Dietary limonene promotes gastrointestinal barrier function via upregulating tight/adherens junction proteins through cannabinoid receptor type‐1 antagonistic mechanism and alters cellular metabolism in intestinal epithelial cells

Abstract Limonene, a dietary monocyclic monoterpene commonly found in citrus fruits and various aromatic plants, has garnered increasing interest as a gastrointestinal protectant. This study aimed to assess the effects of limonene on intestinal epithelial barrier function and investigate the involvement of cannabinoid receptor type‐1 (CB1R) in vitro. Additionally, the study focused on examining the metabolomic changes induced by limonene in the intestinal epithelial cells (Caco‐2). Initial analysis of transepithelial electrical resistance (TEER) revealed that both l ‐limonene and d ‐limonene, isomers of limonene, led to a dose‐ and time‐dependent increase in TEER in normal cells and those inflamed by pro‐inflammatory cytokines mixture (CytoMix). Furthermore, both types of limonene reduced CytoMix‐induced paracellular permeability, as demonstrated by a decrease in Lucifer yellow flux. Moreover, d ‐limonene and l ‐limonene treatment increased the expression of tight junction molecules (TJs) such as occludin, claudin‐1, and ZO‐1, at both the transcriptional and translational levels. d ‐Limonene upregulates E‐cadherin, a molecule involved in adherens junctions (AJs). Mechanistic investigations demonstrated that d ‐limonene and l ‐limonene treatment significantly inhibited CB1R at the protein, while the mRNA level remained unchanged. Notably, the inhibitory effect of d ‐limonene on CB1R was remarkably similar to that of pharmacological CB1R antagonists, such as rimonabant and ORG27569. d ‐limonene also alters Caco‐2 cell metabolites. A substantial reduction in β‐glucose and 2‐succinamate was detected, suggesting limonene may impact intestinal epithelial cells' glucose uptake and glutamate metabolism. These findings suggest that d ‐limonene's CB1R antagonistic property could effectively aid in the recovery of intestinal barrier damage, marking it a promising gastrointestinal protectant.