been reported to have significant neuroprotective properties. Nevertheless, the precise mechanisms underlying its action have not been fully clarified. The microglial imbalance of polarization is regarded as one of the key determinants in the etiology of neurodegenerative conditions, in the contribution of neuroinflammation. The recovery of M1/M2 balance and the inhibition of over-production of the pro-inflammatory effects have become major topics in modern studies of preventing and treating neurodegenerative diseases. Methods: Therefore, the present study aimed to explore the effects of DDB on the Lipopolysaccharide (LPS)-induced neuroinflammatory model in BV2 microglial cells and the potential molecular mechanisms of microglial M1/M2 polarization. Result: The results showed that DDB significantly suppressed Nitric Oxide (NO) release and ROS levels in LPS-induced BV2 cells. ELISA, qPCR, Western blot, and immunofluorescence results indicated that DDB reduced pro-inflammatory mediators Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL-6), and nterleukin-1 beta (IL-1β) and increased anti-inflammatory mediators Interleukin-10 (IL-10) and Arginase-1 (Arg-1). Consistently, it decreased M1-like markers Inducible Nitric Oxide Synthase (iNOS) and Cluster of Differentiation 16/32 (CD16/32) while increasing M2-like/repair-associated markers (CD206 and Arg-1), suggesting a shift toward a more anti-inflammatory microglial activation profile based on the assessed marker pane. Conclusions: These results suggested that DDB can suppress the production of inflammatory cytokines and modulate microglial polarization, which indicated that DDB can be used as an effective compound in the prevention of neuroinflammation-related disorders.

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1. Introduction

The neurodegenerative ailments are distinguished by a progressive weakening of neurons or myelin sheaths [1]. The etiology of them includes a combination of different factors, such as oxidative stress, mitochondrial dysfunction, glutamate excitotoxicity, and immunological inflammation, and the pathological changes that often accompany them are irreversible [2]. With the aging population, neurodegenerative disorders like Alzheimer’s, depressive disorder, and Parkinson’s disease are becoming a major cause of physical and mental harm to patients because of a lack of preventive and therapeutic interventions [3]. Neuroinflammation plays a dynamic part in the progression as well as development of neurodegenerative diseases [4].

The neuroinflammation persistence can be explained by a number of factors, such as long-term psychological stress, infections, and metabolic diseases [5]. Here, microglia, as the key resistant effector cells of the CNS (central nervous system), play the role of a bridge and amplifier, and their activation and polarization are closely associated with the pathogenic changes in neurodegenerative diseases [6]. It has been shown that in the case of microglia activation due to inflammation, they switch their stationary state to a pro-inflammatory M1-type, releasing large amounts of inflammatory mediators (IL-1, TNF-α, IL-6, etc.), leading to neuronal injury, impairment of synaptic plasticity, and neurotransmitter impairment, causing or worsening depressive-like behaviors [7]. On the other hand, when it is polarized to the M2 anti-inflammatory phenotype, it is able to produce anti-inflammatory cytokines like IL-10 and Arg-1, suppress neuroinflammation, and repair neurons and restore function [8,9]. The microglial imbalance of polarization is regarded as one of the key determinants in the etiology of neurodegenerative conditions, in the contribution of neuroinflammation. The recovery of M1/M2 balance and the inhibition of over-production of the pro-inflammatory effects have become major topics in modern studies of preventing and treating neurodegenerative diseases.

Dendrobium is an early and revered Chinese medicine, which is used to improve gastric function, prompt the secretion of bodily fluids, nourish Yin, and get rid of heat [10]. Recent studies show that dendrobium has antioxidant, anti-inflammatory, immune-regulating, and neuroprotective effects [11]. Dendrobium major active compounds include polysaccharides, alkaloids, and phenols. One of the most potent dendrobium alkaloid products is dendrobine, which has attracted the attention of researchers in recent years because of its neuroprotective activity [12]. It has been shown that DDB could regulate monoamine neurotransmitters and the HPA axis to alleviate the symptoms of depression in rat subjects, and alleviate NLRP3-dependent apoptosis and LPS-caused neuron damage [13,14]. In our previous research, is shown that dendrobine inhibits the formation of sensitive oxygen species and production of inflammatory cytokines in SH-SY5Y cells through the Nrf2/Keap1 pathway, which is neuroprotective [15]. Although the neuroprotective effects of DDB are documented, the effect it has on microglial polarization and on the mitigation of neuroinflammation has not been sufficiently studied. Lipopolysaccharide (LPS) is a pathogen-associated molecular pattern (PAMP) that is produced by Gram-negative bacteria and triggers Toll-like receptor 4 (TLR4), resulting in a substantial activation of microglia [16]. It has been widely applied as an agent of neuronal stimulation in in vitro neuroinflammatory conditions, especially to promote M1 polarization in BV2 microglia. There is still a lack of direct experimental evidence as to whether dendrobiine can intervene in neuroinflammation and exert protective effects by regulating the polarization state of microglia.

Therefore, a BV2 microglial model of neuroinflammation induced by LPS was conducted to define the molecular pathogenesis of DDB and determine the impacts of DDB on microglial polarization, the expression of inflammatory mediators, as well as the possible mechanism of regulation.

2. Materials and Methods

2.1. Materials

The mouse-derived microglia BV2 cells were bought from Wuhan Saien Biotechnology Co., Ltd. (Wuhan, China). Dendrobine, as well as Lipopolysaccharide (LPS), was obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Nitric Oxide (NO), Reactive Oxygen Species (ROS), and Lactate Dehydrogenase (LDH) were from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The Cell Counting Kit-8 (CCK-8) was from Solarbio Science & Technology Co., Ltd. (Beijing, China). The ELISA kits for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and arginase-1 (Arg-1) were obtained from Jianglai Biotechnology Co., Ltd. (Shanghai, China). Antibodies against Notch1, Hes1, and Hes5 were supplied by Proteintech (Rosemont, IL, USA), Abcam (Cambridge, UK), and Cell Signaling Technology (Danvers, MA, USA).

2.2. Cell Culture

BV2 microglial cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C with 5% CO_2_. Cells were used for experiments when they reached 75–85% confluence and showed normal morphology and viability.

2.3. Cell Survival Assessment

To determine the appropriate concentration of DDB, BV2 cells (≈80% confluence) were treated with DDB at 12.5, 25, 50, 100, 200, or 400 μmol/L for 24 h. Cell viability was assessed using the CCK-8 assay according to the manufacturer’s instructions. For the establishment of the neuroinflammation model, cells were stimulated with LPS (1 μg/mL) for 24 h, after which the optimal DDB concentration was selected based on the viability results.

2.4. Measurement of Lactate

After cell attachment, cells were treated according to the experimental grouping. Culture supernatants were collected after treatment, and lactate levels were measured using a commercial kit following the manufacturer’s protocol. Absorbance was read at 490 nm using a microplate reader.

2.5. Nitric Oxide (NO) Measurement

After cell attachment, the corresponding treatments were applied according to the experimental groups. Following treatment, the culture supernatants were collected. Subsequently, 50 µL of Griess Reagent I and 50 µL of Griess Reagent II were sequentially added to each well, and the transmission density was measured at 540 nm using a microplate reader.

2.6. Reactive Oxygen Species (ROS) Assay

Intracellular ROS production was assessed using the DCFH-DA fluorescent probe. After treatment, cells were washed with PBS and incubated with DCFH-DA diluted in DMEM according to the manufacturer’s instructions. Fluorescence images were captured using an inverted fluorescence microscope with a blue excitation light source.

2.7. ELISA for Cytokine Measurement

After cell attachment, cells were treated according to the experimental design. Following 12 h of incubation, culture supernatants were collected. Levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as the anti-inflammatory cytokine IL-10 and Arg-1, were quantified using ELISA kits in accordance with the manufacturers’ instructions.

2.8. Western Blot Analysis

Protein samples were obtained from the cells utilizing the lysis buffer. The protein illustrations were separated via SDS-PAGE and were relocated to a PVDF membrane afterward. The membrane was subsequently congested with 5% skimmed milk for 2 h, shadowed by overnight incubation with primary antibodies targeting Iba-1, CD206, iNOS, Notch1, Hes1, and Hes5 at 4 °C. Succeeding primary antibody incubation, the membrane was cleaned once with TBST and then treated with secondary antibodies. Protein signals were identified by enhanced chemiluminescence (ECL), and band intensity was measured utilizing Image Pro software (Image-Pro Plus 6.0).

2.9. Real-Time Quantitative PCR

After 24 h of treatment, culture supernatants were removed and cells were washed three times with pre-chilled PBS. Total RNA was isolated according to the manufacturer’s instructions, and cDNA was synthesized by reverse transcription. Quantitative PCR was performed using cDNA as a template, with GAPDH as the internal control. Relative mRNA expression levels of iNOS, CD206, TNF-α, IL-1β, IL-6, IL-10, and Arg-1 were calculated. Primer sequences are listed in Table 1. GAPDH was used as the housekeeping gene for normalization. Its suitability was verified by confirming stable Ct values across all experimental groups and treatments (no significant variation among groups). Primer specificity was confirmed by melting-curve analysis showing a single peak and by agarose gel electrophoresis yielding a single band of the expected size. Amplification efficiency for each primer pair was determined using a standard curve generated from serially diluted cDNA, and only assays with acceptable efficiency and linearity (R^2^ ≥ 0.99) were included. Quantitative PCR was performed with (technical) triplicates for each sample. Relative gene expression was calculated using the 2^−ΔΔCt^ method with GAPDH as the internal reference, and results were expressed as fold change relative to the control group.

2.10. Immunofluorescence (IF)

After treatment, cells were fixed with 4% paraformaldehyde at room temperature. Cells were permeabilized with 0.1% Triton X-100 on ice and blocked with 2.5% bovine serum albumin (BSA) at room temperature. Cells were then incubated overnight at 4 °C with primary antibodies diluted in blocking solution. After washing, cells were incubated with fluorescent secondary antibodies (1:500) for 2 h in the dark. Nuclei were counterstained with DAPI. Images were acquired using a confocal microscope (LSM 880), and samples were mounted with an anti-fade reagent prior to imaging.

2.11. Flow Cytometry

Subsequent to cell attachment, the relevant treatments were administered in accordance with the experimental groups. Post-treatment, cells were cleaned with PBS and subsequently resuspended. PE-conjugated anti-CD16/CD32 (clone 93) and PE-conjugated anti-CD206 (clone MR6F3) antibodies were added individually, then incubated at room temperature for 30 min within dark. Subsequently, flow cytometric analysis was conducted on the cells.

2.12. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10.0. Data are presented as mean ± standard deviation (SD). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). Statistical significance was defined as p < 0.05. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the LPS group; ^#^ p < 0.05, ^##^ p < 0.01, and ^###^ p < 0.001 compared to the control group.

3. Results

3.1. Effect of DDB on BV2 Cell Viability

The concentrations of dendrobine (DDB) were selected based on a preliminary dose–response cytotoxicity screening using CCK-8. BV2 cells were exposed to DDB (12.5–400 μmol/L) for 24 h, and concentrations that did not significantly reduce cell viability were considered non-cytotoxic and eligible for subsequent anti-inflammatory evaluation in Figure 1A,B. LPS was used to establish an in vitro neuroinflammation model; the concentration of 1 μg/mL for 24 h was chosen because it reliably induces a robust inflammatory response in BV2 cells, consistent with commonly used conditions reported in previous studies, and was further confirmed in our pilot experiments by increased inflammatory readouts (e.g., NO production and pro-inflammatory cytokines) in Figure 1C. NO increases the inflammatory process of neurons as well as glial cells, leading to the release of cytokines and thus intensifying cell injury. The final working concentration(s) of DDB (e.g., 50, 100 and 200 μmol/L) were selected to represent a low and a high non-cytotoxic dose within the effective range observed in the pilot study.

3.2. Effects of DDB on LPS-Induced NO and LDH Release in BV2 Cells

In order to assess the possible protective properties of DDB, a BV2 cell inflammation model was developed under the influence of LPS, and alterations in the levels of LDH were investigated as revealed in Figure 2. The exposure of LPS to LDH release was significantly increased, whereas the pretreatment with DDB (50, 100, and 200 μmol/L) produced a strong effect of reducing the concentration of LDH in comparison to the LPS group (p < 0.001; p < 0.01). Taken together, these outcomes recommend that DDB is an effective inhibitor of the release of LDH in BV2 cells and indicate its cytoprotective effects.

3.3. The Effect of DDB on ROS Production in LPS-Induced BV2 Cells

As shown in Figure 3, the CON group exhibited minimal fluorescence signals, indicating low ROS levels under normal conditions. Compared to the CON group, LPS (1 μg/mL) meaningfully amplified fluorescence intensity in BV2 cells (p < 0.001), indicating the generation of large amounts of ROS and suggesting oxidative stress-induced excessive ROS accumulation in the cells. Treatment with dissimilar absorptions of DDB meaningfully abridged the fluorescence intensity in a dose-dependent manner. The fluorescence signal was slightly reduced in the 25 μmol/L DDB group, gradually diminished in the 50 μmol/L and 100 μmol/L groups, and reached levels similar to the control group at 200 μmol/L. This suggests that high concentrations of DDB effectively inhibit ROS generation. These results indicate that DDB exerts a great protective effect on LPS-induced oxidative damage.

3.4. The Effect of DDB on the Release of Inflammatory Cytokines in LPS-Induced BV2 Cells

To gauge the regulatory properties of different concentrations of DDB (50, 100, and 200 μmol/L) on LPS (1 μg/mL)-stimulated BV2 cell in regard to pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and anti-inflammatory cytokines (IL-10, Arg-1) release into the culture supernatant, we performed ELISA to measure the concentration of pro-inflammatory qPCR was used to analyze the corresponding mRNA levels of expression. The findings showed that (Figure 4A–C) the secretion of TNF-α, IL-6 and IL-1β was greater in the LPS stimulation group than in the control group, and the expression of IL-10 and Arg-1 was significantly reduced (p < 0.01), indicating that the inflammatory model was successfully developed. Likened to the LPS group, DDB treatment had an important effect in inhibiting TNF-A release, IL-6 release, and IL-1 release within a dose-dependent relationship, with the strongest effects observed at the level of 100 and 200 μmol/L (p < 0.01). Moreover, in DDB, the activity of IL-10 and Arg-1 was greatly increased to be practically under control (p < 0.01). On the transcriptional scale, DDB selectively suppressed the expression of TNF-α, IL-6 and IL-1β mRNAs (Figure 4D–F), Arg-1 and IL-10 mRNAs (Figure 4I,J), and exhibited a similar dose-dependent pattern. All these results demonstrate that DDB is a very strong anti-inflammatory and cytoprotective agent since it is able to suppress the LPS-induced pro-inflammatory response as well as encourage the expression of the anti-inflammatory cytokines.

3.5. The Effect of DDB on the Expression of Iba-1 and Related Proteins in LPS-Induced BV2 Cells

Western blot examination was conducted to examine the protein expression levels of the microglial marker Iba-1, the M1 marker iNOS, as well as the M2 marker CD206 in BV2 microglial cells, to characterize the activation status of microglia under various physiological or pathological settings. Figure 5 illustrates that LPS treatment markedly elevated Iba-1 protein expression (p < 0.01), while DDB intervention considerably diminished Iba-1 expression (p < 0.01). The results demonstrate that DDB efficiently suppresses LPS-induced initiation of BV2 microglia, thereby mitigating excessive microglial activation during the inflammatory response and producing anti-inflammatory benefits. Further analysis revealed that, similar to the control cluster, LPS treatment meaningfully amplified iNOS expression (p < 0.01) and significantly decreased CD206 expression (p < 0.01), indicating that LPS induces M1 polarization of microglia and enhances the inflammatory response. In contrast, DDB treatment significantly decreased iNOS expression (p < 0.01), with the most significant inhibitory effect observed at 100 μmol/L DDB; meanwhile, CD206 expression was significantly upregulated (p < 0.01), suggesting that DDB suppresses LPS-induced M1 polarization and promotes M2 polarization in microglia. By modulating the balance between M1 and M2 microglia, DDB may exert its anti-inflammatory effects and regulate microglial immune responses.

3.6. The Consequence of DDB on the Polarization Phenotype of LPS-Induced BV2 Cells

CD16/32, as well as iNOS, are commonly utilized markers to characterize the M1 polarization phenotype of microglia, whereas CD206 and Arg-1 are representative markers for M2 polarization. In this study, immunofluorescence and flow cytometry were employed to assess the expression changes in M1/M2 polarization-related indicators in BV2 cells treated with different conditions. As shown in Figure 6A–H, immunofluorescence analysis revealed that LPS stimulation meaningfully amplified the fluorescence intensity of M1 markers CD16/32 and iNOS (p < 0.01), indicating M1 polarization of BV2 cells. Following DDB treatment, the expression of CD16/32 and iNOS was significantly condensed (p < 0.01), suggesting an inhibitory effect on M1 polarization. However, the fluorescence of M2 markers CD206 and Arg-1 in the group receiving LPS was meaningfully lower (p < 0.01), whereas their expression in the cluster receiving DDB was meaningfully higher (p < 0.01). Flow cytometry of the CD16/32 and CD206 (Figure 6I–L) supported the results of the immunofluorescence test. To recap it all, DDB has a good inhibitory effect in preventing LPS-induced M1 polarization of BV2 cells and is able to induce M2 polarization of the cells, indicating its possible anti-inflammatory action.

4. Discussion

Neuroinflammation is an inflammatory response induced by various pathological stimuli within the central nervous system (CNS), and it is also a significant pathological feature of various neurodegenerative diseases [17]. Given that inhibiting microglial activation is a key strategy to delay the progression of neurodegenerative diseases, in this study, dendrobine (DDB) attenuated LPS-induced inflammatory and oxidative stress-related responses in BV2 microglia. Specifically, DDB did not reduce cell viability within the tested range and decreased NO/LDH release and ROS generation, accompanied by reduced pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and increased anti-inflammatory mediators (IL-10 and Arg-1). In addition, DDB modulated the assessed activation markers (reduced iNOS/CD16/32 and increased CD206/Arg-1), consistent with a shift toward a more anti-inflammatory/repair-associated profile based on the selected marker panel.

Nitric oxide (NO) is a hallmark molecule of activated microglia and a key effector in neuroinflammation [18]. Under inflammatory conditions, the inducible nitric oxide synthase (iNOS) pathway is activated and generates excessive NO, which not only amplifies the inflammatory cascade but also induces neuronal toxicity through mechanisms such as oxidative/nitrative stress. More importantly, the continuous excessive production of NO can drive the inflammatory state to transform from a transient acute response to an irreversible chronic inflammation [19]. Similarly, lactate dehydrogenase (LDH), as an intracellular enzyme, leaks out of the cytoplasm when the plasma membrane is damaged, or its permeability increases [20]. Therefore, it is often used as a classic indicator of membrane integrity loss and cytotoxicity. According to the above biological role, NO and LDH could be employed as the significant readout parameters in assessing the extent of neuroinflammatory damage and neuroprotective activities. This experiment indicated that pretreatment with DDB had a significant effect to inhibit LPS-induced NO and LDH release, and suggested that DDB prevents early inflammatory reactions as well as prevents cell membrane integrity, thus having a strong cytoprotective activity.

Oxidative stress and inflammation are closely related to each other. Preceding studies have established that reactive oxygen species (ROS) are produced in response to inflammatory inducements; in addition to that, ROS production exacerbates oxidative damage, including phospholipid peroxidation, protein oxidation, and DNA impairment, thereby contributing to neural destruction and weakened microglial role [21].

The destruction of cells, in its turn, promotes the publication of provocative mediators that further stimulate the initiation of microglial as well as aging [22]. In line with this paradigm, we found that LPS and DDB have significant effects on ROS production levels, with the highest effect recorded at 200 μmol/L. These results indicate the antioxidant and cytoprotective effect of DDB under oxidative inflammatory conditions.

Although microglial responses are frequently described using the M1/M2 framework, accumulating evidence indicates that microglial activation occurs along a continuum of context-dependent states rather than a strict binary classification. Therefore, in this study, we interpret changes in the assessed markers as a shift in pro-inflammatory versus anti-inflammatory/repair-associated features (i.e., M1-like and M2-like signatures), rather than definitive polarization into discrete M1 or M2 phenotypes. Microglial polarization is also a dynamic process that defines the course of neuroinflammatory responses. M1-type microglia are potent pro-inflammatory effectors, which release TNF-α, IL-6 and IL-1β when exposed to injury or endotoxin stimuli and that promote and amplify neuroinflammation. The continuous activation of M1 creates a vicious cycle of its own, which worsens neuronal damage. On the contrary, anti-inflammatory cytokines induce M2-type microglia that help to clear the debris, repair and preserve the survival of the neurons [23]. Recent findings point to the idea that M2 polarization can be encouraged and alleviate cognitive impairment and synaptic dysfunction by increasing the publication of anti-inflammatory as well as neurotrophic factors, and the phagocytic elimination of 8 -amyloid deposits [24]. Consequently, the rapid transition of microglia amid M1 as well as M2 phenotypes is considered a hopeful healing approach for neuroinflammatory-related illnesses [25]. In accordance with these findings, our data indicate that LPS stimulation elevated the appearance of TNF-α, IL-6, and IL-1β; DDB administration mitigated these pro-inflammatory cytokines as well as enhanced the expression of IL-10 and Arg-1. Consequently, it can be asserted that DDB possesses significant anti-inflammatory properties by reestablishing the equilibrium between pro-inflammatory and anti-inflammatory mediators.

In recent years, as a traditional resource of food and medicine sharing the same origin, DDB has great potential in the fields of medical treatment, health care and the food industry [26]. According to our research and literature, DDB mainly inhibits the release of inflammatory mediators by suppressing the polarization of microglia, thereby alleviating neuroinflammation. This provides a reliable basis for the future protection against neurodegenerative diseases. It should be pointed out that this study still has certain limitations: As an immortalized cell line, the phenotype of BV2 still differs from that of primary or human microglia; meanwhile, LPS-induced responses are more inclined towards acute inflammatory reactions and may not be able to fully simulate the long-term and complex chronic inflammatory microenvironment of neurodegenerative diseases. The current conclusions on “neuroprotection” are mainly based on indirect inferences of indicators related to inflammation and cell damage, and there is still a lack of direct functional evidence provided by experiments such as neuron co-culture or conditioned medium damage. In addition, the key upstream signaling pathways of DDB action and the verification of their causality. Such as the Notch signaling pathway, a canonical regulator of cell fate determination that has recently been implicated in microglial polarization. Under inflammatory conditions, activation of the Notch1–Hes1 axis maintains microglia in a pro-inflammatory state, promoting M1 polarization and sustaining cytokine release through a positive feedback loop [27]. The optimal dose and intervention time window also need to be systematically optimized and further verified in combination with in vivo models. At the same time, research on the correlation between blood–brain barrier permeability and transformation should be supplemented.

5. Conclusions

In an LPS-stimulated BV2 microglial model, dendrobine (DDB) attenuated inflammatory and oxidative stress-related responses, as evidenced by reduced NO/LDH release and ROS generation, decreased pro-inflammatory cytokines, and increased anti-inflammatory mediators. DDB also modulated the assessed microglial activation markers, consistent with a shift toward a more anti-inflammatory/repair-associated profile based on the selected marker panel. Overall, these findings provide preliminary in vitro evidence supporting DDB as a potential modulator of microglia-associated inflammatory responses; however, its neuroprotective or therapeutic relevance requires further validation in more complex systems (e.g., primary/human microglia, neuron–glia co-culture, and in vivo models).