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Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway

  • Authors:
    • Min Li
    • Shu-Li Zhang
    • Feng Yuan
    • Dan Feng
  • View Affiliations / Copyright

    Affiliations: Department of Pain Management, Wuhan Hospital of Traditional Chinese and Western Medicine, Wuhan, Hubei 430000, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 183
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    Published online on: April 28, 2026
       https://doi.org/10.3892/mmr.2026.13893
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Abstract

Ventilator‑induced lung injury (VILI) is a serious complication of mechanical ventilation (MV). The mechanosensitive ion channel Piezo1 converts mechanical forces into biochemical signals; however, its specific role in the pathogenesis of VILI remains unclear. The present study aimed to investigate the role of Piezo1 in lung epithelial cells in mediating VILI and its downstream signalling mechanisms. To this end, the current study utilized a murine VILI model established by high tidal volume MV, lung epithelial‑specific Piezo1 knockout mice, and in vitro cyclic stretch of mouse lung epithelial (MLE‑12) cells combined with genetic knockdown or pharmacological inhibition of Piezo1. Immunofluorescence and immunohistochemical analyses revealed that Piezo1 protein expression was significantly upregulated in the lung epithelium in vivo. Lung epithelial‑specific Piezo1 knockout mice exhibited markedly attenuated MV‑induced lung injury, barrier dysfunction and inflammatory responses. In vitro, cyclic mechanical stretch similarly upregulated Piezo1 expression in mouse lung epithelial MLE‑12 cells, accompanied by cytoskeletal disruption, and release of proinflammatory cytokines IL‑6, TNF‑α and IL‑1β, as assessed using ELISA. Genetic knockdown or pharmacological inhibition of Piezo1 effectively alleviated these injury phenotypes. Mechanistically, Piezo1 activation mediated stretch‑induced Ca2+ influx, which triggered calcineurin activation and subsequent nuclear translocation of the transcription factor NFATc3, ultimately driving the release of proinflammatory cytokines, including IL‑6, TNF‑α and IL‑1β. In conclusion, the results of the present study revealed a novel Piezo1/Ca2+/calcineurin/NFATc3 signalling axis that drives pulmonary epithelial inflammation and barrier dysfunction in VILI, suggesting that Piezo1 and its downstream signalling molecules are potential therapeutic targets.
View Figures

Figure 1

Piezo1 expression is upregulated in
the lung epithelium during ventilator-induced lung injury. (A)
Representative immunofluorescence images of lung sections from
sham-operated control mice and mice subjected to 6 h of mechanical
ventilation. Piezo1 protein (red) is constitutively expressed and
colocalizes (yellow in merged panels) with the epithelial cell
marker CK8 (green). Nuclei were counterstained with DAPI (blue).
Scale bar, 100 µm. (B) Representative images of immunohistochemical
staining for Piezo1 expression in lung epithelium from
sham-operated mice and mice subjected to 6 h of mechanical
ventilation. Scale bar, 20 µm (original magnification, ×400). (C)
Representative haematoxylin and eosin-stained lung sections from
sham-operated and HTV-ventilated mice. Scale bar, 50 µm. (D)
Quantitative lung injury score was determined based on histological
evaluation of alveolar congestion, haemorrhage, leukocyte
infiltration and alveolar wall thickness, each graded from 0
(normal) to 3 (severe). Data are presented as the median
(interquartile range); n=6 mice/group and Pvalues were determined
by Mann-Whitney U test. Lung injury was further evaluated on the
basis of (E) protein concentration, (F) cell number in BALF and (G)
wet/dry weight ratio. (H) MPO activity in lung tissue.
Proinflammatory cytokines in BALF were evaluated using the
following ELISA kits: (I) IL-6, (J) TNF-α and (K) IL-β. (E-K) Data
are presented as the mean ± SD, n=6 mice/group. Pvalues were
determined by Student's t-test. *P<0.05, **P<0.01,
***P<0.001. BALF, bronchoalveolar lavage fluid; CK8, cytokeratin
8; HTV, high tidal volume; MPO, myeloperoxidase.

Figure 2

CS induces Piezo1 expression and
epithelial injury markers in lung epithelial cells in vitro.
(A) Representative western blot analysis of Piezo1 protein
expression in lung epithelial cells under control (unstretched)
conditions, and after 3 or 6 h of CS. (B) Semi-quantification of
Piezo1 protein expression normalized to that of GAPDH. (C) Piezo1
mRNA expression in sham and stretched cells (after 3 and 6 h). (D)
Representative fluorescence images of F-actin (phalloidin, green)
and nuclei (DAPI, blue) in control and stretched cells. Scale bar,
20 µm. Proinflammatory cytokine levels of (E) IL-6, (F) TNF-α and
(G) IL-1β protein levels in sham versus stretched cells. Data are
presented as the mean ± SD, n=6 each. Pvalues were determined by
oneway ANOVA. *P<0.05, **P<0.01, ***P<0.001. CS, cyclic
stretch.

Figure 3

Lung epithelial-specific Piezo1
deletion attenuates ventilator-induced lung injury. (A) Validation
of Piezo1 knockout efficiency in the lung epithelium.
Representative immunofluorescence images of lung sections from
control and Piezo1CKO mice showing the expression of the
Piezo1 protein (red), the epithelial cell marker CK8 (green), and
nuclei (DAPI, blue). Scale bar, 50 µm. (B) Representative
haematoxylin and eosin-stained lung sections from control and
Piezo1CKO mice after 6 h of mechanical ventilation.
Scale bar, 50 µm. (C) Quantitative histopathological lung injury
score. Data are presented as the median (IQR); n=6/group. Data were
analysed using the Kruskal-Wallis test followed by Dunn's post hoc
test. (D) Total protein concentration in BALF. (E) Total cell
counts in the BALF. (F) Lung wet/dry weight ratio. (G) Lung MPO
activity. BALF concentrations of the inflammatory cytokines (H)
IL-6, (I) TNF-α and (J) IL-1β. (D-J) Data are presented as the mean
± SD, n=6 mice/group. Pvalues were determined by oneway ANOVA.
*P<0.05, **P<0.01, ***P<0.001. BALF, bronchoalveolar
lavage fluid; CK8, cytokeratin 8; CKO, conditional; HTV, high tidal
volume; MPO, myeloperoxidase.

Figure 4

Genetic inhibition of Piezo1
attenuates mechanical stretch-induced cytoskeletal disruption and
the inflammatory response in lung epithelial cells in vitro.
(A) Representative western blotting and (B) semi-quantitative
analysis demonstrating efficient knockdown of Piezo1 protein
expression in MLE-12 cells transfected with shPiezo1 compared with
that in cells transfected with Scr-shRNA. (C) Piezo1 mRNA
expression levels measured by quantitative PCR in cells transfected
with shPiezo1 compared with those in cells transfected with
Scr-shRNA. Proinflammatory cytokine levels in cell culture
supernatants from Scr-shRNA and shPiezo1 cells under control
conditions or after 6 h of CS: (D) IL-6, (E) TNF-α and (F) IL-1β
protein levels. (G) Representative fluorescence images showing
F-actin morphology (phalloidin, green) and nuclei (DAPI, blue) in
Scr-shPiezo1 and shPiezo1 cells under control (unstretched)
conditions or after 6 h of CS. Scale bar, 20 µm. Data are presented
as the mean ± SD, n=6 each. Pvalues were determined by oneway
ANOVA. **P<0.01, ***P<0.001. CS, cyclic stretch; Scr,
scrambled; sh, short hairpin.

Figure 5

Piezo1 mediates CS-induced
Ca2+ influx in epithelial cells. (A) Quantitative
analysis of cytosolic Ca2+ levels (ΔF/F0)
measured using a microplate reader in Fluo-3 AM-loaded MLE-12 cells
treated with the Piezo1 agonist Yoda1 (10 µM) for 0, 15 and 30 min.
(B) Representative fluorescence microscopy images of Fluo-3
AM-loaded MLE-12 cells at 30 min following the onset of CS Scale
bar, 50 µm. (C and D) Both genetic knockdown of Piezo1 (with
shPiezo1) and pharmacological inhibition with GsMTx4 (5 µM)
attenuated the Ca2+ elevation induced by 30-min CS. (E)
Genetic knockdown of Piezo1 (with shPiezo1) attenuated mechanical
stretch-induced calcineurin activation after 6 h. (F-H)
Pharmacological inhibition of calcineurin with CsA (10 µM) or FK506
(10 µM) significantly reduced mechanical stretch -induced cytokine
production after 6 h. (I) Disruption of the F-actin network induced
by 6-h mechanical stretching was reversed by pretreatment with CsA
or FK506. Scale bar, 20 µm. Data are presented as the mean ± SD,
n=6 each. Pvalues were determined by oneway ANOVA. *P<0.05,
***P<0.001. CS, cyclic stretch; CsA, cyclosporine A; Scr,
scrambled; sh, short hairpin.

Figure 6

Piezo1 mediates CS-induced
inflammation via calcineurin/NFATc3 signalling. (A)
Immunofluorescence screening of the nuclear translocation of NFAT
isoforms (NFATc1-4) upon CS. NFATc3 showed the most prominent
nuclear translocation. Scale bar, 10 µm. (B) Representative western
blot images showing NFATc3 protein levels in the nuclear and
cytoplasmic fractions of MLE-12 cells transfected with Scr-shRNA or
shPiezo1, with or without CS (6 h). GAPDH and Lamin B1 were used as
loading controls for cytoplasmic and nuclear fractions,
respectively. (C) Semi-quantitative analysis of cytoplasmic NFATc3
protein expression normalized to GAPDH. (D) Semi-quantitative
analysis of nuclear NFATc3 protein expression normalized to Lamin
B1. (E) Representative western blot images showing NFATc3 protein
levels in the nuclear and cytoplasmic fractions of MLE-12 cells
treated with or without calcineurin inhibitors (CsA, 10 µM; FK506,
10 µM) prior to CS (6 h). GAPDH and Lamin B1 were used as loading
controls for cytoplasmic and nuclear fractions, respectively. (F)
Semi-quantitative analysis of cytoplasmic NFATc3 protein expression
normalized to GAPDH. (G) Semi-quantitative analysis of nuclear
NFATc3 protein expression normalized to Lamin B1. Data are
presented as the mean ± SD, n=6 each. Pvalues were determined by
oneway ANOVA. *P<0.05, **P<0.01, ***P<0.001. CS, cyclic
stretch; CsA, cyclosporine A; Scr, scrambled; sh, short
hairpin.
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Spandidos Publications style
Li M, Zhang S, Yuan F and Feng D: Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway. Mol Med Rep 33: 183, 2026.
APA
Li, M., Zhang, S., Yuan, F., & Feng, D. (2026). Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway. Molecular Medicine Reports, 33, 183. https://doi.org/10.3892/mmr.2026.13893
MLA
Li, M., Zhang, S., Yuan, F., Feng, D."Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway". Molecular Medicine Reports 33.6 (2026): 183.
Chicago
Li, M., Zhang, S., Yuan, F., Feng, D."Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway". Molecular Medicine Reports 33, no. 6 (2026): 183. https://doi.org/10.3892/mmr.2026.13893
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Spandidos Publications style
Li M, Zhang S, Yuan F and Feng D: Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway. Mol Med Rep 33: 183, 2026.
APA
Li, M., Zhang, S., Yuan, F., & Feng, D. (2026). Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway. Molecular Medicine Reports, 33, 183. https://doi.org/10.3892/mmr.2026.13893
MLA
Li, M., Zhang, S., Yuan, F., Feng, D."Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway". Molecular Medicine Reports 33.6 (2026): 183.
Chicago
Li, M., Zhang, S., Yuan, F., Feng, D."Mechanotransducer Piezo1 drives ventilator‑induced lung injury in lung epithelial cells via the calcineurin/NFATc3 pathway". Molecular Medicine Reports 33, no. 6 (2026): 183. https://doi.org/10.3892/mmr.2026.13893
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