Journal: Inflammation

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

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: 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).

Article Snippet: 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.

Techniques: Expressing, TUNEL Assay, Staining, Fluorescence, Microscopy, Western Blot

Journal: Inflammation

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

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: 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).

Article Snippet: 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.

Techniques: Activation Assay, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Confocal Microscopy, Western Blot, Expressing, Permeability

Journal: Inflammation

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

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: 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.

Article Snippet: 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.

Techniques: Inhibition, CCK-8 Assay, Confocal Microscopy, Fluorescence, Microscopy, Flow Cytometry, Membrane, Staining, Western Blot

Journal: Inflammation

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

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: 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).

Article Snippet: 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.

Techniques: CCK-8 Assay, Confocal Microscopy, Fluorescence, Microscopy, Flow Cytometry, Staining, Membrane, Expressing

Journal: Inflammation

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

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: 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).

Article Snippet: 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.

Techniques: Knock-Out, Staining, Expressing, Permeability, Confocal Microscopy, TUNEL Assay, Biomarker Discovery

Journal: Inflammation

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

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: 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.

Article Snippet: 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.

Techniques: Activation Assay, Expressing, Inhibition, Membrane