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Article Open Access

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

  • Authors:
    • Xiangyun Yan
    • Peitao Ma
    • Wen Wang
    • Weijian Zeng
    • Yanqiu Li
    • Yujun Hou
    • Jiangnan Ye
    • Qianhua Zheng
    • Wei Zhang
    • Junpeng Yao
    • Ying Li
  • View Affiliations / Copyright

    Affiliations: School of Acupuncture and Tuina, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan 611137, P.R. China
    Copyright: © Yan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 178
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    Published online on: September 1, 2025
       https://doi.org/10.3892/ijmm.2025.5619
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Abstract

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.
View Figures

Figure 1

Colocalization and expression of
Piezo1 and Piezo2 in EC cells of FC Mice. (A) Immunofluorescence
staining of Piezo1, Piezo2 and EC cells in colonic sections from
each group. DAPI (blue) indicates nuclei, Piezo2 (green), Piezo1
(purple) and EC cell (red). Scale bar, 50 μm (magnification,
×200). Yellow arrows indicate co-localization of Piezo1 and ChgA;
orange arrows indicate co-localization of Piezo2 and ChgA; white
arrows indicate triple co-localization of Piezo1, Piezo2 and ChgA.
Percentage of (B) Piezo1+ChgA+, (C)
Piezo2+ChgA+ and (D)
Piezo1+Piezo2+ChgA+ cells as a
percentage of total ChgA+ cells in control and FC
groups; n=5 mice per group. (E) Quantification of Piezo1 and (F)
Piezo2 mRNA levels relative to β-actin in the control and FC group;
n=8 mice per group. (G) Western blot analysis of Piezo1 and Piezo2
protein expression in the control and FC groups; n=3 mice per group
Target proteins and internal controls were detected on separate
membranes processed in parallel. The vertical line indicates
membrane separation. (H) Co-immunoprecipitation of Piezo1 and
Piezo2 in colonic tissues from control and FC model mice, with
Piezo1 immunoprecipitated and Piezo2 detected by western blotting.
Data are presented as mean ± SD and were analyzed using an unpaired
two-tailed Student's t-test. Statistical significance indicated as
*P<0.05, **P<0.01. EC, enterochromaffin
cell; FC, functional constipation.

Figure 2

Piezo1/Piezo2 knockdown attenuates
expression of Piezos in EC cells of FC mice. (A) Schematic of the
experimental timeline for AAV-mediated knockdown. RT-qPCR analysis
of (B) Piezo1 and (C) Piezo2 mRNA levels in the control AAV, AAV
Piezo1-KD, AAV Piezo2-KD and AAV Piezo1/2-KD groups; n=5-7 mice per
group. Data analyzed by one-way ANOVA with Tukey's post hoc test.
(D) Immunofluorescence staining of Piezo1, Piezo2 and EC cells in
colonic sections from each group. DAPI (blue) indicates nuclei,
Piezo2 (green), Piezo1 (purple) and EC cell (red). Scale bar, 50
μm (magnification, ×200). Yellow arrows indicate
co-localization of Piezo1 and ChgA; orange arrows indicate
co-localization of Piezo2 and ChgA; white arrows indicate triple
co-localization of Piezo1, Piezo2 and ChgA. Percentage of (E)
Piezo1+ChgA+, (F)
Piezo2+ChgA+ and (G)
Piezo1+Piezo2+ChgA+ cells as a
percentage of total ChgA+ cells in control and knockdown
groups; n=5 mice per group. Data represent mean ± SD and were
analyzed using Kruskal-Wallis test. Statistical significance is
indicated as *P<0.05, **P<0.01,
***P<0.001. ns, not significant; EC, enterochromaffin
cell; FC, functional constipation; AAV, adeno-associated virus; KD,
knockdown.

Figure 3

Effects of Piezo1 and Piezo2
knockdown on gastrointestinal motility and colonic sensitivity in
FC Mice. (A-H) General effects of the FC model compared with
control mice. Data were analyzed using an unpaired two-tailed
Student's t-test. (I-P) Effects of individual or simultaneous
Piezo1 and Piezo2 knockdown on gastrointestinal motility and
sensitivity in FC mice. Data were analyzed by one-way ANOVA with
Tukey's post hoc test or Kruskal-Wallis test. (A and I) TGITT, (B
and J) Gastric emptying rate, (C, K, E and L) Small intestinal
transit rate, (D and M) Gas volume required to reach an AWR score
of 3 and AWR scores at gas volumes of 0.25, 0.35, 0.50 and 0.65 ml.
(F and N) Histological analysis of colonic tissue in each group;
n=3 mice per group. Red arrows highlight regions of glandular
necrosis with structural disorganization and green arrows indicate
nuclear pyknosis and cytoplasmic dissolution. Scale bar, 100
μm (left, magnification, ×100) and 10 μm (right,
magnification, ×400). (G and O) Serum levels of VIP in each group,
(H and P) Serum levels of SP in each group. Data were analyzed
using an unpaired two-tailed Student's t-test or one-way ANOVA with
Tukey's post hoc test. n=6-9 mice per group. Data represent mean ±
SD. Statistical significance is indicated as *P<0.05,
**P<0.01, ***P<0.001. FC, functional
constipation; TGITT, total gastrointestinal transit time; AWR,
abdominal withdrawal reflex VIP, vasoactive intestinal peptide; SP,
substance P.

Figure 4

Effects of Piezo1 and Piezo2
knockdown on 5-HT synthesis, release and SERT expression in FC
Mice. (A) Representative immunohistochemical staining of
5-HT3 and SERT in colonic sections from control and FC
mice. Scale bar, 40 μm (magnification, ×400). Quantification
of (B) serum 5-HT and (C) colonic TPH-1 levels in control and FC
mice. Data were analyzed using an unpaired two-tailed Student's
t-test; n=4-6 mice per group. (D) Immunohistochemical staining of
5-HT3 and SERT in colonic sections from control AAV, AAV
Piezo1-KD, AAV Piezo2-KD and AAV Piezo1/2-KD groups. n=3 mice per
group. Scale bar, 40 μm (magnification, ×400). (E and F)
Serum and colonic 5-HT levels in each group, comparing knockdown
groups to the control AAV group. (G and H) Relative mRNA levels of
SERT and colonic TPH-1 levels in each group, comparing knockdown
groups to the control AAV. Data were analyzed by one-way ANOVA with
Tukey's post hoc test. n=4-6 mice per group. Data are presented as
mean ± SD, with statistical significance indicated as
*P<0.05, **P<0.01,
***P<0.001. 5-HT, 5-hydroxytryptamine; SERT,
serotonin transporter; FC, functional constipation; TPH-1,
tryptophan hydroxylase-1; AAV, adeno-associated virus; KD,
knockdown.

Figure 5

Expression changes of Piezo1 and
Piezo2 in EC cells following individual and combined knockdown.
Representative Western blot analysis of (A) Piezo1 and (B) Piezo2
protein expression in the NC, VC, LV-Piezo1 KD (Piezo1 knockdown),
LV-Piezo2 KD (Piezo2 knockdown) and LV-Piezo1/2 KD (combined Piezo1
and Piezo2 knockdown) groups. GAPDH served as the loading control.
Target proteins and internal controls were detected on separate
membranes processed in parallel. The vertical line indicates
membrane separation. Quantification of (C) Piezo1 and (D) Piezo2
mRNA levels relative to GAPDH; n=3 cells per group. Data are
presented as mean ± SD and were analyzed by one-way ANOVA with
Tukey's post hoc test. Statistical significance indicated as
*P<0.05, **P<0.01,
***P<0.001. EC, enterochromaffin cell; NC, negative
control; VC, vector control; KD, knockdown.

Figure 6

Effect of Piezo1 and Piezo2 knockdown
on intracellular Ca2+ levels in ECs. (A) Representative
immunofluorescence images of intracellular Ca2+ levels
in different groups. DAPI (blue) was used to stain nuclei and
Ca2+ indicator (green) shows intracellular calcium
levels. Merged images display co-localization of nuclei and
Ca2+ fluorescence. Scale bar, 50 μm
(magnification, ×400). (B) Quantification of Ca2+
fluorescence intensity in each group. (C) Quantification of
Ca2+ fluorescence counts in each group; n=3 cells per
group. Data are presented as mean ± SD and were analyzed by one-way
ANOVA with Tukey's post hoc test. Statistical significance
indicated as *P<0.05, **P<0.01,
***P<0.001. ns, not significant; EC, enterochromaffin
cell.

Figure 7

Effect of Piezo1 and Piezo2 knockdown
on 5-HT and TPH-1 levels in enterochromaffin cells. (A)
Quantification of 5-HT levels (ng/l) in the different groups. (B)
Quantification of TPH-1 enzyme activity (U/l) in each group. n=3
cells per group. Data are presented as mean ± SD and were analyzed
by one-way ANOVA with Tukey's post hoc test. Statistical
significance indicated as *P<0.05,
**P<0.01, ***P<0.001. ns, not
significant; 5-HT, 5-hydroxytryptamine; NC, negative control; VC,
vector control; KD, knockdown; TPH-1, tryptophan hydroxylase-1.

Figure 8

Mechanism diagram of the study. In
functional constipation, Piezo1 and Piezo2 expression is
downregulated, reducing Ca2+ influx and impairing 5-HT
synthesis. Consequently, diminished 5-HT release reduces activation
of 5-HT3 receptors, SP levels decrease and VIP levels
increase, leading to smooth muscle relaxation. Additionally,
heightened SERT activity, exacerbating the deficiency in 5-HT
signaling. These disruptions collectively induce pathological
alterations in intestinal structure and impaired motility, leading
to functional constipation symptoms. (created by figdraw.com). 5-HT, 5-hydroxytryptamine; SP,
substance P; VIP, vasoactive intestinal peptide; SERT, serotonin
transporter; EC, enterochromaffin cell.
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Copy and paste a formatted citation
Spandidos Publications style
Yan X, Ma P, Wang W, Zeng W, Li Y, Hou Y, Ye J, Zheng Q, Zhang W, Yao J, Yao J, et al: Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation. Int J Mol Med 56: 178, 2025.
APA
Yan, X., Ma, P., Wang, W., Zeng, W., Li, Y., Hou, Y. ... Li, Y. (2025). Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation. International Journal of Molecular Medicine, 56, 178. https://doi.org/10.3892/ijmm.2025.5619
MLA
Yan, X., Ma, P., Wang, W., Zeng, W., Li, Y., Hou, Y., Ye, J., Zheng, Q., Zhang, W., Yao, J., Li, Y."Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation". International Journal of Molecular Medicine 56.6 (2025): 178.
Chicago
Yan, X., Ma, P., Wang, W., Zeng, W., Li, Y., Hou, Y., Ye, J., Zheng, Q., Zhang, W., Yao, J., Li, Y."Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation". International Journal of Molecular Medicine 56, no. 6 (2025): 178. https://doi.org/10.3892/ijmm.2025.5619
Copy and paste a formatted citation
x
Spandidos Publications style
Yan X, Ma P, Wang W, Zeng W, Li Y, Hou Y, Ye J, Zheng Q, Zhang W, Yao J, Yao J, et al: Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation. Int J Mol Med 56: 178, 2025.
APA
Yan, X., Ma, P., Wang, W., Zeng, W., Li, Y., Hou, Y. ... Li, Y. (2025). Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation. International Journal of Molecular Medicine, 56, 178. https://doi.org/10.3892/ijmm.2025.5619
MLA
Yan, X., Ma, P., Wang, W., Zeng, W., Li, Y., Hou, Y., Ye, J., Zheng, Q., Zhang, W., Yao, J., Li, Y."Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation". International Journal of Molecular Medicine 56.6 (2025): 178.
Chicago
Yan, X., Ma, P., Wang, W., Zeng, W., Li, Y., Hou, Y., Ye, J., Zheng, Q., Zhang, W., Yao, J., Li, Y."Piezo knockdown reduces 5‑hydroxytryptamine release from enterochromaffin cells and exacerbates intestinal dyskinesia in mice with functional constipation". International Journal of Molecular Medicine 56, no. 6 (2025): 178. https://doi.org/10.3892/ijmm.2025.5619
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