Lycium barbarum Polysaccharides Suppress Radiation-Induced Microglial Inflammation by Inhibiting the IKKβ/IκBα/NF-κB Pathway

Abstract

Background

Radiation therapy for brain tumors often leads to radiation-induced brain injury, which is closely linked to microglial hyperactivation and neuroinflammation. Lycium barbarum polysaccharide (LBP), the primary active component of Lycium barbarum, may provide neuroprotection by suppressing microglial overactivation and reducing neuroinflammation.

Methods

BV2 microglial cells were pretreated with LBP for 12 hours (h), exposed to 10 Gy X-ray irradiation, and then post-treated with LBP for another 12 h. We assessed microglial polarization and measured levels of nitric oxide (NO), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and key proteins in the IKKβ/IκBα/NF-κB pathway.

Results

LBP treatment shifted microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype and significantly decreased the release of NO, IL-1β, and TNF-α following irradiation.

Conclusion

Our findings demonstrate that LBP mitigates radiation-induced microglial inflammation by inhibiting the IKKβ/IκBα/NF-κB pathway, suggesting its potential as a radioprotective agent against radiotherapy-induced neuroinflammation.

Introduction

Cranial radiotherapy is a highly effective treatment for primary and metastatic brain tumors.^1-3 However, it inevitably damages normal brain tissue, leading to cognitive dysfunction, reduced quality of life, and significant societal burden.^4-6 These adverse effects are associated with impaired neuronal function, diminished hippocampal neurogenesis, increased oxidative stress, and chronic neuroinflammation.^7-10 While the exact pathogenic mechanisms remain unclear, substantial evidence implicates sustained radiation-induced inflammatory responses—characterized by excessive pro-inflammatory factor release and microglial hyperactivation—as key contributors.^11-13

Microglia, the brain’s resident immune cells, account for 10-15% of the glial population. In their resting state, they maintain central nervous system (CNS) homeostasis by monitoring the microenvironment and secreting neurotrophic factors. ¹⁴ Upon activation by CNS insults, microglia polarize into either the pro-inflammatory M1 phenotype (releasing cytotoxic cytokines) or the anti-inflammatory M2 phenotype (promoting tissue repair and neuroprotection).^15,16

Following irradiation, microglia rapidly activate, exhibiting morphological changes (e.g., soma enlargement, process retraction), upregulated transcription factors (nuclear factor kappa-B (NF-κB), activator protein-1 (AP-1)), and increased secretion of inflammatory mediators (inducible nitric oxide synthase (iNOS), NO, IL-1, IL-6, TNF-α).^17,18 Rodent studies confirm that brain irradiation triggers microglial proliferation, cytokine overexpression, and hippocampal neurogenesis impairment.^19-21 Thus, shifting microglia from the M1 to the M2 phenotype and dampening neuroinflammation could mitigate radiation-induced brain damage.

Lycium barbarum (goji berry), a traditional Chinese herb, exhibits diverse pharmacological properties, including antioxidant, anti-inflammatory, anti-tumor, and immunomodulatory effects.^10,22 Its primary bioactive component, LBP, regulates immune responses by activating macrophages, dendritic cells, and T cells. ²³ Notably, LBP has been shown to ameliorate radiation-induced injuries in the intestine, testes, and bone marrow by modulating apoptosis and microbiome balance.^24-27 However, its potential to protect against radiation-induced brain injury—specifically via microglial modulation—remains unexplored. This study investigates the effects of LBP on radiation-induced inflammatory responses in BV2 microglial cells and elucidates the underlying molecular mechanisms.

Materials and Methods

Inclusion and Exclusion Criteria

Inclusion criteria:

1. Cell type and characteristics: This experiment exclusively uses mouse microglia cell lines purchased from Wuhan Purisell Biotechnology Co., Ltd. (catalog number: CL-0493), which are non-human cell lines in good condition.

2. Cell culture conditions: The complete culture medium used in this experiment consists of 89% high-glucose dulbecco’s modified eagle medium (DMEM), 10% fetal bovine serum, and 1% penicillin-streptomycin antibiotic. Cells are cultured in a cell culture incubator at 5% CO₂ and 37°C, and must maintain good growth status throughout the culture period.

3. Relevance to experimental objectives: The microglia selected for this experiment must be relevant to the experimental objectives and sensitive to ionizing radiation and LBP to achieve the experimental objectives.

Exclusion criteria:

1. Cell health status: Unhealthy cells (e.g., contaminated cells or cells with a high proportion of dead cells) will be excluded.

2. Genetic background: Cells with specific genetic backgrounds will be excluded to avoid interfering with experimental results.

3. Inappropriate culture conditions: Cells that do not meet the specific culture conditions required for the experiment will be excluded.

4. Irrelevance to the experimental purpose: Cells unrelated to the experimental purpose will be excluded.

X-Ray Irradiation of BV2 Cells

BV2 cells were irradiated using a linear accelerator (Elekta, UK) at a dose rate of 2 Gy/min until reaching the target dose. The irradiation field was set to 20 × 20 cm² with a source-to-cell distance of 100 cm. The experiment was performed from September 2021 to January 2023.

Cell Viability Assessment

BV2 cell viability was evaluated using Cell Counting Kit-8 (CCK-8) assay (Biosharp, China) under two conditions: (1) 12 h post-irradiation (1-20 Gy) to determine optimal X-ray dose, (2) 24 h after LBP treatment in irradiated and non-irradiated cells. Following treatment, 10 µL CCK-8 reagent was added per well and incubated for 1 h at 37°C. Absorbance was measured at 450 nm (Molecular Devices microplate reader, USA).

NO Concentration Determination

We strictly followed the operating instructions provided by the reagent supplier, adding the test samples and reagents in sequence. After the reagents in the Griess kit (Beyotime, China) reacted with the test samples to produce a colored product, we measured the absorbance of the product in the sample wells at 540 nm. Based on the relationship between the concentration of the standard sample and its absorbance, we plotted a NO standard curve to calculate the concentration of NO in the samples.

Immunofluorescence Staining

BV2 cells cultured on glass coverslips (12-well plates) were fixed with 4% paraformaldehyde (30 minutes (min)), permeabilized with 0.3% Triton X-100 (15 min; Kerui, China), and blocked with 5% goat serum (2 h; Servicebio, China). Primary antibodies against iNOS (1:300), chitinase-like protein 3 (Ym-1) (1:200), arginase-1 (Arg-1) (1:100), and NF-κB p65 (p65) (1:500; all from CST/USA or Abcam/UK) were applied overnight at 4°C. After phosphate buffered saline with tween-20 (PBST) washes, cells were incubated with Cy3-conjugated secondary antibodies (2 h; Servicebio, China), counterstained with 4',6-diamidino-2-phenylindole (DAPI), and imaged using a Leica fluorescence microscope.

Western Blot Analysis

Protein fractions (total, cytoplasmic, nuclear) were extracted using radioimmunoprecipitation assay (RIPA) buffer and fractionation kit (Beyotime, China), followed by bicinchoninic acid (BCA) assay quantification. Samples (24 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8-12% gels), transferred to polyvinylidene fluoride (PVDF) membranes, and blocked with 5% bovine serum albumin (BSA) or non-fat milk (2 h). Membranes were probed overnight at 4°C with primary antibodies against p65, (phospho (p)- p65, inhibitor kBα (IκBα), p-IκBα, IκB kinaseβ (IKKβ), p-IKKβ (CST, USA), IL-1β, TNF-α (Abcam, UK), Lamin B (Proteintech, China), and β-actin (CST, USA). HRP-conjugated secondary antibodies were applied (50 min, RT), and bands were visualized using electrochemiluminescence (ECL) (Meilunbio, China).

Statistical Analysis

Data were analyzed using GraphPad Prism 8.0 and expressed as mean ± SEM. Intergroup comparisons were performed using Student’s t-test or one-way ANOVA with post hoc tests. P < .05 was considered statistically significant.

Results

Effect of LBP and Radiation on BV2 Cell Viability

To establish an appropriate irradiation dose for BV2 cells, we performed CCK-8 assays 12 h post-irradiation (1-20 Gy). Results showed that doses ≥ 10 Gy significantly reduced cell viability (Figure 1A), prompting our selection of 10 Gy for subsequent experiments.

Figure 1.

LBP and Radiation Effects on BV2 Cell Viability. (A) Cell Viability 12 h Post-irradiation with Doses Ranging From 0 to 20 Gy. (B) Cell Viability after 24 h LBP Treatment (0-1600 µg/mL). (C) Cell Viability with 12 h LBP Pre-treatment Followed by 10 Gy Irradiation and Then 12 h LBP Treatment after Irradiation. Data = Mean ± SEM (n = 3). ^####P < .0001 vs Control

We next evaluated LBP cytotoxicity in BV2 cells. Treatment with LBP (10-800 µg/mL) for 24 h showed no significant effect on cell viability (Figure 1B), whereas 1600 µg/mL significantly reduced viability. Notably, pretreatment with LBP (100-800 µg/mL) for 12 h failed to prevent radiation-induced viability loss (Figure 1C).

Based on these findings, we selected LBP concentrations of 100, 200, and 400 µg/mL for further studies.

LBP Attenuates Radiation-Induced Inflammatory Responses

Radiation exposure significantly increased the production of inflammatory mediators in BV2 cells, elevating NO levels (Figure 2D) and upregulating TNF-α and IL-1β protein expression (Figure 2A–C) compared to sham-irradiated controls. LBP pretreatment dose-dependently suppressed these effects, markedly reducing NO, TNF-α, and IL-1β levels.

Figure 2.

LBP Suppresses Radiation-Induced Inflammatory Mediators in BV2 Cells. (A) IL-1β and TNF-α Expression in Total Cell Lysates. (B, C) Quantitative Analysis of TNF-α and IL-1β Protein Levels by Western Blot. (D) NO Concentration in Culture Medium Measured by Griess Assay. Data Represent Mean ± SEM (n = 3 Independent Experiments, Triplicate Samples). ^####P < .0001 vs Control; ^*P < .05, ^**P < .01, ^***P < .001, ^****P < .0001 vs Irradiation Group

LBP Promotes M2 Phenotype Polarization in Irradiated Microglia

To assess LBP’s effect on microglial polarization, we evaluated the expression of phenotypic markers (iNOS for M1; Ym-1 and Arg-1 for M2) via immunofluorescence. Compared to radiation alone, LBP pretreatment significantly reduced iNOS expression (Figure 3A) while upregulating Ym-1 (Figure 3B) and Arg-1 (Figure 3C), suggesting the LBP drives irradiated BV2 cells toward the anti-inflammatory M2 phenotype.

Figure 3.

LBP Drives M2 Polarization in Irradiated Microglia. (A–C) Representative Immunofluorescence Images of iNOS (M1 Marker), Ym-1, and Arg-1 (M2 Markers) in BV2 Cells (Scale bar = 50 µm). (D–F) Quantitative Analysis of Fluorescence Intensity (Experimental/Control Ratio). Data Represent Mean ± SEM (n = 3). ^####P <.0001 vs Control; *P <.05, ^**P <.01, ^***P < .001, ^****P <.0001 vs Irradiation Group

LBP Suppresses Radiation-Induced NF-κB Pathway Activation

To elucidate how LBP inhibits microglial pro-inflammatory polarization, we examined NF-κB p65 localization and pathway activation. Immunofluorescence and Western blot analysis revealed that while p65 was predominantly cytoplasmic in untreated cells, irradiation triggered its nuclear translocation. LBP pretreatment effectively blocked this radiation-induced p65 nuclear shift (Figure 4A–D).

Figure 4.

LBP Inhibits Radiation-Induced NF-κB Pathway Activation. (A) p65 Subcellular Localization (Scale bar = 50 μm). (B) Western Blot Analysis of Nuclear and Cytoplasmic p65 (Lamin B and β-actin as Loading Controls). (C–D) Quantitative Analysis of Nuclear and Cytoplasmic p65 Expression. (F-K) Expression and Phosphorylation Levels of NF-κB Pathway Components (p65, IκBα, IKKβ) in Total Cell Lysates (β-actin as Loading Control). Data Represent Mean ± SEM (n = 3). ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 vs Control; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 vs Irradiation Group

Further investigation of NF-κB pathway components (IKKβ, IκBα, p65) demonstrated that radiation significantly increased phosphorylation of IKKβ, IκBα, and p65, along with elevated total p65 levels. Importantly, LBP pretreatment concentration-dependently reversed these radiation-induced effects (Figure 4F–K).

Discussion

Lycium barbarum polysaccharide (LBP), the primary bioactive component of Lycium barbarum, comprises antioxidant monosaccharides (e.g., galactose, glucose, rhamnose) capable of crossing the blood-brain barrier. ²⁸ In this study, we investigated its effects on BV2 microglia, which exhibit phenotypic plasticity—shifting between pro-inflammatory M1 (NO, TNF-α, IL-1β, iNOS) and anti-inflammatory M2 (Ym-1, Arg-1) states.^15,16 Radiation typically drives M1 polarization, exacerbating neuroinflammation.^17-30 Our results demonstrate that LBP reverses radiation-induced M1 polarization while upregulating M2 markers, suggesting it promotes a phenotype shift. This transition is critical, as microglial function hinges on interactions between cytokines, transcription factors, and neighboring cells.

NF-κB activation underpins radiation-induced inflammation: in resting cells, NF-κB (p50/p65) remains inactive in the cytoplasm bound to IκB. Upon irradiation, IKKβ phosphorylates IκBα, triggering its degradation and enabling NF-κB nuclear translocation to initiate pro-inflammatory gene transcription.^17,18,31,32 Notably, we found LBP inhibits p65 phosphorylation and nuclear translocation while suppressing IKKβ/IκBα phosphorylation revealing its mechanism via IKKβ/IκBα/NF-κB pathway downregulation.

Key limitations include the in vitro focus (lacking in vivo validation of microglial morphology), partial marker analysis (omitting CD206, CD86, and CD32), and absence of pathway modulators (e.g., NF-κB inhibitors/activators). Future studies should address these gaps to fully elucidate LBP’s regulatory role.

Since the experimental subjects in this study were BV2 cells and did not involve animals or clinical trials, the calculation and rationale for the sample size (animals) selected in this study were not included in the “Materials and Methods” section.

Conclusion

LBP attenuates radiation-induced neuroinflammation by shifting microglia from M1 to M2 phenotypes and suppressing pro-inflammatory mediators (NO, IL-1β, TNF-α) via IKKβ/IκBα/NF-κB pathway inhibition. These findings highlight LBP’s potential as a radioprotective agent to mitigate radiotherapy-associated neurotoxicity. However, the anti-inflammatory mechanism of LBP, via NF-κB pathway inhibition, could theoretically confer radioprotection to tumor cells and diminish therapeutic outcomes. We explicitly acknowledge this as a limitation of our current study and a central challenge in the field of radioprotectants. Subsequent investigations will be essential to determine if LBP’s effects are selective for normal microglia or if its tumorigenic potential necessitates strategies like targeted delivery to the healthy brain parenchyma to circumvent any oncoprotective effects.

Summary

Key gaps filled by this study: (1) Mechanistic specificity gap: prior research lacked evidence on how LBP modulates specific signaling pathways in radiation-triggered neuroinflammation, this study identifies the IKKβ/IκBα/NF-κB pathway as the precise molecular target of LBP, demonstrating that LBP blocks NF-κB nuclear translocation via IKKβ/IκBα suppression, curtailing neuroinflammation at the transcriptional level; (2) Cell-type-specific action gap: The role of LBP in regulating microglia (key drivers of neuroinflammation) post-radiation was unexplored, this study shows that LBP inhibits radiation-induced microglial M1 polarization (pro-inflammatory state) and promotes M2 (anti-inflammatory) transition; (3) Radiation-specific therapeutic validation gap: while LBP’s general anti-inflammatory effects were known, its efficacy against radiation-specific neuroinflammation (distinct from other insults) was unproven. This study proves LBP mitigates neuroinflammation specifically triggered by ionizing radiation, not just generic inflammation. Future research must prioritize evaluating the selective protection of LBP, focusing on mechanisms that spare tumor tissue while safeguarding healthy neuroinflammatory pathways.