Pentamidine Alleviates Inflammation and Lipopolysaccharide-Induced Sepsis by Inhibiting TLR4 Activation via Targeting...

Area of Science:

• Immunology
• Pharmacology
• Molecular Biology

Background:

• Toll-like receptor 4 (TLR4) recognizes lipopolysaccharides (LPS) and initiates immune responses.
• Excessive TLR4 activation by LPS can cause cytokine storms and severe sepsis.
• Current sepsis therapies have limitations, necessitating novel therapeutic agents.

Purpose of the Study:

• To identify novel TLR4 antagonists for sepsis therapy.
• To investigate pentamidine's mechanism of action as a TLR4 antagonist.
• To evaluate pentamidine's therapeutic potential in a mouse model of sepsis.

Main Methods:

• Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) spectroscopy to assess pentamidine-MD2 interaction.
• Cellular Thermal Shift Assay (CETSA) to evaluate pentamidine's effect on MD2 stability.
• In silico simulations to analyze pentamidine binding to MD2.
• Western blotting to assess TLR4 signaling pathway activation (NF-κB, MAPKs).
• In vivo studies in septic mice to evaluate survival rates and anti-inflammatory effects.

Main Results:

• Pentamidine directly binds to myeloid differentiation protein 2 (MD2), a TLR4 co-receptor.
• Pentamidine binding decreases MD2 stability and flexibility, inhibiting TLR4/MD2/MyD88 complex formation.
• Pentamidine blocks LPS-induced activation of NF-κB and MAPKs signaling pathways.
• Pentamidine reduces pro-inflammatory mediators (NO, TNF-α, IL-1β) and improves survival in septic mice.
• Structural modification of pentamidine abolished its TLR4 antagonist activity, confirming the importance of its chemical structure.

Conclusions:

• Pentamidine functions as a TLR4 antagonist by interacting with MD2.
• Pentamidine effectively inhibits TLR4-mediated inflammatory signaling.
• Pentamidine demonstrates therapeutic potential for alleviating inflammation and treating sepsis in vivo.