Inhibition of TRPV4 Regulates Mitophagy Through the Sirt1/FoxO1 Signaling Pathway To Alleviate Acute Lung Injury

Abstract

The transient receptor potential vanilloid 4 (TRPV4) channel has emerged as a key mediator of calcium dysregulation in acute lung injury (ALI), but its role in mitophagy—the selective autophagic clearance of dysfunctional mitochondria—and crosstalk with the Sirtuin 1(Sirt1)signaling axis remain unclear. Lipopolysaccharide (LPS) induced the upregulation of TRPV4, oxidative stress (ROS), and apoptosis in both in vivo and in vitro models. TRPV4 activation (GSK1016790A) exacerbated ALI by impairing mitophagy, as evidenced by reduced LC3/Translocase of the outer mitochondrial membrane 20 (TOMM20) co-localization and decreased PTEN induced kinase 1(PINK1)/PARK2 expression. Conversely, TRPV4 inhibition (GSK2193874) or knockout attenuated lung injury, enhanced mitophagic flux, and reduced mitochondrial damage. Mechanistically, TRPV4 inhibition upregulated Sirt1/Forkhead box O1(FoxO1) signaling, driving PINK1/PARK2-dependent mitophagy. Sirt1 inhibition abrogated these protective effects, confirming its critical role in the TRPV4-mitophagy axis. TRPV4 knockout༈Trpv4⁻/⁻༉mice exhibited reduced pulmonary inflammation, apoptosis, and improved mitochondrial ultrastructure compared to wild-type controls.TRPV4 exacerbated LPS-induced ALI by suppressing Sirt1/FoxO1-mediated mitophagy. Genetic or pharmacological inhibition of TRPV4 restored mitophagic clearance of dysfunctional mitochondria, offering a promising therapeutic strategy for septic ALI. These findings highlighted the TRPV4-Sirt1/FoxO1 axis as a novel target for improving outcomes in critical care settings.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02433-y.

Introduction

Sepsis-induced acute lung injury (ALI) remains a cardinal challenge in critical care, contributing significantly to morbidity and mortality in intensive care unit (ICU) patients. Despite advances in supportive ventilation and antimicrobial therapy, the mortality rate of sepsis-associated ALI persists at 34.9–46.1% [1, 2], primarily due to the lack of targeted therapies addressing its complex pathophysiology. The alveolar-capillary barrier, composed of pulmonary epithelial and endothelial cells, is disrupted by septic insults, triggering mitochondrial dysfunction, calcium overload, and excessive apoptosis-key drivers of hypoxemia and respiratory failure [3].

The transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable ion channel activated by inflammatory cytokines, mechanical stretch, and lipid metabolites [4, 5], has emerged as a critical player in sepsis-induced lung injury [6–9]. Preclinical studies have demonstrated that TRPV4 activation exacerbates pulmonary edema, endothelial barrier breakdown [10], and neutrophil infiltration [11], while genetic knockout or pharmacological inhibition mitigates calcium overload and improves survival in murine models [12, 13]. However, the role of TRPV4 in regulating mitochondrial quality control—particularly mitophagy-in alveolar epithelial cells remains poorly understood, despite its relevance to critical care interventions targeting cellular stress responses.

Multiple studies have established the critical role of mitochondrial dysfunction in the pathogenesis of ALI [14]. Mitophagy, the selective autophagic clearance of dysfunctional mitochondria [15], is essential for maintaining cellular bioenergetics and limiting the release of pro-inflammatory mitochondrial damage-associated molecular patterns (DAMPs), such as mitochondrial DNA (mtDNA); it also helps reduce oxidative stress mediated by reactive oxygen species (ROS) [16, 17]. In endotoxin-induced ALI, mitophagy not only enhances the clearance of damaged mitochondria but also reduces reactive ROS production, thereby mitigating ALI [18, 19]. Upon LPS stimulation, the expression of PINK1 is upregulated, and PARK2 is recruited from the cytoplasm to the mitochondria, which in turn activates PINK1/PARK2-mediated mitochondrial autophagy. PARK2 gene knockout studies have shown that the reduction of mitochondrial autophagy in ALI is due to PARK2’s regulatory effects on the expression of inflammation-associated proteins and downstream signaling pathways [20, 21].In the context of critical care, where intervention timing is critical, defining how TRPV4 influences mitophagic flux in epithelial cells could unlock novel therapeutic windows.

The NAD⁺-dependent deacetylase Sirt1 is a central regulator of mitochondrial health, linking metabolic stress to autophagic control [22]. Sirt1 deacetylates Forkhead box O1(FoxO1), a transcription factor that upregulates PINK1/PARK2 to promote mitophagy [23–25], while also inhibiting pro-apoptotic pathways via deacetylation of p53 and NF-κB [26–30]. Dysfunctional Sirt1 activity, observed in septic patients, correlates with mitochondrial hyperacetylation, calcium dysregulation, and enhanced apoptosis. Given that TRPV4 activation induces calcium influx and Sirt1 inhibition recapitulates mitochondrial phenotypes of ALI, we hypothesized that TRPV4 suppresses Sirt1/FoxO1-mediated mitophagy, thereby exacerbating epithelial injury in sepsis.

Against this backdrop, we aimed to: (1) determine whether TRPV4 activation impairs mitophagic flux in alveolar epithelial cells during Lipopolysaccharide (LPS) challenge; (2) elucidate the molecular mechanism by which TRPV4 interacts with the Sirt1/FoxO1 pathway; (3) evaluate the therapeutic potential of TRPV4 inhibition in restoring mitochondrial quality control and attenuating ALI severity. These insights could inform the development of time-sensitive, cell-type-specific interventions for septic patients, a critical unmet need in critical care medicine.

Methods

The Culture and Treatment of MLE12 Cells

The mouse lung epithelial cell line MLE12 was bought from American Type Culture Collection (ATCC, CRL-2110, USA). Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified incubator at 37 °C with 5% CO₂.All cell lines were tested for mycoplasma contamination. The culture medium was replaced every two days, and cells were used at the third passage.

To model sepsis in vitro, MLE12 cells were treated with various pharmacological agents prior to experimentation. Cells were seeded at a density of 1 × 10⁴ cells/mL in 96-well plates. Experiments were conducted once cells reached 70–80% confluence. To investigate the role of TRPV4 in LPS (Sigma-Aldrich, L2880, USA) stimulated pulmonary epithelial cells, the cells were cultured in 12-well and 6-well plates and randomly divided into four experimental groups: control group, LPS(5 µg/mL) group, GSK1016790A(GSK101) (5 µmol) + LPS group, and GSK2193874 (GSK219)(10 µmol) + LPS group [31, 32]. To clarify the important role of Sirt1 in the regulation of mitochondrial autophagy by TRPV4, cells were treated with the Sirt1 inhibitor EX-527 (10 µmol) in this experiment.

Cell Viability

Cell viability following treatment with various pharmacological agents or dimethyl sulfoxide (DMSO) vehicle was assessed using a CCK-8 assay kit (ELabscience, E-CK-A362, China) according to the manufacturer’s protocol.

ROS and Mitochondrial Membrane Potential (MMP) Measurement

Cells were seeded into 24-well plates and cultured for 24 h prior to drug administration. Fluorescent dye was added following the manufacturer’s instructions for ROS detection. After a 20-minute incubation, the fluorescence intensity was measured using a fluorescence microscope 24 h later.

Cells were stained using a JC-1 assay kit (Solarbio, M8650, China), and fluorescence changes were analyzed (monomer: 515 nm excitation, 529 nm emission; polymer: 585 nm excitation, 590 nm emission) via laser confocal microscopy. The results were analyzed using ImageJ software.

Ca²⁺ Measurement

Cells were cultured in 35 mm glass-bottomed Petri dishes (NEST Scientific, China) for 24 h, followed by group-specific treatments prior to subsequent experiments.Fluo-4AM was added following the protocol of the Fluo-4 Calcium Fluorometric Assay Kit (Elabscience, E-BC-F-100, China). Intracellular calcium levels in live cells were visualized and imaged using a laser confocal microscope at an excitation wavelength of 490 nm.

Apoptosis Assay

Cells were harvested, washed, resuspended in buffer, and stained with Annexin V-FITC and propidium iodide (PI) (Elabscience, E-CK-A211, China). Apoptosis was assessed using a flow cytometer (Attune NxT, Thermo, USA), and data were analyzed using Flow Jo software.

Animals and Treatments

Male C57BL/6 mice (6–8 weeks old, 20–25 g) were purchased from Shandong Pengyue Laboratory Animal Technology Co., Ltd. All procedures adhered to the guidelines specified in the National Research Council’s Guide for the Care and Use of Laboratory Animals and were pre-approved by the Animal Ethics and Welfare Committee of the Affiliated Hospital of Qingdao University. Trpv4⁻/⁻ mice were generated by Shanghai Southern Model Biotechnology Co., Ltd. Animals were caged at a temperature of 23–25 °C and a humidity of 30–70%, with access to food and water.

Mice were administered with LPS (15 mg/kg) for 12 h as previously described and then sacrificed [14]. To validate the regulatory role of TRPV4, mice were randomly allocated into four groups (n = 6): control, LPS, LPS + GSK101 (0.1 mg/kg), and LPS + GSK219 (5 mg/kg). Additionally, Trpv4⁻/⁻ mice were utilized to verify the signaling pathway involved in TRPV4-modulated mitophagy. Euthanasia was performed with meticulous attention to minimizing animal distress, in full compliance with ethical regulations. Blood samples and lung tissues were harvested and stored at − 80 °C for subsequent biochemical analyses and experimental assays.

CT Scan

Chest scans were conducted using a PerkinElmer Quantum GX2 CT scanner (PerkinElmer, USA). Mice were positioned head-down, with the scan covering the region from the lung apex to the diaphragm. The scan parameters were as follows: voltage, 90 kV; current, 88 µA; and spatial resolution, 72 μm. Images were analyzed by two technicians, each with over five years of experience in small animal CT imaging.

Histological Analysis

The left upper lung was fixed in 10% neutral buffered formalin and 3-µm paraffin sections were prepared for hematoxylin and eosin staining (H&E). H&E staining was carried out according to a standardized protocol. H&E staining was carried out according to a standardized protocol: sections were deparaffinized, rehydrated, stained, dehydrated, and mounted in neutral resin before being examined under an optical microscope. The severity of lung injury was assessed based on alveolar edema, neutrophilic infiltration, airway congestion, and parenchymal lesions [33]. Lung injury assessments were performed by blinded pathologists.

Determination of Pulmonary Edema

The left lower lung lobe was weighed to determine the wet tissue weight. The tissue was then dried in an oven at 80 °C for 48 h, and the dry weight was recorded. Lung edema was calculated as the wet-to-dry weight ratio multiplied by 100%.

Evans Blue Staining

Evans blue dye (30 mg/kg)(Sigma-Aldrich, L2880, USA) was injected via tail vein one hour before euthanasia, as previously described [34, 35]. The pulmonary circulation was perfused by phosphate-buffered saline (PBS). Lungs were excised and homogenized in ice-cold PBS. The homogenate was incubated with formamide at 60 °C for 24 h, followed by centrifugation at 13,000 × g for 20 min. The absorbance of the supernatant was measured at 620 nm (A620) and 740 nm (A740). Evans blue content in the tissue, expressed as micrograms per gram of lung tissue per minute, was calculated using the formula: A620 (corrected) = A620 - (1.1649 × A740 + 0.004). The Evans blue index was defined as the ratio of dye content to lung tissue weight.

Bronchoalveolar Lavage Fluid (BALF) Collection

The right main bronchus was ligated in each mouse. A 20-G catheter was inserted into the left main bronchus. The trachea was flushed with 0.5 mL PBS to collect BALF, and cell lysates were added to isolate proteins [35]. Protein concentrations were quantified using a BCA protein assay kit.

Enzyme-linked Immunosorbent Assay (ELISA)

Pro-inflammatory cytokine levels, including TNF-α, IL-1β, and IL-6 in serum, were quantified using ELISA kits according to the manufacturer’s instructions (Servicebio, GB12188, China; Servicebio, GB300002, China; Servicebio, GB30000, China). Cytokine concentrations were quantified using ELISA and calculated based on a standard curve.

Mitochondrial ROS Detection

Single-cell suspensions were prepared from tissues following the manufacturer’s protocol using the Reactive Oxygen Species Assay Kit (Solarbio, CA1410, China). ROS levels were quantified by fluorescence spectrometry (excitation/emission: 488 nm/525 nm) and normalized against a standard calibration curve.

TUNEL Assay

Apoptosis in 3-µm lung tissue sections was assessed using a TUNEL Apoptosis Detection Kit (Elabscience, E-CK-A-320, China) following the manufacturer’s protocol.

Transmission Electron Microscopy

Sample processing and electron microscopy analysis were conducted at the electron microscopy core laboratory of Qingdao University, following established protocols [36]. Each mouse was evaluated by a blinded pathologist based on images from at least three randomly selected regions.

Immunofluorescence

After overnight incubation with primary antibodies at 4 °C, sections were incubated for one hour at room temperature with fluorescently labeled secondary antibodies. The following reagents were used: anti-LC3B (CST, 12741 T, 1:100, USA), anti-TOMM20 (Proteintech, 66777–1-Ig, 1:100, China), and DAPI (Solarbio, s2100, China) for labeling autophagosomes, mitochondria, and nuclei, respectively. After washing three times with TBST, images were acquired using a laser scanning confocal microscope (Olympus U-25ND25 model, Tokyo, Japan).

Western Blot Analysis

To extract total proteins from MLE12 cells and mouse lung tissues, we adhered to the protocol provided by the manufacturer. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes, which were then blocked with tris-buffered saline with 0.1% tween-20(TBST) containing 5% skim milk for one hour at room temperature.

Membranes were incubated overnight at 4 °C with rabbit primary antibodies.Then the membranes underwent incubation with the second antibody. Protein bands were visualized using enhanced chemiluminescence reagent (Elabscience, E-IR-R307, China), and captured using a digital imaging system (Nanjing Zhongke Tongyi, Nanjing, China).

Statistical Analysis

To ensure the precision of the results, every experiment was repeated three times. All data were confirmed to be in normal distribution, and results were presented as means ± standard deviation (SD) and analyzed using Graph Pad Prism 9.0. The t-test and non-parametric test were used for comparisons between two groups. One-way analysis of variance(ANOVA) was applied for comparisons among multiple groups, followed by the Tukey’s honestly significant difference (HSD) test. A P-value threshold of 0.05 was considered statistically significant.

Results

LPS Induced ALI and Promoted the Overexpression of TRPV4

In this study, a murine model of sepsis-induced acute lung injury was established via tail vein injection of LPS (15 mg/kg) for 12 h. High-resolution CT imaging revealed that LPS-treated mice exhibited a marked increase in lung texture complexity, characterized by uneven thickness, a disordered network pattern, and scattered or patchy inflammatory exudates with indistinct boundaries in the lung parenchyma (Fig. 1A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed a marked increase in apoptotic cells in LPS-challenged pulmonary tissues compared to saline-treated controls (Fig. 1B/1 C). H&E staining demonstrated significant damage in the LPS group (Fig. 1D/1E). Western blot analysis demonstrated that LPS challenge significantly elevated expression of TRPV4, Bax and Cleaved-caspase3, while reducing Bcl-2 compared to vehicle controls (Fig. 1F).

Fig. 1.

LPS induced ALI and elevated TRPV4 expression. A Thoracic CT images of mices were observed after 12 h of LPS stimulation (n = 3) B, C TUNEL staining was used to observe the apoptosis rate of lung tissue cells by fluorescence microscopy (original magnification, ×40, scale bar: 20 μm) (n = 6) D, E H&E staining detected the pathological changes of lung tissue and analyze them according to the lung injury score (original magnification,×20, scale bar: 50 μm) F Western blot images showed the expression of apoptosis-related proteins (Bcl-2, Bax, Caspase 3, Cleaved-Caspase 3) (n = 6).

Activation of TRPV4 Exacerbated Lung Injury and Decreased Mitophagy

As a polymodal nonselective cation channel, TRPV4 plays a critical role in pulmonary homeostasis [37]. To elucidate the role of TRPV4 in LPS-induced ALI, mice were pretreated with the TRPV4 agonist GSK101 and the inhibitor GSK219. More apoptotic cells were found in the GSK101 group (Fig. 1B/1 C). H&E staining demonstrated more severe lung tissue damage in the GSK101 group (Fig. 2A/2B). Levels of IL-6, IL-1β, TNF-α, albumin contents in BALF and the wet-to-dry weight ratio revealed that they were significantly elevated in the GSK101 group but markedly reduced in the GSK219 group (Fig. 2C/2D/2E/2F/2G).

Fig. 2.

Lung injury aggravated after activation of TRPV4. A, B HE staining indicated that in the TRPV4 agonist group, lung tissue injury was aggravated, and the lung injury score was high (original magnification, ×20; scale bar: 50 μm) (n = 6) C-E The inflammatory factors in mouse serum (IL-6,IL-1β,TNF- α) were detected by ELISA(n = 6) F The BALF of mice was collected to measure the albumin concentration G The dry-wet ratio measured the edema and exudation in the lung tissue H-J Laser confocal microscopy was used to visualize the co-localization of LC3 (red) and TOMM20 (green) in lung tissue sections. Cell nuclei were stained with DAPI (blue) (original magnification, ×40; scale bar: 20 μm) (n = 6) K Western blot images showed the expression of apoptosis-related proteins and autophagy-related proteins (TRPV4, LC3, and TOMM20) in mouse lung tissues (n = 6) L Evans blue examined the permeability of pulmonary blood vessels in each group (n = 6).

Co-localization studies of LC3 and the outer mitochondrial membrane 20 (TOMM20) provided visual evidence of the interaction between autophagosomes and mitochondria during autophagy [25]. Building upon established links between TRPV4 and autophagic regulation [29], we quantitatively assessed LPS-induced mitophagy in pulmonary tissues through LC3-II/TOMM20 co-localization analysis, combining confocal microscopy with computational image analysis. Immunofluorescence co-localization of LC3 with TOMM20 confirmed autophagosome-mitochondria’s interaction, indicative of ongoing mitophagy (Fig. 2H/2I/2J). Protein detection results were consistent with the TUNEL assay outcomes. Specifically, the expressions of the pro-apoptotic proteins Bax and Cleaved - caspase3 were upregulated. Moreover, an elevation in the expressions of LC3 and TOMM20 was observed in the TRPV4 inhibition group (Fig. 2K). The assessment of pulmonary vascular permeability via Evans Blue dye indicated a remarkable increase in the agonist group. This finding was consistent with the albumin concentration measured in BALF, thus validating the consistency of the experimental results (Fig. 2L).

Inhibition of TRPV4 Could Alleviate the Damage and Mitochondrial Dysfunction of MLE12 Cells

Considering that alveolar epithelial cells play an important role in acute lung injury [38]and TRPV4 also exerts significant functions in alveolar epithelial cells [6], we used MLE-12 cells as the research object to explore the effect of TRPV4 on mitophagy in vitro. MLE-12 cells were pretreated with GSK101 or GSK219 for 30 min, followed by stimulation with LPS for 24 h. Compared to the control group, the LPS-treated cells showed a significant 25% reduction in viability (P < 0.001, CCK-8 assay). In contrast, treatment with GSK219 (10 µM) could reverse this effect, increasing cell viability by 6% and reducing calcium overload (detected by Fluo-4 staining), decreasing ROS production (DCFH-DA assay), and lowering the apoptotic cell ratio (Annexin V/PI staining) compared to the LPS group. Conversely, GSK101 (5 µM) exacerbated cellular damage, as evidenced by a 36% decrease in viability, elevated intracellular calcium levels and ROS generation, which was in line with the detrimental effects of TRPV4 activation (Fig. 3A 3B/3 C/3D/3E/3F).

Fig. 3.

Inhibition of TRPV4 increased mitochondrial autophagy and attenuated damage of MLE12 cells. A CCK-8 assay was detected to evaluate cell viability in each group (n = 6) B, C Intracellular calcium ion concentrations were measured using laser confocal microscopy (original magnification,×64; scale bar: 50 μm) D, E Fluorescence microscopy was used to assess ROS levels F Cell apoptosis in each group was detected by flow cytometry (original magnification,×64; scale bar: 50 μm) (n = 6) G, H Changes in mitochondrial membrane potential were detected using laser confocal microscopy with JC-1 staining(original magnification, ×64; scale bar: 50 μm) (×64; scale bar: 20 μm) (n = 6). Nuclei were revealed using DAPI staining I The expressions of autophagy-related proteins PINK1 and PARK2 and their upstream regulatory factor Sirt1、FoxO1 in mitochondria were measured by Western Blot.

To investigate mitochondrial damage, we assessed MMP using JC-1 staining. As shown in Fig. 3G and H, LPS stimulation significantly elevated MMP (red/green fluorescence ratio) and induced mitochondrial damage, whereas GSK219 treatment decreased MMP and alleviated injury, as confirmed by laser confocal detection. To examine the impact of TRPV4 on mitophagy, we performed western blot analysis to quantify the protein expression levels. Consistent with these findings, TRPV4 inhibition markedly increased the expression levels of Sirt1, FoxO1, PINK1 and PARK2 in MLE12 cells (Fig. 3I). These results demonstrated that TRPV4-regulated mitochondrial dysfunction was linked to Sirt1 signaling pathway activity.

Sirt1 Served as a Pivotal Node in TRPV4-governed Mitophagy

TRPV4 inhibition not only enhanced Sirt1 expression, but also upregulated the Sirt1/FoxO1 signaling pathway and its downstream mitophagy-related proteins. To elucidate the functional involvement of Sirt1 in TRPV4-dependent mitophagy modulation, we suppressed Sirt1 expression and examined changes in mitophagy-related indices. MLE-12 cells were incubated with the Sirt1 inhibitor EX-527 for 1 h before being treated with GSK219. Experimental results demonstrated that, in comparison with the GSK219-treated group, EX-527 treatment significantly impaired MLE-12 cell viability, accompanied by calcium overload, elevated ROS production, and increased apoptotic rates (Fig. 4A/4B/4 C/4D/4E/4F). JC-1 staining analysis revealed heightened mitochondrial membrane potential in EX-527-treated cells, indicating exacerbated mitochondrial dysfunction (Fig. 4G/4H). Notably, Sirt1 inhibition suppressed the expression of key mitophagy regulators (FoxO1, PINK1, and PARK2) in MLE12 cells (Fig. 4I). These data suggest that mitochondrial autophagy decreases following Sirt1 inhibition, indicating that TRPV4 may act on pulmonary epithelial cell mitochondrial autophagy through the important Sirt1/FoxO1 pathway.

Fig. 4.

SIRT1 is an important factor for trpv4 to regulate mitochondrial autophagy. A Cell viability in each group was assayed using the CCK-8 method (n = 6) B, C Intracellular calcium ion concentrations were measured by laser confocal microscopy (original magnification,×64; scale bar: 50 μm) (n = 6) D, E Fluorescence microscopy measured ROS levels (original magnification,×64; scale bar: 50 μm) (n = 6) F Flow cytometry was utilized to quantify cell apoptosis across groups (n = 6) G, H With JC-1 staining, laser confocal microscopy detected changes in mitochondrial membrane potential (original magnification,×64; scale bar: 20 μm) (n = 6).Nuclei were revealed using DAPI staining I Protein detection measured the expression levels of mitochondrial proteins (n = 6).

TRPV4 Knockout Activated Mitophagy Mediated the Sirt1/FoxO1 Pathway and Alleviated Acute Lung Injury

To further explore the role of TRPV4 in regulating mitophagy via the Sirt1/FoxO1 pathway in vivo, we generated Trpv4⁻/⁻ for study (Fig. 5A/5B). High-resolution CT imaging of Fig. 1A showed that the Trpv4^–/– group exhibited significantly diminished pulmonary tissue markings, reduced exudative changes, and alleviated lung pathology compared with LPS-challenged wild-type animals. The H&E staining results showed that the degree of inflammatory infiltration and lung injury in the Trpv4^–/– group was significantly reduced (Fig. 5C/5D).

Fig. 5.

Knockout of TRPV4 alleviated LPS induced acute lung injury. A, B displayed the identification results of the knockout mice (n = 6) C, D HE staining revealed the lung tissue injury (original magnification,×20; scale bar: 50 μm) (n = 6) The mitochondrial ROS levels were shown in E (n = 6) F–H The expression levels of inflammatory factors including TNF-α, IL-1β, and IL-6 in each group were measured I Evans blue staining was utilized to assess the vascular permeability within lung tissue J, K Laser confocal microscopy detected apoptosis in TUNEL-stained lung tissues (original magnification, ×40; scale bar: 20 μm) (n = 6) L The expression levels of apoptosis-associated proteins and their co-localization patterns were detected. (n = 6) M Validation of TRPV4 protein expression in TRPV4 knockout (Trpv4⁻/⁻) mice (n = 3).

Genetic ablation of TRPV4 significantly diminished ROS production, decreased levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and alleviated pulmonary vascular permeability (Fig. 5E/5F/5G/5H/5I). TUNEL analysis revealed markedly decreased apoptosis rates in the Trpv4^–/– group, indicating that TRPV4 knockout conferred substantial protection against alveolar injury (Fig. 5J/5K). The depletion of TRPV4 elicited a marked decrease in the expression of apoptosis-associated proteins (Fig. 5L). To confirm the reliability of our findings, we verified via western blotting that TRPV4 expression was markedly diminished in TRPV4-knockout mice relative to controls. (Fig. 5M).

The LC3/TOMM20 co-localization analysis demonstrated enhanced protein interaction following TRPV4 ablation compared with the WT LPS group, suggesting mitophagy activation might mediate the protective effects of Trpv4 deletion (Fig. 6A/6B/6 C).Western blot measurements showed that the protein expression of Sirt1, FoxO1, PINK1 and PARK2 increased, whereas the WT LPS group suppressed these proteins (Fig. 6D). Transmission electron microscopy (TEM) revealed that Trpv4 knockout substantially ameliorated LPS-induced mitochondrial damage and increased autophagosome formation (Fig. 6E).

Fig. 6.

Knockout of TRPV4 activated the SIRT1/FOXO1 pathway and mitochondrial autophagy. A,B, C Laser confocal microscopy visualized the co-localization of LC3 (red) and TOMM20 (green) in lung tissue sections, with cell nuclei counterstained by DAPI (blue) (original magnification, ×40; scale bar: 20 μm) (n = 3) D Western blot analysis examined mitochondrial autophagy-related proteins, including PINK1, PARK2, Sirt1, FoxO1, and Acetyl-FoxO1 (n = 6) E Transmission electron microscopy (TEM) revealed the morphological changes of mitochondria. The lower panel provided a magnified view of mitochondria from the upper panel. Detached, small, rounded mitochondria with ill-defined cristae were indicated by red arrows, while the yellow arrow pointed to an autophagosome (n = 3).

Discussion

Our study identified a critical role of TRPV4 in driving LPS-induced acute lung injury (ALI) through suppression of the Sirt1/FoxO1-mediated mitophagy pathway, providing a mechanistic link between ion channel dysfunction, mitochondrial quality control, and inflammatory pathogenesis in sepsis-related lung injury (Fig. 7).

Fig. 7.

TRPV4 modulates mitophagy via the Sirt1/FoxO1 signaling axis. Activation of TRPV4 triggered intracellular calcium overload, exacerbated oxidative stress, suppressed Sirt1 expression, diminished FoxO1 deacetylation, inhibited mitochondrial autophagy, and induced cell apoptosis. By contrast, pharmacological inhibition or genetic ablation of TRPV4 alleviated intracellular calcium overload, promoted the co-localized expression of proteins including LC3 and TOMM20 on the mitochondrial membrane, activated the Sirt1/FoxO1 signaling cascade, enhanced mitochondrial autophagic flux, and mitigated cellular injury.

In both murine models and pulmonary epithelial cells, TRPV4 activation exacerbated LPS-induced tissue damage, apoptosis, and inflammatory cytokine release (e.g., TNF-α, IL-6), whereas genetic or pharmacological inhibition of TRPV4 conferred robust protection. All data prompt us to explore its relevance in human patients. These findings aligne with clinical observations of TRPV4 upregulation in human ALI/ARDS patients, where elevated TRPV4 expression correlates with disease severity and poor outcomes [39, 40]. The functional significance of TRPV4 in mitochondrial dysfunction was highlighted by fluorescence co-localization and TEM analyses, which revealed that TRPV4 activation disrupted autophagosome-mitochondria interaction and enhanced mitochondrial damage, whereas inhibition restored mitophagic flux. This disruption and restoration pattern is consistent with the known role of TRPV4 in regulating calcium - related processes. This aligns with prior studies linking TRPV4 to pathological calcium overload in respiratory diseases, including COPD and asthma [13], where TRPV4-mediated Ca²⁺ influx drives airway hyperresponsiveness and epithelial dysfunction.

A key finding of our study is that Sirt1 acts as a critical downstream effector of TRPV4 in mitophagy regulation. Pharmacological inhibition of Sirt1 (EX-527) abrogated the protective effects of TRPV4 antagonism, leading to exacerbated calcium dysregulation, ROS production, and apoptosis—phenotypes reversed by TRPV4 knockout. Mechanistically, Sirt1 deacetylated FoxO1, enhancing its transcriptional activity to upregulate PINK1/PARK2, critical drivers of the mitophagic pathway [41]. This signaling axis was conserved across cell types, as demonstrated in MLE-12 epithelial cells and Trpv4⁻/⁻ mouse lungs, where Sirt1/FoxO1 activation was correlated with increased autophagosome formation and reduced mitochondrial damage.

Notably, the Sirt1/FoxO1 pathway’s role in mitophagy aligned with its established functions in metabolic stress and aging [28]. For example, Sirt1-mediated deacetylation of PARK2 enhances its E3 ligase activity [42], facilitating ubiquitination of damaged mitochondria for autophagic clearance, a mechanism that is disrupted in neurodegenerative diseases [43]. In the context of ALI, our data suggest that TRPV4 suppresses Sirt1 activity and impairs mitophagic clearance of dysfunctional mitochondria and promotes inflammatory signaling. This is further supported by the observation that Sirt1 agonists (resveratrol) protected against lung injury in preclinical models, thereby reinforcing the therapeutic potential of this pathway [44].

TRPV4’s role in ALI is closely linked to intracellular calcium homeostasis [45]. As a non-selective cation channel, TRPV4 mediates LPS-induced Ca²⁺ influx, which activates CaMKII and promotes phosphorylation of the mitochondrial calcium uniporter(MCU) at Ser92, thereby enhancing mitochondrial Ca²⁺ uptake and membrane potential depolarization [46]. Concurrently, Sirt1 deacetylates MCU at Lys332 to reduce its activity [24]. So, the suppression of Sirt1 by TRPV4 may disrupt this balance; thus, TRPV4-induced suppression of Sirt1 likely exacerbates MCU hyperacetylation, creating a vicious cycle of calcium overload and mitochondrial dysfunction. This mechanism is reminiscent of cardioprotective pathways where Sirt1-MCU crosstalk regulates ischemia-reperfusion injury [47], suggesting conserved mechanisms across organ systems.

The anti-inflammatory effects of mitophagy observed in our study stem from the selective elimination of damaged mitochondria, which serve as sources of pro-inflammatory DAMPs (e.g., mtDNA, ROS) [17]. In Trpv4⁻/⁻ mice, enhanced mitophagy was correlated with reduced BALF cytokine levels and attenuated vascular permeability, mirroring findings in models of Parkinson’s disease where PINK1/PARK2-dependent mitophagy suppresses mtDNA-induced neuroinflammation [48]. Clinically, increased LC3/TOMM20 co-localization (a marker of mitophagy) in human ALI lung tissues correlates with improved survival, supporting the hypothesis that enhancing mitophagic flux may represent a novel therapeutic strategy for sepsis-related lung injury [49].

Some studies indicate that the TRPV4 channels in different types of lung cells play distinct roles in lung injury, which may contribute to different pathological processes. For instance, following LPS stimulation, TRPV4 activation triggers the activation of cAMP response element-binding protein (CREB) in macrophages, thereby upregulating interleukin-10 (IL-10) and mitigating mitochondrial damage [50]. By contrast, in endothelial cells, researchers have demonstrated either endothelial-specific deletion of TRPV4 or pharmacological blockade of this channel preserved pulmonary function—assessed via clinically relevant metrics including arterial oxygen partial pressure (PaO₂) and lung compliance—and reduced inflammation and edema following ischemia-reperfusion (IR) injury [51]. This observation aligns with our own findings. The contradictory roles of TRPV4 in different cells indicate that further in-depth research is still needed in the future to explore its role in lung injury, thereby providing strong evidence for confirming TRPV4 as a therapeutic target.

Despite their progress in pain and itch research, while TRPV4 antagonists (GSK219) are in early-phase clinical trials for pain and itch [31], their potential in ALI/ARDS remains unexplored. Our preclinical data suggest that targeting TRPV4 can offer dual benefits: reducing calcium overload-driven inflammation while enhancing Sirt1-mediated mitophagy. However, challenges remain, including off-target effects of pan-TRPV4 inhibitors and the need for lung-specific delivery. These challenges are interrelated; for example, the off - target effects may be exacerbated by the lack of lung-specific delivery. Additionally, the temporal window for intervention (early vs. late inflammation) and cell-type specificity require further clarification. Our findings suggest that inhibition or knockout of TRPV4 in ALI mice results in increased expression of PINK1/PARK2 and enhanced LC3/TOMM20 co-localization—observations indicative of a close association with mitophagy (Fig.7). This aligns with previous research [52, 53]; however, direct evidence of autophagic flux remains lacking. Further validation of this phenomenon will be conducted in future studies.

Conclusion

Our study demonstrates that TRPV4 is a critical driver of ALI by suppressing the Sirt1/FoxO1/mitophagy axis and provides a mechanistic foundation for developing TRPV4-targeted therapies. Since TRPV4’s suppression of the Sirt1/FoxO1/mitophagy axis leads to mitochondrial dysfunction and enhanced inflammation in ALI, by restoring mitochondrial quality control and dampening inflammatory signaling, TRPV4 inhibition offers a promising strategy to improve outcomes in sepsis - related lung injury.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

All authors contributed to the study conception and design.\u0000Xiuyun Wu, Jianbo Yu, Shu’an Dong conducted the conceptualization and the methodology. Data collection was performed by Shasha Liu and Qin Zhao. Formal analysis and investigation were performed by Mu Xu, Changxin Jia, Jia Shi .\u0000Xiuyun Wu wrote the original draft. Jianbo Yu and Shu’an Dong reviewed and revised the article. All authors read and approved the final manuscript.

Funding

This study was funded by the Tianjin Key Medical Discipline Construction Project (Grant No. TJYXZDXK-3-013B), the National Natural Science Foundation of China (Grant No. 82002069), and the Joint General Program of Tianjin Natural Science Foundation (Grant No. 25JCLMJC00110).

Data Availability

All data, materials and software application information are included in the manuscript and supplementary materials, and can also be obtained from the corresponding author upon reasonable request.

Declarations

Ethics Approval and Consent to Participate

All procedures followed the guidelines of the National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics and Welfare Committee of the Affiliated Hospital of Qingdao University (Approval No. AHQU-MAL20230730WX).

Competing Interests

The authors declare no competing interests.

Clinical Trial Number

Not applicable.