Amelioration of astrocyte-mediated neuroinflammation by EI-16004 confers neuroprotection in an MPTP-induced Parkinson’s disease model

Alleviating Effect of EI-16004 on Astrocyte-Mediated Neuroinflammation in MPTP-Induced Parkinson’s Disease Model

Background Introduction

Parkinson’s Disease (PD) is a neurodegenerative disorder primarily affecting the elderly, characterized by tremors, bradykinesia, rigidity, and loss of postural reflexes, mainly due to the loss of dopaminergic neurons in the nigrostriatal pathway. Neuroinflammation is a prominent pathological feature of PD, manifested as glial cell activation. Chronic inflammation in the central nervous system (CNS) exacerbates neuroinflammation, releasing toxic pro-inflammatory factors leading to surrounding neuronal degeneration. Astrocytes, as a type of glial cell, play a crucial role in maintaining brain homeostasis through energy metabolism supply and neurotransmitter cycling with neurons.

Research Motivation and Questions

In the treatment of Parkinson’s disease, controlling inflammatory responses has become a potential therapeutic approach. The authors conducted this study to identify compounds with anti-inflammatory effects and verify whether they could alleviate neuroinflammation in PD. Additionally, the research aimed to explore the CNS penetration of the compound EI-16004 and attempt to inhibit astrocyte activation by targeting the NF-κB and MAPK signaling pathways, thereby providing neuroprotection for PD.

Paper Source

This research was conducted by Professor Jaewon Lee and his team, mainly from the School of Pharmacy at Pusan National University, Korea Brain Research Institute, Department of Nanoscience and Nanotechnology at Pusan National University, and Tokyo Metropolitan Institute of Gerontology. The research results were published in the journal “Neuromolecular Medicine” in 2024.

Research Process and Details

Compound Screening and Identification

The research team screened compounds with anti-inflammatory effects from Pusan National University’s proprietary chemical library. These compounds underwent preliminary screening through luciferase assays, mainly aimed at identifying compounds that could inhibit NF-κB activity in Mpp+-induced astrocytes. Among them, EI-16004 showed significant anti-inflammatory effects.

Experimental Procedures and Analysis

Initial Compound Screening

After screening the proprietary chemical library, researchers found that EI-16004 could effectively inhibit Mpp+-induced NF-κB activity. The specific process involved seeding primary astrocytes in 96-well plates, adding 4×NF-κB plasmids, pre-treating with EI-16004, followed by co-treatment with Mpp+, and performing luciferase assays.

Cell Viability Assessment

To rule out the cytotoxicity of EI-16004, the research team conducted MTT assays to evaluate its effects on primary astrocytes at different concentrations. Results showed that EI-16004 had no significant impact on cell viability at 1 µM concentration, which was chosen for further experiments.

Verification of Anti-inflammatory Effects

Immunocytochemistry and Western Blotting

The research verified the anti-inflammatory effects of EI-16004 in primary astrocytes through immunocytochemistry and Western blotting. Results showed that EI-16004 could significantly inhibit Mpp+-induced NF-κB activity, reduce the expression of inflammatory factors such as IL-1β, TNF-α, and IL-6, as well as the levels of chemokine CCL2.

Molecular Docking Simulation and Binding Affinity Analysis

Through molecular docking simulation, the study found that EI-16004 could bind to the ATP binding site of Erk with higher affinity than ATP. Further Western blot analysis showed that this binding inhibited Erk phosphorylation, thereby reducing NF-κB activity.

Animal Experiments and Behavioral Tests

To verify the in vivo effects of EI-16004, experiments were conducted on MPTP-induced PD mouse models. MPTP treatment led to neuronal loss and motor deficits. In the experiment, mice received EI-16004 treatment, and motor function recovery was assessed through Rota-rod tests. Results showed that mice in the EI-16004 treatment group recovered motor function more quickly, with significant improvement compared to the control group.

Histological Analysis

Through immunohistochemistry and double fluorescence immunolabeling, the research team further verified the protective effects of EI-16004 on astrocytes and dopaminergic neurons. Results showed that EI-16004 could significantly reduce the markers of glial fibrillary acidic protein (GFAP) and Iba-1, indicating effective inhibition of astrocyte and microglia activation, thus providing protection for dopaminergic neurons.

Research Results and Conclusions

Main Research Findings

  1. EI-16004 has significant anti-inflammatory effects, effectively inhibiting Mpp+-induced NF-κB activity and the expression of related pro-inflammatory factors and chemokines.
  2. Molecular docking simulation showed that EI-16004 inhibits Erk phosphorylation by binding to the ATP binding site of Erk, thereby reducing NF-κB activity.
  3. Animal experiment results showed that EI-16004 not only improved MPTP-induced motor deficits but also effectively reduced the loss of dopaminergic neurons.
  4. Histological analysis further confirmed the anti-inflammatory protective effects of EI-16004, alleviating glial cell activation.

Scientific Significance and Application Value

This study demonstrates that EI-16004, as a potential compound for treating Parkinson’s disease, mainly exerts neuroprotective effects by inhibiting astrocyte-mediated neuroinflammation. By targeting the Erk-p65 pathway, EI-16004 can effectively reduce neuroinflammation, thereby providing protection for PD. This provides a new direction and potential therapeutic approach for future treatment of Parkinson’s disease.

Research Highlights

  • EI-16004 exhibits unique anti-inflammatory effects, capable of crossing the blood-brain barrier (BBB) and showing specific anti-inflammatory effects on astrocytes.
  • The regulation of the Erk-NF-κB signaling pathway in astrocytes provides a new therapeutic mechanism, enhancing the therapeutic potential for PD.

Conclusion

EI-16004, as a novel 3-benzyl-N-phenyl-1H-pyrazole-5-carboxamide compound, has shown significant anti-neuroinflammatory and neuroprotective effects. It reduces NF-κB activity by inhibiting Erk phosphorylation, thereby lowering the expression of pro-inflammatory factors, which provides a potential new approach for PD treatment. This study further enriches our understanding of the role of neuroinflammation in Parkinson’s disease and opens up new avenues for effective treatment of this disease.