Novel anti-inflammatory compounds that alleviate experimental autoimmune encephalomyelitis

Abstract

Background

Multiple sclerosis (MS) is an autoimmune disease primarily characterized by inflammatory demyelination. Despite significant research efforts, effective therapies for MS remain limited. Drug screening offers a promising approach to rapidly identifying potential therapeutic compounds.

Purpose

This study aimed to screen compounds that can exert anti-inflammatory effects and alleviate experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Study design

A fundamental research in vitro and in vivo. A high-throughput screen was performed to screen drugs that can mitigate EAE and the molecular mechanism was explored.

Methods

Based on our previous research highlighting the crucial role of AXL, a receptor tyrosine kinase, in microglial function, we constructed an AXL-GFP reporter gene in BV2 microglia cells. A high-throughput screen of an FDA-approved compound library was performed to identify potential AXL-targeting compounds. The effects of candidate compounds on cellular morphology, cell cycle, apoptosis, mitochondrial function, inflammatory cytokine production, polarization, and phagocytic activity of BV2 cells were assessed. To investigate the in vivo effects of AXL modulation, EAE mice were generated. AXL was either upregulated using recombinant Gas6 protein or knocked out using CRISPR/Cas9. The impact of AXL modulation on disease progression and underlying molecular mechanisms was explored.

Results

Primary and secondary screenings identified three potential AXL-targeting compounds: Betulin, Clofibric acid, and Isosorbide. Molecular docking analysis revealed that Isosorbide exhibited poor binding affinity with AXL at the molecular level and was excluded from further studies. Betulin and Clofibric acid were found to promote M2 polarization, reduce inflammation, enhance phagocytosis, extend the S phase of the cell cycle, inhibit apoptosis, and improve mitochondrial structure in BV2 cells. In vivo studies demonstrated that Betulin (20 mg/kg) alleviated EAE, while AXL gene knockout reversed its protective effects.

Conclusion

This study elucidates the molecular mechanism underlying Betulin's therapeutic effects in MS, both in vitro and in vivo. Betulin exerts its beneficial effects by upregulating the AXL/SOCS3 pathway and inhibiting the JAK2/STAT1 signaling pathway. These findings suggest that Betulin holds significant promise as a potential therapeutic agent for multiple sclerosis.