Overactivation of TRPV4 by toosendanin induces intracellular calcium overload and colonic toxicity

Ethnopharmacological relevance Fructus Meliae Toosendan (FMT) is a widely utilized natural medicine across various traditional medical systems, with a long history of extensive application. However, excessive use of FMT may result in severe intestinal toxicity. The specific components responsible for this toxicity and the underlying mechanisms remain unclear. Materials and methods The compositional differences between FMT and stir-fried FMT (SFFMT) were identified using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Subsequently, bioinformatics analysis, molecular docking, kinetic modeling and microscale thermophoresis (MST) were employed to ascertain that toosendanin (TSN) targets intestinal transient receptor potential vanilloid receptor 4 (TRPV4). Calcium ion (Ca 2+ ) fluorescent probes, flow cytometry, immunofluorescence analysis and western blotting were employed to assess the elevation of calcium ions induced by TSN, which subsequently resulted in intestinal barrier damage. Finally, H&E staining, mouse endoscopy and immunofluorescence analysis confirmed that excessive oral administration of TSN leads to intestinal barrier damage in mice. Results Firstly, the content of TSN in SFFMT significantly decreased compared to that in FMT. Secondly, we present that TSN activates TRPV4, and that excessive TSN intake leads to elevated Ca 2+ levels and overload in colonic epithelial cells, which induces acute colonic toxicity by damaging the colonic barrier. It has been demonstrated in mouse models that high doses of TSN induce colonic barrier damage. Conclusions Excessive TSN induces abnormal activation of TRPV4, leading to an elevation of intracellular calcium ions. This phenomenon is recognized as the primary cause of colonic toxicity associated with FMT.