Ligustilide improves functional constipation by non-covalently activating TRPA1 in colon tissue

Abstract

Ethnopharmacological relevanceAngelica sinensis (Oliv.) Diels (AS), a medicinal plant renowned for its constipation-relieving properties, lacks comprehensive studies on its active pharmaceutical ingredients (APIs) and underlying mechanisms. In the gastrointestinal tract, TRP channels enhance colonic mucus secretion, expedite intestinal motility, and regulate gastrointestinal hormones; however, few reports have systematically established the relationship between TRPs and ligustilide (Lig), a key API of AS. Aim of the studyThis study aimed to explore the pharmacodynamic properties of AS in alleviating functional constipation, assess the potential of Lig for activating TRPs, and elucidate its mechanism of action. MethodsThe therapeutic efficacy of AS was assessed in a mouse model of loperamide hydrochloride-induced functional constipation. The APIs were screened via integrated activity-based UPLC profiling through periodic acid-Schiff (PAS) staining of the colon and immunofluorescence staining of HT-29 cells. The potential target was identified via target fishing and colocalization imaging via an alkynyl-modified Lig probe (AM-Lig). Molecular docking, microscale thermophoresis (MST), fluorescence quenching (FQ), and Fluo-4/Ca2+ influx assays were employed to reveal the interaction mode between Lig and the target protein. Finally, we assessed the efficacy of Lig in alleviating constipation in an animal model. ResultsThe efficacy of AS in improving functional constipation was demonstrated in a mouse constipation model, with Lig identified as the primary constituent responsible for inducing colon mucus secretion. Lig specifically targets TRPA1 in the colon, leading to calcium influx and subsequent mucus secretion, ultimately ameliorating functional constipation. Furthermore, a binding mode study revealed that Lig attaches to Thr684, located in the pre-S1 region, triggering TRPA1 channel activation. ConclusionsOur findings demonstrate that Lig, the API in AS for constipation treatment, activates TRPA1 through non-covalent interactions, increasing mucus secretion and improving functional constipation. These findings advance our understanding of the therapeutic mechanism of AS and Lig on constipation and suggest a new approach for developing TRPA1 agonists.