Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation

Enterochromaffin (EC) cell dysfunction decreases 5‑hydroxytryptamine (5‑HT) secretion, contributing to functional constipation (FC). However, the underlying mechanisms remain unclear. Piezo ion channels mediate 5‑HT release from EC cells. The present study investigated the roles and mechanisms of Piezo1 and Piezo2 in the pathogenesis of FC and explored possible interactions. In a loperamide‑induced FC mouse model, Piezo1 and Piezo2 were singly or simultaneously knocked down using adeno‑associated viruses. In vitro, their function in EC cells was assessed via lentiviral‑mediated knockdown in the QGP‑1 cell line. In FC mice, the expression of Piezo1 and Piezo2, along with their colocalization with EC cells, was significantly reduced. Knockdown of either channel impaired intestinal motility, prolonged gastrointestinal transit time, delayed gastric emptying and reduced small intestinal propulsion. Correspondingly, 5‑HT, 5‑HT₃ receptor and tryptophan hydroxylase‑1 (TPH‑1) levels were decreased. Dual knockdown exacerbated these effects, resulting in colon structural abnormalities, decreased substance P expression and increased serotonin transporter levels. Knockdown of Piezo1 or Piezo2 reduced ERK and protein kinase C (PKC) phosphorylation in colonic tissues, with combined knockdown producing a more pronounced suppression of PKC phosphorylation. Consistently, dual knockdown in EC‑like cells led to more pronounced reductions in intracellular calcium, 5‑HT and TPH‑1 compared with single knockdowns. These findings demonstrated that Piezo1 and Piezo2 play critical and cooperative roles in maintaining intestinal homeostasis in FC mice by jointly inducing calcium ion influx in EC cells, thereby coordinating 5‑HT signaling homeostasis. Targeting Piezo channels may offer novel therapeutic avenues for managing functional constipation.