Naringenin Ameliorates LPS-Induced Neuroinflammation Through NF-κB Signaling in Human Microglia and Protects Neuronal Cells

TL;DR

Naringenin, a citrus flavonoid, reduces neuroinflammation by blocking NF-κB signaling in microglial cells and protecting neurons from inflammation.

Contribution

This study demonstrates naringenin's novel anti-neuroinflammatory effects via NF-κB suppression in human microglia and neuronal protection.

Findings

Naringenin reduces ROS and proinflammatory cytokines (TNF-α, IL-6, IL-1β) in LPS-treated microglial cells.

Naringenin inhibits NF-κB activation and nuclear translocation in microglia.

Naringenin protects neuronal cells from inflammatory microglial secretome.

Abstract

What are the main findings?
Naringenin significantly attenuates LPS-induced neuroinflammation by reducing ROS generation and proinflammatory cytokine (TNF-α, IL-6, IL-1β) expression in human microglial cells.Naringenin suppresses NF-κB activation and nuclear translocation in microglia, thereby protecting neuronal cells exposed to an inflammatory microglial secretome.

Naringenin significantly attenuates LPS-induced neuroinflammation by reducing ROS generation and proinflammatory cytokine (TNF-α, IL-6, IL-1β) expression in human microglial cells.

Naringenin suppresses NF-κB activation and nuclear translocation in microglia, thereby protecting neuronal cells exposed to an inflammatory microglial secretome.

What are the implications of the main findings?
Targeting microglial NF-κB signaling with dietary flavonoids such as naringenin represents a promising strategy to mitigate neuroinflammation-driven neuronal damage.These findings support the therapeutic potential of naringenin for preventing or slowing neuroinflammatory processes associated with neurodegenerative disorders.

Targeting microglial NF-κB signaling with dietary flavonoids such as naringenin represents a promising strategy to mitigate neuroinflammation-driven neuronal damage.

These findings support the therapeutic potential of naringenin for preventing or slowing neuroinflammatory processes associated with neurodegenerative disorders.

Background: Engagement of the NF-κB signaling pathway is crucial for controlling immune and inflammatory gene expression within the central nervous system (CNS). Naringenin, a flavonoid derived from citrus fruits, is known for its anti-inflammatory and antioxidant effects; however, its impact on LPS-induced neuroinflammation in HMC3 (human microglial) and SH-SY5Y (neuronal) cell lines has not been thoroughly studied. Objectives: We sought to ascertain the neuroprotective role of Naringenin in LPS-induced neuroinflammation in microglia and neuronal cell lines with a focus on modulation of the NF-κB signaling pathway. Methods: LPS treatment was given to HMC3 cells to induce an inflammatory response, besides the secretome of HMC3 cells was transfered to SH-SY5Y cells with the administration of Naringenin. A cell viability assay, ROS level measurements, Western blotting, and immunocytochemistry were employed to quantify and localize NF-κB and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). Nuclear fractions of NF-κB were analyzed to screen its activation and translocation. Results: Naringenin treatment led to a dose-dependent decrease in LPS-induced reactive oxygen species (ROS) production. It significantly reduced the expression of pro-inflammatory cytokines and inhibited NF-κB activation in HMC3 cells. The nuclear translocation of NF-κB was notably diminished after treatment, as demonstrated by both Western blot and immunocytochemistry. These results suggest that Naringenin exerts an anti-inflammatory effect by suppressing the NF-κB signaling pathway. Conclusions: The findings suggest the potential therapeutic role of Naringenin using in vitro models in mitigating neuroinflammation through modulation of the NF-κB signaling pathway.

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Full text

1. Introduction

Neurodegenerative diseases are characterized by neuronal loss and diminished cognition, representing a global health concern with socioeconomic impact. The precise molecular mechanisms underlying such conditions, despite extensive research, remain poorly understood, and effective disease-modifying therapies remain limited. Accumulating evidence suggests neuroinflammation and oxidative stress as central processes in the pathogenesis and progression of neurodegenerative disorders. Stimulation of the CNS innate immune system, particularly microglial cells, is now recognized as a crucial factor in the genesis of neuronal dysfunction and death. This activation can be triggered by pathological stimuli such as infection, trauma, or the buildup of misfolded proteins. While microglial activation initially serves a protective role by releasing reactive oxygen species and proinflammatory cytokines, prolonged or dysregulated activation can become harmful [1]. Chronic microglial activation is widely recognized to cause neuronal damage and accelerate the progression of neurological diseases. NF-κB, a transcription factor, is the central mediator of microglial activation and neuroinflammatory signaling. It regulates the expression of several genes involved in the immune and inflammatory response, cell proliferation, and cell survival [2]. Under resting conditions, NF-κB remains in the cytoplasm in an inactive state, bound to inhibitory proteins termed IκBs. The translocation of NF-κB typically occurs upon stimulation by pro-inflammatory signals, such as LPS, which degrades or phosphorylates IκB. The binding of NF-κB with the recognition site of DNA initiates transcription of target genes like tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) within microglial cells, contributing to neurotoxicity combined with oxidative stress [3].

Lipopolysaccharide (LPS) is widely used to induce neuroinflammation in both in vitro and in vivo models. LPS activates toll-like receptors on microglia, leading to NF-κB signaling and subsequent release of pro-inflammatory mediators, making it a well-established model for evaluating the anti-inflammatory potential of therapeutic candidates. Naringenin (4′,5,7-trihydroxyflavanone-7-rhamnoglucoside), a flavonoid abundant in citrus fruits, has been proven against inflammation, carcinogenesis, oxidative stress, etc. The effect of naringenin has not been tested in LPS-stimulated HMC-3 and SHSY cell lines, and the mechanisms underlying its neuroprotective effects remain poorly understood. In this study, we aimed to investigate the effect of Naringenin against LPS-induced neuroinflammation using human microglial (HMC-3) and SHSY5Y cell lines. By treating HMC3 cells with LPS to induce an inflammatory phenotype and subsequently exposing SH-SY5Y nerve cells to conditioned media extracted from LPS-treated microglial cells, we focused on elucidating the action of Naringenin on the production of an inflammatory microenvironment, ROS generation, and the action of the NF-κB signaling pathway [4]. These observations provide new insights into the mechanism by which Naringenin may confer neuroprotection and support its potential as a therapeutic agent for the prevention or treatment of neuroinflammatory and neurodegenerative disorders.

2. Materials and Methods

2.1. Material

Naringenin was procured from Sigma-Aldrich, Saint Louis, MO, USA, and a stock solution of 250 mM was formulated by dissolving it in DMSO, stored at −20 °C. We used 0.04% DMSO as a vehicle control throughout the study. Modified Dulbecco’s Eagle’s medium (DMEM F12) was acquired from Thermofisher Scientific, Waltham, MA, USA, and stored at 4 °C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired from Sigma. The fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) was obtained from Invitrogen. 4′,6-diamino-2′-phenylindoledihydrochloride (DAPI) and protease inhibitor were sourced from Thermofisher Scientific. Antibodies against Rabbit anti-IL-6, anti-TNF alpha, anti-IL1beta, anti-beta actin, rabbit anti-Gapdh, and Horseradish peroxidase-linked IgG antibodies were acquired from Cell Signaling Technology Inc. (Danvers, MA, USA). All other chemicals used were of high grade and commercially available.

2.2. Cell Culture

Human microglial cells HMC3 were procured from ATCC (American Tissue Culture Collection, Manassas, VA, USA). Initially, the cells were cultivated in EMEM in a Petri dish in an incubator under a humidified atmosphere of 95% air and 5% CO_2_, with 1% penicillin/streptomycin. Media changes were performed every 2 days with a DPBS wash until cells reached 80% confluence. Subsequently, cells were trypsinized with 0.25% trypsin-EDTA, followed by centrifugation, sediment collection, and subculturing at a density of 1 × 10^4^ cells/well and 2 × 10^5^ cells/well on 96- and 6-well plates, respectively. Plates were initially treated with poly D-Lysine (ThermoFisher Scientific-Waltham, MA, USA) for 3 h to facilitate proper cell adhesion. The subsequently selected media consisted of Dulbecco’s Modified Eagle Medium (DMEM/F12), 10% horse serum, and 5% FBS. SH-SY5Y cells (undifferentiated) were cultured on Eagle’s Minimum Essential Medium (EMEM) with 10% FBS and 1% streptomycin/penicillin in an incubator with a humidified atmosphere containing 95% air and 5% CO_2_ in a Petri dish. The cells were subjected to media changes every 2 days, with DPBS washes, until they reached 80% confluence. These cells were used for cell viability assays and ROS staining.

2.3. MTT Assay

This assay is based on the enzymatic reduction of the MTT dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which produces a measurable absorbance that correlates directly with the number of viable cells, as described previously [5]. The HMC3 cells were treated with various concentrations of Naringenin (25, 50, 100, 200, 300, 400 and 500 µM) for 24 h in 100 µL of media. Re-incubation for 3 h was performed after adding 10 µL of MTT dye until the formation of formazan blue crystals. Afterward, the media was removed, and 100 µL of DMSO was added for 10 min at 37 °C to dissolve the formazan crystals. The absorbance was measured at 550 nm using a microplate reader; cytotoxicity was calculated and expressed as a percentage change [6].

2.4. Preparation of LPS

Lyophilized lipopolysaccharide (LPS) derived from E. coli, strain O111:B4, was obtained from Sigma. One milligram of LPS was dissolved in 1 mL of sterile, cell culture-grade distilled water (ThermoFisher), dispensed in aliquots, and stored at −20 °C until further use.

2.5. Cell Viability Assay

This assay facilitates convenient testing using Dojindo’s highly water-soluble tetrazolium salt, WST-8 {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt}, which generates soluble formazan dye upon reducing under the influence of an electron mediator [7]. This assay was performed using the CCK-8 Kit (Dajinoo Laboratories). HMC3 cells were seeded in 6-well plates and divided into two groups: control (no treatment) and LPS (100 ng/mL LPS added and incubated for 24 h in a CO_2_ incubator). The media of HMC3 cells were collected and added to SH-SY5Y cells using 96-well plates along with treatments of Naringenin at 15, 20, 25, and 30 µM in different groups. The cells were cultured for 24 h, and then 10 µL of Cell Counting Kit-8 (CCK-8) solution was added to each well of the plate. The plate was incubated for 1 h at 37 °C and absorbance was measured at 450 nm using a microplate reader.

2.6. H2DCFDA-ROS Staining

H_2_DCFDA-ROS levels were estimated by using the standard protocol [8]. HMC3 cells were plated in 6-well plates and divided into two experimental groups: an untreated control group and an LPS group exposed to 100 ng/mL LPS, which was subsequently incubated for 24 h in a CO_2_ incubator. Culture media from HMC3 cells were collected and transferred to SH-SY5Y cells in 96-well plates, along with Naringenin at concentrations of 15, 20, 25, and 30 µM for different groups. Subsequently, cells were cultured for an additional 24 h on conditioned media containing the secretome. Following this incubation, the cells were stained with 20 μM DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) from the DCFDA Assay Kit (Abcam, Waltham, MA, USA) for 30 min at 37 °C in the dark. Changes in fluorescence intensity were observed using a fluorescent microscope.

2.7. Western Blotting Analysis

HMC3 cells were initially seeded in EMEM with 10% FBS and 1% penicillin/streptomycin in an incubator with a humidified atmosphere containing 95% air and 5% CO_2_ in a Petri dish, grown for 5 days, and then washed with 1x PBS. The trial was conducted at various time points as biological triplicates. The plates were kept in a CO_2_ incubator (5% CO_2_) until 60–70% confluency was achieved. The media were discarded from each well before treatment. The 6-well plates (each group set as triplicate) were assigned to the following groups: control receiving DMSO (0.04%); LPS at 100 ng/mL; Naringenin at 100 µM dissolved in DMEM HS. The plates were incubated in a CO_2_ incubator for 24 h. The plates were removed from the incubator, and the media from each well were collected separately into labeled tubes and stored for further evaluation.

Ice-cold lysis buffer (20 mM Tris, pH 8.0; 137 mM NaCl; 1% NP-40; 10% glycerol) containing protease inhibitors was used to prepare the cell lysates (1 mM phenylmethyl sulfonyl fluoride (PMSF), 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 3 mM Na_3_VO_4_). Following centrifugation to clear the sample, protein concentration was determined using the Bradford assay (Bio-Rad; Hercules, CA, USA). A quantity of 25 μg of total protein was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a gel concentration of 10–12% and then transferred to a polyvinylidene difluoride (PVDF) membrane. Following a 30 min block with blocking buffer (composed of 5% BSA, PBS, 0.05% Tween-20), the membranes were left to incubate overnight at 4 °C with primary antibodies obtained from Cell Signaling Technology, MA, USA: anti-NF-κB (1:1000), anti-IL-6 (1:1000), anti-IL-1β (1:1000), anti-TNF-α (1:1000), GAPDH and β-actin (1:5000). Subsequently, secondary antibodies, including anti-rabbit and anti-mouse HRP IgG (1:5000), were applied. β-actin and GAPDH (both diluted 1:3000, Cell Signaling Technology) were used as loading controls.

2.8. Cell Fractionation

A Nuclear Cytosolic Fraction of NF-κB was collected by running a similar fresh experiment on HMC3 cells, and the cell lysates were prepared in ice-cold buffer A (composed of 20 mM Hepes, 10 mM KCl, 2 mM MgCl_2_, 0.5% Nonidet P40, and 1 mM Na_3_VO_4_) with PIC. The cell scrapings were transferred to 1.5 mL microfuge tubes and then centrifuged at 1500× g for 5 min. The supernatant was centrifuged in a cooling centrifuge, and the resultant supernatant was stored as a cytosolic fraction. The nuclear pellet underwent three washes with lysis buffer A before being added to the same buffer supplemented with 0.5 M NaCl to facilitate nuclear protein extraction. The sample was vortexed 4–5 times at 10 min intervals, then centrifuged at 1500× g for 10 min. The collected supernatants yielded a nuclear fraction that contained NF-κB.

2.9. Immunocytochemistry

Immunocytochemistry was performed following standardized protocols. HMC-3 cells were maintained in EMEM supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (PenStrep), seeded onto poly-D-lysine (PDL)-coated coverslips in a 24-well plate, and incubated for 24 h or until reaching the desired confluency. The cells were then treated with lipopolysaccharide (LPS) and Naringenin for an additional 24 h. Following treatment, the cells were gently washed with 1X phosphate-buffered saline (PBS, pH 7.4), fixed with 4% paraformaldehyde, and subsequently permeabilized with 0.25% Triton X-100. To prevent nonspecific antibody binding, cells were blocked for 30 min using a solution of 1% bovine serum albumin (BSA) and 22.52 mg/mL glycine in PBST (PBS supplemented with 0.1% Tween 20) in the incubator. After the blocking step, the cells were incubated overnight at 4 °C with gentle agitation in rabbit anti-NF-κB primary antibody (1:250; Cell Signaling Technology, Danvers, MA, USA). This was followed by 1 h dd hyphen incubation with Texas red-conjugated secondary antibody (anti-rabbit IgG (H+L), F(ab’)2 fragment (1:1000; Cell Signaling Technology)) the next day. Following three 5 min washes with PBS, the coverslips were mounted onto glass slides using Fluoromount-G™ mounting medium containing DAPI (Invitrogen™), and the edges were sealed. Fluorescence images were then captured at 40× magnification using a fluorescence microscope.

2.10. Statistical Analysis

Prism6 software-10.6.1(GraphPad Software) was utilized for statistical analyses. The data were analyzed using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests for multiple comparisons. Mean values ± standard error of the mean are presented, and significant differences are denoted as *, **, and ***, **** corresponding to p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

3. Results

3.1. Cytotoxicity Effect of the Naringenin on HMC Cells

As shown in Figure 1, the MTT assay demonstrates a decrease in HMC3 cell proliferation at Naringenin concentrations ranging from 25 to 500 µM. The estimated IC-50 for HMC 3 cells was found to be 485 ± 8 µM after 24 h of treatment. Safe concentrations of no more than 150 µM were selected based on the MTT assay for HMC3 cells.

3.2. Naringenin Protects Neuronal Cells

The effects of different concentrations of Naringenin (15 µM, 20 µM, 25 µM, and 30 µM) on SH-SY5Y cells treated with conditioned media (enriched in the secretome) collected from HMC3 cells after LPS (100 ng/mL) treatment for 24 h were investigated. A significant reduction in cell viability was observed in LPS group compared to control. Treatment with naringenin significantly attenuated the reduction in cell viability, and all tested concentrations demonstrated a protective effect against LPS-induced neurotoxicity (Figure 2).

3.3. Naringenin Reduced the ROS Production in Neuronal Cells

The generation of ROS, as determined by using the fluorogenic probe 2′, 7′-dichlorodihydrofluorescein, revealed the lipopolysaccharide (LPS)-initiated reactive oxygen species (ROS) generation in the LPS group. Naringenin reduced ROS production triggered by lipopolysaccharide (LPS). The effect of various concentrations of Naringenin (15 µM, 20 µM, 25 µM, and 30 µM) on SH-SY5Y cells treated for 24 h with the conditioned media extracted from LPS (100 ng/mL)-treated HMC3 cells revealed a dose-dependent decrease in ROS production triggered by lipopolysaccharide (Figure 3).

3.4. Effect of Naringenin on the Level of Proinflammatory Cytokines and NF-κB Activity

The proinflammatory cytokines TNF-α, IL-1β, and IL-6, and the transcription factor NF-κB, are key markers of the neuroinflammatory response. Their quantification and levels showed a significant increase in the LPS-treated (100 ng/mL) group (Figure 4). Our results demonstrate the anti-inflammatory role of Naringenin given at 100 µM to HMC3 cells by significantly decreasing the activity of TNF-α (p < 0.001), IL-6 (p < 0.05), IL-1β (p < 0.01), and transcription factor NF-κB (p < 0.0001) compared to control (Figure 4).

3.5. Effect of Naringenin on Nuclear Fraction of NF-κB Activity

The transcription factor NF-κB, a contributor to inflammation, is expressed in the nucleus upon activation. Our study detected NF-κB in the nuclear fraction, which increased significantly in LPS-treated HMC3 cell lines. Treatment of Naringenin delivered to the LPS-treated cells significantly inhibited NF-κB levels at doses of 100 µM (p < 0.05) and 150 µM (p < 0.05) (Figure 5). The expression was measured using the marker protein Histone H3, which is a core nucleosome protein with stable expression, making it a reliable marker.

3.6. Naringenin Reduced the Nuclear NF-κB in Microglia

Immunocytochemistry was performed on HMC-3 cells subjected to the following treatment conditions: (1) control, (2) LPS (100 ng/mL), and (3) LPS (100 ng/mL) + Naringenin (100 μM). After 24 h of treatment, cells were stained with an NF-κB antibody conjugated to Texas Red (red fluorescence) and counterstained with DAPI (blue) to visualize the nucleus. The LPS-treated group exhibited a marked increase in red fluorescence intensity, indicating elevated NF-κB expression relative to the control group. Conversely, cells co-treated with Naringenin (100 µM) showed reduced NF-κB fluorescence. These findings indicate that Naringenin effectively suppresses LPS-induced NF-κB activation in HMC-3 microglial cells (Figure 6).

4. Discussion

Oxidative stress and neuroinflammation contribute to the pathogenesis of neurodegenerative disorders. Microglia, the resident brain macrophages, on the one hand, phagocytose non-self cells and debris, thereby maintaining brain homeostasis; on the other hand, they exacerbate neuronal injury in neurodegenerative diseases by contributing to chronic inflammation. Our study aimed to investigate the role of naringenin in mitigating LPS-induced neuroinflammation in HMC3 cells. Furthermore, our study involved the use of conditioned culture (LPS-treated microglial cell extract) on the nerve cells (SH-SY5Y) for understanding the action of this compound in a more homogeneous in vitro model. The results of this study demonstrated that LPS induces the production of inflammatory cytokines by activating the NF-κB signaling pathway. In contrast, Naringenin exhibited significant anti-inflammatory and antioxidative effects by downregulating the NF-κB transcription factor, as evident from Western blot and ICC. The ICC results, being highly significant, represent a more sensitive approach for assessing NF-κB nuclear translocation in this experimental context, and the Western blot results support the same directional trend. Thus, Naringenin exerts neuroprotective effects by mitigating microglial-mediated inflammatory responses and reducing neuronal cell death. Promoting an anti-inflammatory M2 phenotype may offer a new approach for combating neuroinflammation and enhancing neuroprotection. Neuroinflammation driven by microglia is widely acknowledged as the key mechanism leading to neuronal loss [9,10]. They function as guardians of the central nervous system (CNS), consistently surveying the environment for detrimental threats or pathogens. Distributed throughout the CNS like sentinels, they activate in response to local danger signals, adopting their cell morphology by enlarging their soma and retracting cytoplasmic processes. These activated microglia stimulate A1 subtype astrocytes, which attract neuronal damage by releasing inflammatory cytokines such as IL-6, tumor necrosis factor α (TNF-α), IL-1β, reactive oxygen species, and excitotoxins such as glutamate via activation of nuclear factor κB (NF-κB)-dependent mechanisms [11]. Conversely, activation of reactive astrocytes by M2 microglia induces the brain to produce anti-inflammatory factors via the STAT6 pathway [12]. The LPS-induced neuroinflammation in our study, mediated by immune cell activation and the release of pro-inflammatory molecules via NF-κB signaling, is attributable to microglial activation. Our study demonstrated that Naringenin reduced lipopolysaccharide-induced ROS production in a dose-dependent manner. Our study is consistent with many studies suggesting a promising effect of Naringenin on LPS-induced inflammatory and oxidative responses [13,14,15,16]. There is wider evidence in support of ROS production and the resultant oxidative damage as contributing factors to incur neuronal damage. In neurodegenerative diseases, overproduction of ROS to levels that exceed the reductive capacity of the intact antioxidant system is responsible for neuronal cell damage and apoptosis. At the cellular level, mitochondrial damage from ROS leakage disrupts mitochondrial function, which can be mediated by mitochondrial fission, apoptosis, and energy depletion, thereby contributing to the etiopathology of neurodegenerative diseases [17].

Proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, along with the transcription factor NF-κB, serve as key indicators of a neuroinflammatory response. In this study, their expression levels were significantly elevated in HMC3 cells following LPS stimulation. Treatment with 100 µM Naringenin markedly reduced the expression of these inflammatory markers, demonstrating its potent anti-inflammatory effect. Specifically, Naringenin significantly downregulated TNF-α, IL-1β, and NF-κB levels, critical mediators of the inflammatory cascade, when compared to the control group.

Several studies have highlighted the pro-inflammatory role of cytokines in the pathogenesis of neuroinflammatory conditions. TNF-α, a pleiotropic proinflammatory cytokine activated by PAMPs such as LPS in this study, is a key proinflammatory cytokine produced by microglia in response to injury, infection, or neurodegeneration. Toll-like receptors (TLRs) function as microglial receptors, thereby initiating signal transduction pathways. TNF-α increases IL-6 and IL-1β levels, which are involved in the further progression of inflammatory reactions [18]. Targeting cytokine trafficking and degradation would support the development of new anti-cytokine formulations. Thus, our study confirms that Naringenin promotes cytokine degradation, which may offer a new avenue for drug development in this area.

This study demonstrated that lipopolysaccharide (LPS) stimulation induced a pronounced increase in nuclear NF-κB levels in HMC3 microglial cells, indicating activation of the inflammatory signaling cascade. However, treatment with 100 µM Naringenin significantly attenuated this LPS-induced nuclear translocation of NF-κB. As a pivotal transcription factor, NF-κB governs the inflammatory response by regulating the expression of genes that drive the production and activation of various proinflammatory mediators. LPS, as a PAMP, tends to activate HMC3 cells to produce a spectrum of cytokines and chemokines. NF-κB released from the cytoplasm translocates to the nucleus, binds DNA, and activates transcription of target genes such as IL-6, IL-1β, IL-12, and TNF-α [19]. Thus, our study demonstrated that Naringenin is a potent agent in ameliorating LPS-induced inflammation by inhibiting NF-κB, a key regulator of pro-inflammatory gene induction and function.

Despite the promising findings, several limitations of the present study should be acknowledged. First, this work was conducted exclusively using in vitro models based on immortalized human microglial (HMC3) and neuronal (SH-SY5Y) cell lines. While these systems are well established and provide mechanistic insight, they do not fully recapitulate the complex cellular interactions of the brain’s neurovascular unit, including neuron–astrocyte–microglia crosstalk, blood–brain barrier dynamics, and systemic immune influences. Therefore, extrapolation of these findings to in vivo or clinical settings should be performed with caution. The present study focused primarily on the NF-κB signaling pathway as a central mediator of LPS-induced neuroinflammation. Other anti-inflammatory and cytoprotective pathways potentially modulated by naringenin—such as Nrf2, MAPK, JAK/STAT, and PPAR signaling—were not explored and remain important avenues for future investigation.

5. Conclusions

The present study demonstrates that naringenin suppresses LPS-induced neuroinflammatory signaling in HMC3 microglial cells by downregulating the NF-κB pathway and reducing LPS-induced oxidative stress in SH-SY5Y neuronal cells.

This suppression results in decreased production of proinflammatory cytokines and oxidative stress markers, highlighting Naringenin’s potential as a neuroprotective agent. The neuroprotective potential of Naringenin is established, thus suggesting its promise as a therapeutic agent for combating neuroinflammatory processes and neuronal damage associated with neurodegenerative disorders. By promoting an anti-inflammatory response and mitigating microglial activation, Naringenin offers a valuable strategy for enhancing neuroprotection and potentially slowing the progression of neurodegenerative diseases. Further research is warranted to clarify the underlying molecular mechanisms and to investigate the translational potential of these findings in preclinical and clinical settings.