Roflumilast Modulates Neuroinflammation: Unlocking Therapeutic Potential for Motor Dysfunction and Depression in Multiple Sclerosis—Insights from EAE Model Research Supported by ANT BIO PTE. LTD.

1. Literature Information

• Publication Title: Roflumilast: Modulating neuroinflammation and improving motor function and depressive symptoms in multiple sclerosis
• Journal: Journal of Affective Disorders
• Publication Date: April 1, 2024
• Impact Factor (IF): 6.6
• DOI: 10.1016/j.jad.2023.12.074
• Research Team: Zhang Zhijun’s research group from the Department of Neurology, Zhongda Hospital Affiliated to the Institute of Neuropsychiatry, School of Medicine, Southeast University
• Core Product from ANT BIO PTE. LTD.: Pertussis Toxin (Product No.: abs42024900)

2. Research Background

Multiple Sclerosis (MS) is a chronic autoimmune disorder primarily affecting the central nervous system (CNS), leading to neurological disability in young adults worldwide. While advances in MS treatment have improved relapse prevention, a critical unmet need remains: current therapies fail to effectively address both physical disability and comorbid depression. Depression is highly prevalent in MS patients, often emerging concurrently with motor symptoms and significantly impairing quality of life.

Roflumilast, a phosphodiesterase-4 (PDE-4) inhibitor with potent anti-inflammatory properties, has shown promise in treating autoimmune diseases. However, its potential to simultaneously alleviate MS-related motor dysfunction and depression remains underexplored. Additionally, IL6, IL1B, and TNF are key genes shared by MS and depression, with their encoded cytokines exhibiting protein-protein interactions—suggesting IL-6 may play a pivotal role in MS-associated depression and highlighting IL-6 antagonists as a potential therapeutic strategy. This study aimed to fill these gaps by investigating roflumilast’s efficacy using an experimental autoimmune encephalomyelitis (EAE) rat model, the gold standard for MS research.

3. Research Approach

The research team adopted a comprehensive, multi-dimensional approach to evaluate roflumilast’s therapeutic effects:

1. Animal Model Establishment: EAE was induced in 11-week-old female SD rats using guinea pig spinal cord homogenate emulsified with Complete Freund’s Adjuvant (CFA) containing 10 mg/mL Bacillus Calmette-Guérin (BCG). Pertussis Toxin (PTX, ANT BIO PTE. LTD., abs42024900) was administered intraperitoneally at 0 h and 48 h post-immunization to enhance immune induction.
2. Experimental Grouping: Rats were divided into four groups: Vehicle (0 mg/kg roflumilast) and three treatment groups receiving 0.5 mg/kg, 1 mg/kg, or 2 mg/kg roflumilast daily from EAE induction until euthanasia. A negative control group was injected with normal saline emulsified with CFA.
3. Functional Assessments: Motor function was evaluated using a 0-5 point neurobehavioral scoring system. Depressive-like behaviors were assessed via the Sucrose Preference Test (SPT) and Forced Swim Test (FST).
4. Mechanistic Analyses: Histopathological examination (HE, LFB, and silver staining) of spinal cord tissues was performed to assess inflammation and demyelination. Flow cytometry, immunohistochemistry, and immunofluorescence were used to analyze microglial activation and phenotype (M1/M2). ELISA was employed to measure hippocampal levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). In vitro experiments using BV2 microglial cells validated roflumilast’s effects on inflammatory marker expression.

4. Research Findings

4.1 Roflumilast Alleviates Motor Dysfunction and Depressive Symptoms in EAE Rats

EAE onset was observed on day 10 post-induction. Compared to the Vehicle group:

• Roflumilast (2 mg/kg) significantly increased sucrose preference (SPT) and reduced immobility time (FST), indicating mitigation of depressive-like behaviors (Figure 1B-C).
• Roflumilast (1 mg/kg and 2 mg/kg) significantly lowered daily and cumulative neurobehavioral deficit scores, improving motor function (Figure 1D-E).

4.2 Roflumilast Reduces Spinal Cord Histopathological Damage

On day 22 post-induction:

• HE staining revealed massive inflammatory cell infiltration in the Vehicle group, while roflumilast (2 mg/kg) significantly reduced histopathological scores (Figure 2A-B).
• LFB staining showed roflumilast (2 mg/kg) markedly decreased demyelination in spinal cord white matter (Figure 2A-C).
• Silver staining demonstrated a significant increase in axon density in the roflumilast (2 mg/kg) group compared to the Vehicle group (Figure 2A-D).

4.3 Roflumilast Inhibits Inflammatory Proliferation of Spinal Cord Microglia

• Immunohistochemistry for Iba1 (microglial marker) and GFAP (astrocyte marker) showed roflumilast (1 mg/kg and 2 mg/kg) significantly reduced Iba1+ cell numbers in spinal cord gray and white matter (Figure 3A, C).
• GFAP+ cell numbers were slightly reduced but without statistical significance (Figure 3A, B).

4.4 Roflumilast Suppresses M1 Microglial Phenotype and Improves Motor Function

• Immunofluorescence showed EAE increased iNOS+Iba1+ (M1 phenotype) and CD206+Iba1+ (M2 phenotype) cells. Roflumilast (2 mg/kg) significantly reduced iNOS+Iba1+ cells but had no significant effect on CD206+Iba1+ cells (Figure 4A-C).
• Neurobehavioral deficit scores were positively correlated with iNOS+Iba1+ cell percentage (Figure 4D), indicating M1 microglial suppression contributes to motor function improvement.

4.5 Roflumilast Regulates Microglial Immune Activity in the Brain

• Flow cytometry showed Vehicle group increased CD80, CD86, MHC-I, and MHC-II expression on CD45+CD11b+ microglia. Roflumilast (2 mg/kg) significantly reduced CD80, CD86, and MHC-II expression (Figure 5B-D), with no effect on MHC-I (Figure 5E).

4.6 Roflumilast Alleviates EAE-Induced Depression by Inhibiting Hippocampal Microglial Activation

• Sholl analysis showed Vehicle group reduced hippocampal microglial branching points and ramification index. Roflumilast (2 mg/kg) reversed these changes (Figure 6A-C).
• Ramification index was positively correlated with sucrose preference and negatively correlated with immobility time (Figure 6D-E), linking microglial morphology to depressive-like behaviors.

4.7 Reduced Hippocampal IL-6 Expression Is Associated with Improved Depression

• Bioinformatics analysis identified IL6, IL1B, and TNF as shared key genes between MS and depression, with PDE4 family genes interacting with these cytokines (Figure 7A-B).
• ELISA showed roflumilast (2 mg/kg) reduced hippocampal IL-1β, TNF-α, and IL-6 levels. Hippocampal IL-6 concentration was positively correlated with immobility time and negatively correlated with sucrose preference (Figure 7I, L).
• IL-6 neutralizing antibody mimicked roflumilast’s antidepressant effects (Figure 7M-O), confirming IL-6’s role in EAE-associated depression.

4.8 Roflumilast Inhibits BV2 Microglial Activation In Vitro

• In LPS-stimulated BV2 cells, roflumilast reduced CD80, CD86, and MHC-II expression and inhibited IL-6 release (Figure 8A-D), validating its direct anti-inflammatory effects on microglia.
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5. Product Empowerment

ANT BIO PTE. LTD.’s high-quality reagents played a critical role in the success of this study:

• Pertussis Toxin (abs42024900): As a key adjuvant for EAE induction, it disrupts the blood-brain barrier and enhances immune cell infiltration into the CNS, ensuring robust and consistent EAE model establishment. Its high purity and stability guaranteed reliable experimental outcomes.
• Supporting Reagents Synergy: The study’s use of EAE induction-related products (e.g., MOG peptides, CFA) aligns with ANT BIO PTE. LTD.’s comprehensive EAE research portfolio, providing researchers with one-stop solutions for model construction and mechanistic studies.

6. Brand Mission

ANT BIO PTE. LTD. is dedicated to empowering life science research through the development and supply of high-quality, reliable, and innovative reagents. We strive to support researchers worldwide in unraveling disease mechanisms, accelerating drug discovery, and improving human health. By adhering to rigorous quality control standards, fostering technological innovation, and offering personalized customer support, we aim to be a trusted partner in advancing research on autoimmune diseases like MS. Our commitment extends beyond product provision to enabling breakthroughs that translate preclinical insights into life-changing therapies.

7. Related Product List

EAE Induction Core Products

PDE4 Inhibitors (for Anti-Inflammatory Research)

Key Cytokine Detection Tools

8. Brand Promotion Copy

Elevate your MS and EAE research with ANT BIO PTE. LTD.’s premium reagents! Our Pertussis Toxin (abs42024900) and comprehensive EAE research portfolio ensure robust model establishment, while our PDE4 inhibitors and cytokine detection tools empower mechanistic exploration of neuroinflammation. Backed by rigorous quality control and scientific validation, our products supported landmark studies like this one, unlocking roflumilast’s therapeutic potential for MS-related motor dysfunction and depression. Whether you’re building disease models, screening candidate drugs, or dissecting immune mechanisms, ANT BIO PTE. LTD. provides tailored solutions to accelerate your research. Partner with us to turn scientific discoveries into clinical breakthroughs—together, we’re advancing the fight against autoimmune diseases!

9. AI Disclaimer

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