Chlorogenic acid reduces Lipopolysaccharide-Induced intestinal inflammation through Inhibition of acetylcholinesterase and activation of muscarinic receptors

Background: Chlorogenic acid (CGA) is a dietary polyphenol with well-documented anti-inflammatory property. However, its effects on cholinergic signaling during inflammation remain under-explored.

Aim: To investigate the role of CGA in modulating lipopolysaccharide (LPS) induced inflammation and epithelial barrier dysfunction in human intestinal cells, with a focus on cholinergic pathways involving acetylcholinesterase (AChE) and muscarinic receptors.

Methods: Caco-2 cell monolayers were treated with LPS, with or without CGA. Inflammatory markers, tight junction proteins, Mucin secretion, and cholinergic gene expression were assessed via qRT-PCR, ELISA, immunofluorescence, and Alcian Blue staining. AChE activity was measured in cell supernatants. Kinetic studies were done with pure human and electric eel Enzymes. Inhibition kinetics and molecular docking was performed. Darifenacin was used to probe the role of muscarinic receptors.

Results: CGA significantly suppressed LPS-induced expression of TNF-α, IL-6, and COX-2, and restored tight junction protein expression (ZO-1, occludin) and sucrase-isomaltase mRNA levels. CGA attenuated LPS-induced Mucin hypersecretion without affecting MUC2 gene or protein expression. It also reversed LPS-induced upregulation of α7 nicotinic ACh receptors and further elevated choline acetyltransferase (ChAT) and M3 muscarinic receptor (mAChR3) expression. These effects were abolished by muscarinic receptor antagonism, indicating CGA's dependence on mAChR signaling. CGA reduced cholinesterase activity in supernatants. Kinetic studies revealed that CGA competitively inhibited HuAChE (IC₅₀ = 225 nM; K_i = 30.7 nM) and EeAChE (IC₅₀ = 150 µM; K_i = 15.5 µM). Molecular docking showed strong interactions with HuAChE catalytic residues and the mAChR3 orthosteric site, supporting its dual role as an AChE Inhibitor and muscarinic receptor agonist. Bioavailability radar analysis confirmed CGA's potential as a drug-like molecule.

Conclusions: CGA protects intestinal epithelial cells from inflammatory damage by enhancing cholinergic anti-inflammatory signaling through specific AChE inhibition and regulation. Future studies should validate these effects in vivo and confirm functional receptor activation and bioavailability.

Acetylcholinesterase inhibition; Chlorogenic acid; Intestinal barrier integrity; Lipopolysaccharide (LPS); Muscarinic receptor.