YAP/Nrf2 suppresses ferroptosis to alleviate acute lung injury induced by intestinal ischemia/reperfusion

Abstract

Intestinal ischemia reperfusion (II/R) injury is a common critical disease with high morbidity and mortality. The mechanism of II/R-induced acute lung injury (ALI) is not fully elucidated. Yes-associated protein (YAP), a downstream transcriptional coactivator of the Hippo signaling pathway, plays a central role in controlling organ development and cell proliferation. However, whether YAP is involved in regulating II/R-induced ALI remains to be further explored. This study aimed to investigate the regulatory role of YAP in ALI and ferroptosis caused by II/R, and to explore whether YAP exerts anti-ferroptosis and anti-inflammatory effects by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear entry and upregulating Nrf2 expression. In vivo models demonstrated that overexpression of YAP inhibited II/R-induced ALI and ferroptosis. This was evident through the upregulation of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and Nrf2 expression, as well as the mitigation of characteristic mitochondrial ferroptosis changes in lung type II epithelial cells. Additionally, YAP overexpression protected against II/R-induced ALI in mice, leading to notable improvements in lung pathology, reduced pulmonary edema, and decreased lung inflammation. Consistent conclusions were also reached in vitro models. It was observed that overexpression of YAP inhibited ferroptosis and oxidative stress by increasing Nrf2 expression and promoting its nuclear translocation. Additionally, it was discovered that knocking down Nrf2 resulted in the abolition of YAP-mediated ferroptosis alleviation in MLE-12 cells. Based on our findings, we can infer that YAP inhibits ferroptosis by upregulating Nrf2 expression and promoting its translocation into the nucleus, thereby ameliorating oxidative stress and lung injury along with the systemic inflammatory response following II/R. Furthermore, we propose that targeting YAP could be a promising approach for the treatment of ALI by suppressing ferroptosis.

Highlights

• •Intestinal ischemia-reperfusion (II/R) induced acute lung injury (ALI) and ferroptosis.
• •YAP overexpression protects against II/R-induced ALI by reducing lung epithelial ferroptosis.
• •YAP suppresses ferroptosis in II/R-induced ALI by enhancing Nrf2 expression and facilitating its nuclear translocation.

1. Introduction

Intestinal ischemia-reperfusion (II/R) is a prevalent critical illness in clinical practice. It occurs when there is a sudden decrease in blood supply to the intestines and is commonly associated with various clinical conditions, including shock, mesenteric artery thrombosis, and intestinal obstruction [1,2]. II/R not only causes local intestinal damage, but also impairs intestinal barrier function and facilitates the translocation of intestinal flora. This can result in systemic inflammation and dysfunction of distal organs. Among these organs, the lung is particularly susceptible to II/R, often leading to the occurrence of ALI [3,4].

Ferroptosis, a newly discovered form of cell death in recent years, is typically characterized by the accumulation of substantial amounts of iron, lipid peroxidation, and reactive oxygen species (ROS) during the cell death process [5]. Ferroptosis occurs in a completely different process from other types of cell death [6]. Morphologically, ferroptosis is characterized by an intact cell membrane, normal nuclear morphology, mitochondrial atrophy, increased density of the mitochondrial membrane, and reduced or disappeared density of mitochondrial cristae [7]. Biochemically, ferroptosis primarily occurs due to the depletion of glutathione (GSH) in the cell and the inactivation of glutathione peroxidase 4 (GPX4), which leads to an increase in lipid peroxidation and lipid ROS [8]. The nuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant and a regulatory molecule for ferroptosis. It functions by binding to the antioxidant response element (ARE) in the nucleus, thereby promoting the transcription of target genes [9]. Previous studies have demonstrated a correlation between ferroptosis and the development of ALI caused by II/R. Furthermore, inhibiting ferroptosis in lung epithelial cells has been found to effectively mitigate lung injury resulting from intestinal II/R [10]. Blocking lung epithelial cell ferroptosis may be a potential treatment for II/R-induced ALI.

Yes-associated protein 1 (YAP1), also known as YAP, functions as a downstream transcriptional coactivator within the Hippo signaling pathway, playing a central role in regulating organ development and cell proliferation [11]. The Hippo pathway operates as a conserved kinase cascade. Activation of the Hippo pathway induces direct phosphorylation of YAP by large tumor suppressor 1/2 (LATS1/2) kinase, rendering phosphorylated YAP inactive and targeting it for cytoplasmic degradation. Conversely, The Hippo pathway inactivation permits unphosphorylated YAP to translocate to the nucleus, where it binds with TEA domain family (TEAD) transcription factors to drive downstream gene expression [[12], [13], [14]]. Recent evidence further implicates the Hippo pathway in cerebral and myocardial ischemia/reperfusion injury [15,16]. Specifically, YAP activation exerts protective effects against inflammation and oxidative stress in intestinal I/R [17] and liver I/R models [18], and crucially, that Nrf2 is necessary for YAP to mediate these protective effects. Moreover, a growing body of evidence suggests involvement of the Hippo signaling pathway in respiratory system pathologies. Nevertheless, the specific role and mechanistic underpinnings of YAP in modulating lung injury induced by II/R remain to be elucidated.

The objective of our study was to investigate the involvement of YAP in ferroptosis of lung epithelial cells induced by II/R and to explore the underlying mechanism of ferroptosis in ALI induced by II/R.

2. Materials and methods

2.1. Animals

Adult male C57BL/6 mice (6–8 weeks old, body weight 20–25 g) were purchased from SiPeiFu Experimental Animal Co., Ltd. (Beijing, China). Animals were housed at temperatures of 22 ± 1 °C, 12 h/12 h light-dark cycle, had free access to food and drinking water, and acclimated for a week. Before the study, the animals fasted for 8 h but were allowed water ad libitum. All animal experiments were performed in compliance with the guidelines provided by the Institutional Animal Care and Use Committee's guidelines of Southwest Medical University, Luzhou, China. This study was approved by the Ethics Committee on Animal Experiments at Southwest Medical University (Approval No. 20221026-002).

2.2. II/R model

Mice were fasted for 8 h prior to the process of establishing the II/R model. Mice were then anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal injection) and allowed to breathe normally during surgery. A midline abdominal incision was made, and the superior mesenteric artery (SMA) was gently exposed and occluded by a micro clip. After 1 h of ischemia, the micro clip was removed, and the abdominal wound was closed. The mice in the Sham group received only anesthesia and laparotomy.

Dimethyl sulfoxide (DMSO) was used to dissolve ferrostatin-1 (MedChemExpress, Monmouth Junction, NJ, USA) and ML385 (MedChemExpress, Monmouth Junction, NJ, USA). Ferrostatin-1 is a ferroptosis inhibitor, and ML385 is a Nrf2 inhibitor. To investigate the impact of ferroptosis on II/R-induced ALI, we administered ferrostatin-1 (0.8 mg/kg) through the tail vein one day prior to the surgery. In certain cases, the Nrf2 inhibitor ML385 (5 mg/kg) was also administered via the tail vein 1 h before inducing intestinal ischemia.

2.3. Adeno-associated virus (AAV) administration

For in vivo overexpression of YAP in the lung, male C57BL/6 mice (3–4 weeks old) were randomly assigned to experimental groups. Four weeks prior to II/R surgery, mice were anesthetized with an intraperitoneal injection of pentobarbital sodium. Using a sterile 29-gauge insulin syringe, 50 μL of recombinant adeno-associated virus (AAV), which was packaged from the pcAAV-CMV-Yap1-3 × FLAG-P2A-GdGreen-WPRE vector (OBiO Technology, Shanghai, China), containing 10¹¹plaque-forming units (PFU), was slowly injected intratracheally. Mice in the control group were injected with equal volumes of AAV empty vector following the same procedure.

2.4. Cell culture and OGD/R model

Mouse lung epithelial MLE-12 cells were cultured with 10 % fetal bovine serum (Peak serum, Colorado, USA) and 1 % penicillin/streptomycin antibiotics (Beyotime Biotechnology, Shanghai, China) in DMEM medium (Basalmedia, Shanghai, China) at 37 °C in a 5 % CO₂ constant temperature incubator. OE-YAP (pcSLenti-EF1-EGFP-P2A-Puro-CMV-Yap1-3 × FLAG-WPRE), sh-Nrf2 (pCLenti-U6-shRNA (Nfe212)-CMV-EGFP-F2A-BSR-WPRE) lentivirus was designed and chemically synthesized by (OBiO Technology Shanghai Corp, Ltd, Shanghai, China).

In order to express or inhibit related genes, MLE-12 cells were infected with the lentiviral vector. To stabilize the knockdown and overexpression of the Nrf2 or YAP gene, cells were inoculated at a density of 2.5 × 10⁵ cells per well in a 6-well plate and allowed to adhere overnight. The following day, cells were infected with lentivirus according to the manufacturer's protocol. Stably infected cells were selected using 2 μg/mL puromycin (Beyotime Biotechnology, Shanghai, China). After 72 h of transfection, the efficiency of silencing was confirmed by Western blot (WB).

To model ischemia/reperfusion (I/R) injury in vitro, a model of oxygen-glucose deprivation and reoxygenation (OGD/R) was created. Cells were cultured in glucose-free DMEM (Shanghai Basalmedia Technologies CO.,Ltd, Shanghai, China) and incubated in an anaerobic chamber (5 % CO₂, 1 % O₂, and 94 % N₂) to simulate ischemia by depriving them of oxygen and glucose. After 12 h of incubation, the cells were replaced with normal DMEM medium containing 10 % FBS and placed in an oxygen chamber (37 °C, 5 % CO₂) for 12 h to simulate reperfusion.

2.5. Histological analysis

The lung samples were fixed in 10 % formalin and subsequently embedded in paraffin. The blocks will be cut into 5-μm sections, stained with hematoxylin and eosin (H&E), and analyzed under an optical microscope.

2.5.1. Total protein concentration in BALF

The trachea was exposed by opening the chest and neck after euthanizing the mice. Endotracheal intubation was then performed, and 1 mL of phosphate buffered saline (PBS) was instilled into the lungs and subsequently aspirated three times using a syringe connected to a catheter. The bronchoalveolar lavage fluid (BALF) was centrifuged at 3000 rpm for 10 min at 4 °C, and the resulting supernatant was collected. The protein content in the BALF was quantified using the BCA Protein Assay kit (Beyotime Biotechnology, Shanghai, China) instructions provided by the manufacturer.

2.6. Lung wet/dry (W/D) ratio

The W/D ratio was used to measure the level of pulmonary edema. The median sternotomy was performed and the lobes were removed from the pleural space. The lung surface was then dried with absorptive paper and weighed to obtain the wet weight. The lung was placed in a drying oven at 65 °C for 72 h and weighed again to obtain the dry weight. W/D was calculated using the following equation: W/D = wet weight/dry weight.

2.7. Transmission electron microscopy (TEM)

The samples were prefixed with 3 % glutaraldehyde, then refixed with 1 % osmium tetroxide. They were dehydrated step by step using acetone and embedded with Epon812. For optical positioning, the semi-thin sections were stained with toluidine blue, while ultrathin sections were made using a diamond knife. Sections stained with uranyl acetate and lead citrate, and were examined with JEM-1400-FLASH Transmission Electron Microscope.

2.8. Intracellular ROS and Fe²⁺ production

In vitro, the intracellular oxidative stress was determined by staining with Dihydroethidium (DHE) (Yeasen Biotechnology, Shanghai, China). MLE-12 cells were incubated with 10 μM DHE at 37 °C for 30 min in a humid and dark chamber. The images were captured under a fluorescence microscope.

Intracellular Fe²⁺ levels were detected using the FerroOrange probe (Dojindo, Japan) according to the manufacturer's instructions. Briefly, MLE-12 cells were harvested and incubated with 1 mmol/L FerroOrange for 30 min. After washing with PBS 3 times, the MLE-12 cells were observed under a fluorescence microscope.

2.9. Western blot analysis

The mice lung tissues or MLE-12 cells were homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1 % PMSF (Beyotime Biotechnology, Shanghai, China). The supernatants were collected by sonication and centrifugation at 14,000 g, 4 °C for 20 min. The supernatants were then quantified using a BCA kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF). The membranes were blocked with 5 % skim milk and incubated with primary antibodies. After incubation with secondary antibodies conjugated to horseradish peroxidase, the signals were visualized using enhanced chemiluminescence (Yamay Biomedical Technology, Shanghai, China) as the manufacturer's instructions. The intensity of each band was analyzed using Image J software. The primary antibodies used were anti-GPX4 (1:1000; Abcam, Cambridge, MA, USA); anti-SLC7A11(1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-YAP (1:1000; Cell Signaling Technology, Danvers, MA, USA); anti–HO–1 (1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-Nrf2 (1:2000; Proteintech, Wuhan, China); β-actin (1:5000; Proteintech, Wuhan, China); anti-Bax (1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-Bcl-2 (1:1000; Beyotime Biotechnology, Shanghai, China); anti-rabbit IgG (1:1000; Beyotime Biotechnology, Shanghai, China); anti-mouse IgG (1:1000; Beyotime Biotechnology, Shanghai, China).

2.10. Immunofluorescence analysis

The frozen section of lung tissues and MLE-12 cells were fixed in paraformaldehyde for 30 min. Subsequently, they were permeabilized with 0.2 % Triton X-100 for 15 min, and blocked with 1 % bovine serum albumin at room temperature for 60 min. Afterward, the sections were incubated at 4 °C overnight with the following primary antibodies: anti-YAP (1:200; Abcam, Cambridge, MA, USA); anti-Nrf2 (1:200; Proteintech, Wuhan, China); anti-GPX4 (1:200; Abcam, Cambridge, MA, USA); anti-4-HNE (1:200; Bioss, BeiJing, China). Finally, the sections were washed with PBS and incubated with fluorescence-conjugated secondary antibodies (1:200, Affinity, Changzhou, China) at room temperature for 1 h. After washing, the sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China). The images were captured under a fluorescence microscope. The relative fluorescence intensity was measured using Image J software.

2.11. ELISA

The BALF and serum samples were collected and analyzed for inflammatory factors. The levels of interleukin IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) were determined using an ELISA kit (ZCIBIO, Shanghai, China) following the manufacturer's instructions.

2.12. Measurement of Fe²⁺, glutathione (GSH) and MDA levels

The Fe²⁺ detection kit was used (Meimian, Wuhan, China). The MDA assay kit was used (Beyotime Biotechnology, Shanghai, China). The ratio of reduced glutathione (GSH)/oxidized glutathione disulfide (GSSG) detection kit was used (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All kits were used according to the manufacturer's instructions.

2.13. TUNEL staining

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed according to the manufacturer's instructions (Solarbio, Beijing, China) to quantify cell apoptosis in lung tissue. Images were acquired using a fluorescence microscope.

2.14. Molecular docking

Amino acid sequences of YAP and Nrf2 were downloaded from the Uniprot database (https://www.uniprot.org). Protein molecular modeling and molecular docking were conducted utilizing HDOCK Server (http://hdock.phys.hust.edu.cn/) to achieve the optimal docking outcomes. The prediction results were visualized by PyMol tool.

2.15. Chromatin immunoprecipitation (ChIP) assay

Chromatin immunoprecipitation (ChIP) experiments were conducted utilizing the ChIP Assay Kit (Beyotime Biotechnology, Shanghai, China) under the instruction of the manufacturer. Briefly, cells were cross-linked with 1 % formaldehyde to fix protein-DNA interactions, followed by cell lysis. The cell lysate was sonicated to shear chromatin into fragments ranging from 200 to 1000 bp. Then, the resulting chromatin was immunoprecipitated overnight at 4 °C with anti-YAP antibody (Cell Signaling Technology, Danvers, MA, USA), while normal IgG served as the negative control. After immunoprecipitation, cross-links were reversed and DNA was purified from the precipitated chromatin. The enrichment of specific DNA sequences was analyzed by PCR using the primers targeting the Nrf2 promoter region. The sequences of DNA primers for ChIP-qPCR were listed in the Supplementary Table S1.

2.16. Dual-luciferase assay

The plenti-CMV-YAP plasmid, generously provided by Dr. Yi Zhang (Chongqing University), was co-transfected into 293T cells along with pNRF2-Fluc-hPEST-Neo plasmid (Miaoling Biology, Wuhan, China) or the plasmid pRL-TK control plasmid (Miaoling Biology, Wuhan, China). Forty-eight hours post-transfection, firefly and Renilla luciferase activities were measured using the Dualucif Firefly & Renilla Assay Kit (UElandy, Suzhou, China), following the manufacturer's protocol. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase signals.

2.17. Co-immunoprecipitation (Co-IP) assay

To investigate protein interactions, protein lysates were collected from MLE-12 cells with YAP overexprssion using lysis buffer and incubated on ice for 30 min. After centrifugation to remove cell debris, the supernatant was incubated overnight at 4 °C with either anti-YAP antibody (Cell Signaling Technology, Danvers, MA, USA), anti-Nrf2 antibody (Proteintech, Wuhan, China), or control IgG (Beyotime Biotechnology, Shanghai, China). Protein A/G magnetic beads (Beyotime Biotechnology, Shanghai, China) were then added to the mixtures and incubated for an additional 3 h at 4 °C with gentle rotation. Beads were washed three times with lysis buffer to remove nonspecific binders. The immunoprecipitated proteins were eluted using 2 × SDS loading buffer at 95 °C for 5 min, and analyzed by western blotting.

2.18. Patient samples

From January 1 to June 30, 2025, we consecutively recruited patients undergoing elective heart valve replacement or coronary artery bypass grafting with cardiopulmonary bypass (CPB) at the Department of Cardiac Surgery, the Affiliated Hospital of Southwest Medical University, Luzhou, China. The inclusion criteria were as follows: (1) age ≥18 years; (2) Healthy Physical Examination Group: individuals without a history of cardiopulmonary disease; (3) Cardiopulmonary Bypass Group: patients undergoing cardiac surgery with the CPB duration exceeding 2 h; (4) participants who provided extensive informed consent, explicitly allowing the use of their data for future research. The exclusion criteria included: (1) history of pulmonary diseases; (2) cases with incomplete data or missing key variables.

The protocol received approval from the Ethics Committee of the Affiliated Hospital of Southwest Medical University (approval number KY2025380). This study included a total of 13 patients. All participants provided informed consent prior to their inclusion. Blood samples were collected 6 h after CPB. We measured the primary marker of lung injury, human pulmonary surfactant protein D (SP-D) (ZC-32509w, ZCIBIO, China), along with markers of gastrointestinal injury and intestinal barrier dysfunction, including intestinal fatty acid binding protein (iFABP) (ZC-32609, ZCIBIO, China) and diamine oxidase (DAO) (A088-1-1, Nanjing Jiancheng, China). Additionally, we assessed biochemical parameters such as GSH (A006-1-1, Nanjing Jiancheng, China) and inflammatory factors including TNF-α (YJ064303, MLBio, China), IL-6 (YJ058097, MLBio, China) and IL-1β (ZC-32420w, ZCIBIO, China).

2.19. Statistical analysis

Data were expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8.3 statistical software (GraphPad software, San Diego, CA, USA). The data were statistically analyzed using analysis of variance (ANOVA), followed by Tukey's multiple comparison post hoc test. In addition, the Spearman method was used for correlation statistical analysis. P < 0.05 was considered statistically significant.

3. Results

3.1. Ferroptosis occurs in ALI due to II/R

To verify the presence of ferroptosis in Intestinal ischemia-reperfusion (II/R)-induced ALI, we tested whether ferroptosis inhibitor ferrostatin-1(Fer-1) could rescue II/R-induced ALI. Ferrostatin-1, a specific ferroptosis inhibitor, has previously been shown to attenuate ischemia-reperfusion injury [19,20]. Mice were pretreated with Fer-1 for 24 h. Compared to Sham group, the Histological analysis (HE) staining of lung tissue in the II/R group revealed significant lung injuries, including atelectasis, alveolar and hemorrhage. However, following Fer-1 treatment, the damage to lung epithelial cells caused by II/R was markedly reduced (Fig. 1A). Using Transmission electron microscopy (TEM), lung mitochondria of type Ⅱ alveolar epithelial cells of II/R mice showed characteristics typical of ferroptosis changes, including smaller mitochondria and reduced cristae, compared with the Sham group, and the degree of damage was reduced after Fer-1 administration (Fig. 1B). Malondialdehyde (MDA), glutathione (GSH), Fe²⁺ have been recognized as the key and effective metabolic indicators of ferroptosis [21]. Compared to the Sham group, the levels of MDA and Fe²⁺ were significantly increased, while the ratio of reduced and oxidized glutathione (GSH/GSSG) was decreased in the II/R model. However, these changes were reversed in the Fer-1 pretreatment group (Fig. 1C–E). To further support our preliminary findings, MLE-12 was chosen for in vitro experiments. Intracellular Fe²⁺ levels were measured using FerroOrange, while intracellular ROS levels were assessed using DCFH-DA. OGD/R led to an increase in Fe²⁺ and ROS in MLE-12 cells, which were subsequently reversed by Fer-1 administration (Fig. 1F and G). Moreover, the reduction of the ferroptosis negative regulatory proteins GPX4 and SLC7A11 in the OGD/R model (Fig. 1H–J). These findings highlight the involvement of ferroptosis in the pathophysiology of II/R-induced ALI.

Fig. 1.

Ferroptosis occurs in ALI due to II/R. (A) Representative H&E staining of lung tissues in each group. n = 6 per group. (magnification: × 200; Scale bars = 50 μm). (B) Representative TEM of lung tissue ultrastructure (magnification: 500 nm × 25000; Scale bars = 500 nm). (C–D) Level of Fe²⁺, MDA and GSH/GSSG in lung tissue. n = 6 per group. (F) Levels of Fe²⁺ in MLE-12 were assessed by FerroOrange. n = 3 per group (magnification: × 200, scale = 50 μm). (G) Levels of ROS in MLE-12 were assessed by DHE staining. n = 3 per group (Magnification: × 200, scale = 50 μm). (H–J) SLC7A11, GPX4 representative Western blot bands and quantitative analysis. n = 6 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

3.2. II/R leads to increased apoptosis and inflammatory damage in mouse lung tissue cells

To assess the detrimental effects of II/R on lung injury in mice, we collected lung tissues and bronchoalveolar lavage fluid (BALF) for further analysis. The protein concentration in the BALF and the W/D ratio of mice in the II/R group were significantly elevated compared to those in the Sham group (Fig. 2A and B). Additionally, we employed enzyme linked immunosorbent assay (ELISA) to quantify pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) in both BALF and serum. The findings indicated that the expression levels of pro-inflammatory cytokines in the II/R group were significantly higher than those in the Sham group, while the expression of these inflammatory factors markedly decreased in the Fer-1 pretreatment group (Fig. 2C-H). These results suggest that II/R-induced ALI is characterized by extensive inflammatory cell infiltration and severe pulmonary edema, with Fer-1 demonstrating a significant protective effect against this damage. To investigate the effect of II/R on the apoptotic capacity of lung tissue, lung tissue sections were stained using the TUNEL, and the expression levels of Bax and Bcl-2 were assessed through Western blotting. The results indicated that II/R significantly increased apoptosis in mice lung tissue, an effect that was alleviated by the administration of Fer-1 (Fig. 2I–M). The results of apoptosis in vitro were consistent with those in vivo (Fig. 3J–L). Collectively, our findings indicate that the inhibition of ferroptosis not only diminished inflammatory cell infiltration but also reduced apoptosis.

Fig. 2.

II/R leads to increased apoptosis and inflammatory damage in mouse lung tissue cells. (A–B) Protein concentration in BALF and W/D ratio of lung tissue. n = 6 per group. (C–E) Changes in IL-1β, IL-6 and TNF-α levels in serum. n = 6 per group. (F–H) Changes in IL-1β, IL-6, and TNF-α levels in BALF. n = 6 per group. (I–J) Representative TUNEL staining of lung tissues in each group and quantitative analysis. n = 3 per group. (magnification: × 200; Scale bars = 50 μm). (K–M) Bax, Bcl-2 representative Western blot bands and quantitative analysis. n = 6 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

Fig. 3.

Alterations in lung YAP/Nrf2 after II/R. (A) Molecular docking results of YAP and Nrf2. (B–F) SLC7A11, Nrf2, GPX4, YAP representative Western blot bands and quantitative analysis. n = 6 per group. (G–I) Nrf2, YAP representative Western blot bands and quantitative analysis. n = 6 per group. (J–L) Bax, Bcl-2 representative Western blot bands and quantitative analysis. n = 6 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

3.3. Alterations in lung YAP after II/R

YAP, a downstream transcriptional coactivator of the Hippo pathway, plays a crucial role in various cellular processes. Recent studies have demonstrated that inhibiting the activation of the YAP in non-small cell lung cancer cells can promote ferroptosis [22]. YAP stimulates the expression of GPX4 and SLC7A11 by regulating the binding of TEAD4 to the GPX4/SLC7A11 promoter, thereby protecting cells from ferroptosis in a mouse sepsis model [23]. Numerous studies have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2), as a crucial transcription factor, can activate the expression of various antioxidant genes, including HO-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) [24,25]. These genes encode proteins that mitigate intracellular oxidative stress, consequently inhibiting ferroptosis [26,27]. Meanwhile, studies on TAZ, the paralog of YAP, reveal direct functional interactions with Nrf2 in the context of mitigating inflammation and enhancing antioxidant capacity in microglia [47]. Based on the above findings, we propose that there may be both a direct and indirect link between YAP and Nrf2, which could influence the onset and progression of ferroptosis in the II/R induced ALI model. To test this hypothesis, we initially conducted a molecular docking analysis of YAP and Nrf2, revealing that these proteins possess multiple binding sites (Fig. 3A). We then assessed the expression of YAP and ferroptosis negative regulatory proteins, including GPX4, Nrf2, and SLC7A11 in mouse lung tissues. Our results indicated that the expression levels of YAP, Nrf2, GPX4, and SLC7A11 were significantly reduced in the II/R model, but were partially restored following Fer-1 intervention (Fig. 3B–F). Furthermore, the expression levels of Nrf2 and YAP in the cell model were found to be consistent with those observed in the animal model (Fig. 3G–I). Fer-1 treatment not only restored the expression of ferroptosis negative regulatory proteins but also restored the expression level of YAP. These results suggest that ferroptosis is involved in the II/R-induced lung injury and the YAP may be a potential key regulator in this pathological process.

3.4. Overexpression of YAP inhibits ferroptosis in II/R-induced ALI

To further elucidate the involvement of YAP in ferroptosis and II/R-induced ALI, we overexpressed YAP in mice using adeno-associated virus (AAV). Notably, the overexpression of YAP in the mice led to a noticeable alleviation of lung tissue damage, as evidenced by improved ALI lung tissue pathology characterized by reduced pulmonary hemorrhage and thickened alveolar walls (Fig. 4A). We observed with TEM that YAP overexpression reversed the characteristics typical of ferroptosis changes in mitochondria of lung type Ⅱ alveolar epithelial cells induced by ALI in mice (Fig. 4B). Compared with the II/R group, the II/R + YAP-AAV group exhibited a significant decrease in Fe²⁺ and MDA levels, as well as an increase in the GSH/GSSG ratio (Fig. 4C–E). Meanwhile, the immunofluorescence assay also showed that YAP overexpression reversed the decrease in GPX4 and the increase in 4-hydroxy-2-nonenal (4-HNE) caused by II/R-induced ALI (Fig. 4F–I). MDA and 4-HNE are the primary metabolites of lipid peroxidation, and ferroptosis is typically accompanied by a significant accumulation of iron and lipid peroxidation during cell death [5]. As expected, overexpression of YAP inhibited ferroptosis and alleviated lung injury. This was further evidenced by alterations in the expression levels of GPX4, SLC7A11, and Nrf2, as well as changes in the protein content of BALF and lung W/D ratios (Fig. 5A–G). The overexpression of YAP also mitigated the inflammatory response and apoptosis induced by II/R (Fig. 5H–R). In vitro experiments were consistent with the in vivo findings. Specifically, transfection of MLE-12 cells with a YAP overexpression lentivirus attenuated the OGD/R-induced increases in intracellular Fe²⁺ and ROS (Fig. 6A and B). YAP overexpression restored the expression levels of Nrf2, GPX4, and SLC7A11, subsequently inhibiting apoptosis (Fig. 6C–J).

Fig. 4.

Overexpression of YAP inhibits ferroptosis in II/R-induced ALI. (A) Representative H&E staining of lung tissue in each group. n = 6 per group (magnification: × 200; Scale = 50 μm). (B) Representative TEM of lung tissue ultrastructure. (magnification: 500 nm × 25000 scale). (C–E) Level of MDA, Fe²⁺ and GSH/GSSG in lung tissue. n = 6 per group. (F–I) Representative micrographs of 4-HNE and GPX4 immunofluorescence staining of lung tissue (magnification: × 200; Scale = 50 μm) and quantitative analysis of the IF intensity of 4-HNE and GPX4. n = 3 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

Fig. 5.

Overexpression of YAP inhibits inflammation and apoptosis in II/R-induced ALI. (A–E) SLC7A11, Nrf2, GPX4, YAP representative Western blot bands and quantitative analysis. n = 6 per group. (F, G) Protein concentration in BALF and W/D ratio of lung tissue. n = 6 per group. (H–J) Changes in IL-1β, IL-6 and TNF-α levels in serum. n = 6 per group. (K–M) Changes in IL-1β, IL-6, and TNF-α levels in BALF. n = 6 per group. (O–N) Representative TUNEL staining of lung tissues in each group and quantitative analysis. n = 3 per group. (magnification: × 200; Scale bars = 50 μm). (P–R) Bax, Bcl-2 representative Western blot bands and quantitative analysis. n = 6 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

Fig. 6.

Overexpression of YAP inhibits ferroptosis and apoptosis induced by OGD/R. (A) Levels of Fe²⁺ in MLE-12 were assessed by FerroOrange. n = 3 per group. (magnification: × 200, scale = 50 μm). (B) Levels of ROS in MLE-12 were assessed by DHE staining. n = 3 per group (Magnification: × 200, scale = 50 μm). (C–G) SLC7A11, Nrf2, GPX4, YAP representative Western blot bands and quantitative analysis. n = 6 per group. (H–J) Bax, Bcl-2 representative Western blot bands and quantitative analysis. n = 6 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

Multiple studies have shown that nuclear YAP levels have transcriptional capabilities in regulating gene expression [28]. To analyze nuclear protein, nuclear cytoplasmic separation was performed and further analyzed using western blotting and immunofluorescence for nuclear localization. It has been observed that the overexpression of YAP primarily leads to nuclear proliferation and facilitates the translocation of Nrf2 protein into the nucleus (Fig. 7A–E). The mechanism by which YAP inhibits ferroptosis and reduces lung injury may involve promoting Nrf2 nuclear translocation, thereby upregulating downstream antioxidant factors.

Fig. 7.

Expression of YAP and Nrf2 in the nucleus. (A–B) Representative microscopic images of YAP and Nrf2 immunofluorescence staining of MLE-12 cells. (Magnification: × 400, scale = 50 μm). (C–E) YAP, Nrf2 in the nucleus representative Western blot bands and quantitative analysis. n = 3 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

3.5. Inhibiting the Nrf2 gene weakens the protective effect of YAP on II/R-induced ALI and ferroptosis

To confirm that the protective effect of YAP is due to Nrf2 activation, we pretreated mice with the Nrf2-specific inhibitor ML385 prior to modeling. Firstly, the results from HE indicated that inhibition of Nrf2 reversed the protective effects of YAP against II/R-induced ALI (Fig. 8A). Mitochondria in lung type Ⅱ alveolar epithelial cells using TEM, and found that the protective effect of YAP on ferroptosis was blocked when Nrf2 was inhibited (Fig. 8B). Additionally, the above conclusions were supported by the expression levels of Fe²⁺, GSH/GSSG, MDA, and the expression of 4-HNE and GPX4 detected by immunofluorescence, further confirming that Nrf2 inhibition hindered the anti-lipid oxidation function of YAP (Fig. 8C–I). Secondly, the expression of GPX4, SLC7A11, Nrf2 and its downstream protein HO-1 was detected using western blotting. It was observed that ML385 can effectively inhibit the expression of Nrf2, GPX4, SLC7A11 and HO-1. However, the overexpression level of YAP was not affected by Nrf2 inhibitors (Fig. 9A–F). Finally, the results from inflammatory factors and BALF analysis indicated that inhibition of Nrf2 reversed the protective effects of YAP against II/R-induced ALI (Fig. 9G–N). In summary, the activation of Nrf2 by YAP may improve II/R-induced ALI by suppressing inflammatory responses and inhibiting ferroptosis.

Fig. 8.

Inhibition of Nrf2 gene weakens the protective effect of YAP on II/R-induced ferroptosis. (A) Representative H&E staining of lung tissue in each group. n = 6 per group (magnification: × 200; Scale = 50 μm). (B) Representative TEM of lung tissue ultrastructure (magnification: 500 nm × 25000 scale). (C–E) Level of Fe²⁺, GSH/GSSG and MDA in lung tissue. n = 6 per group. (F–I) Representative micrographs of 4-HNE and GPX4 immunofluorescence staining of lung tissue (magnification: × 200; Scale = 50 μm) and quantitative analysis of the IF intensity of 4-HNE and GPX4. n = 3 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

Fig. 9.

Inhibition of Nrf2 gene weakens the anti-inflammatory and lung-protective effects of YAP. (A–E) SLC7A11, Nrf2, GPX4, HO-1, representative Western blot bands and quantitative analysis. n = 6 per group. (F–G) Protein concentration in BALF and W/D ratio of lung tissue. n = 6 per group. (H–J) Changes in IL-1β, IL-6 and TNF-α levels in serum. n = 6 per group. (K–M) Changes in IL-1β, IL-6, and TNF-α levels in BALF. n = 6 per group. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

3.6. Inhibition of Nrf2 gene weakens the protective effect of YAP against OGD/R-induced ferroptosis and oxidative stress in MLE-12 cells

To further investigate the mechanism of YAP-mediated II/R-induced ALI, in vitro experiments were conducted using MLE-12 cells. These cells were infected with OE-YAP and sh-Nrf2 lentivirus. The findings revealed that YAP-mediated inhibition of ROS and Fe²⁺ in OGD/R-induced MLE-12 cells was abolished by Nrf2 knockdown (Fig. 10A and B). Meanwhile, OE-YAP could effectively reverse the decrease in SLC7A11, Nrf2, and GPX4, which were negative regulatory proteins of ferroptosis caused by OGD/R. However, when sh-Nrf2 was added, the effect of YAP disappeared, and the expression of HO-1 was also significantly inhibited (Fig. 10C–H). Subsequent detection of nuclear protein expression and immunofluorescence localization revealed that overexpression of YAP reversed the reduction of Nrf2 nuclear expression induced by OGD/R. Furthermore, the effect of YAP was nullified after Nrf2 knockdown (Fig. 10I–K,11A-B). Nrf2 is a crucial transcription factor involved in oxidative stress [29]. These findings suggest that YAP plays an anti-oxidative role in preventing ferroptosis by promoting Nrf2 expression in the nucleus.

Fig. 10.

Inhibition of Nrf2 gene weakens the protective effect of YAP against OGD/R induced ferroptosis. (A) Levels of Fe²⁺ in MLE-12 were assessed by FerroOrange. n = 3 per group (magnification: × 200, scale = 50 μm). (B) Levels of ROS in MLE-12 were assessed by DHE staining. n = 3 per group (Magnification: × 200, scale = 50 μm). (C–H) SLC7A11, Nrf2, GPX4, HO-1, YAP representative Western blot bands and quantitative analysis. n = 6 per group. (I–K) YAP, Nrf2 in the nucleus representative Western blot bands and quantitative analysis. n = 3 per group. (Magnification: × 400, scale = 50 μm). (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

3.7. YAP regulates Nrf2 expression by interacting with Nrf2 protein to enter the nucleus

YAP functions as a transcriptional co-activator and exerts its transcriptional regulatory effects primarily through interaction with TEAD transcription factors [30]. To investigate the potential transcriptional regulatory effect of YAP on Nrf2, we utilized the JASPAR database (https://jaspar.genereg.net) to predict transcription factor binding sites for TEAD within the Nrf2 promoter [31]. The analysis identified possible YAP/TEAD-related binding sequences: AAATTCC, TCATACCT, and ACATTTCA in the Nrf2 promoter region. However, ChIP-PCR results demonstrated that YAP did not bind to the Nrf2 promoter via TEAD (Fig. 11C). To further assess whether YAP could regulate Nrf2 transcriptionally, the Nrf2 promoter region was cloned into a luciferase reporter vector (pNrf2-Fluc-hPEST-Neo) and firefly luciferase gene activity was measured upon YAP overexpression. Dual-luciferase assay results showed no significant increase in Nrf2 promoter activity (Fig. 11D). These findings suggest that YAP does not transcriptionally activate Nrf2 expression. Subsequently, we investigated whether YAP might regulate Nrf2 at the protein level through direct interaction. Co immunoprecipitation (Co-IP) analysis revealed a direct binding between YAP and Nrf2 proteins (Fig. 11E and F). Therefore, our results indicate that YAP probably exerts its anti-ferroptotic and lung-protective effect by binding to Nrf2 protein and promoting its nuclear translocation, rather than through transcriptional activation of Nrf2 expression, thereby regulating downstream target genes.

Fig. 11.

YAP regulates Nrf2 expression by interacting with Nrf2 protein to enter the nucleus. (A–B) Representative microscopic images of YAP and Nrf2 immunofluorescence staining of MLE-12 cells. (Magnification: × 400, scale = 50 μm). (C) Chip-PCR verified the transcription of Nrf2 by YAP. (D) 293T cells were infected with difference combination of NC and OE-YAP and then luciferase activity of Nrf2 was detected. n = 3 per group. (E–F) Co-IP representative bands for YAP and Nrf2 interaction analysis. (Data were expressed as average ± SD, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 denotes intergroup significant difference).

3.8. Ferroptosis exists in clinical patients with II/R injury

II/R injury frequently occurs during cardiac surgery involving cardiopulmonary bypass (CPB) [32]. Various pathophysiological factors generated during CPB can lead to inadequate blood perfusion of the intestinal mucosa, compromising the integrity of the intestinal barrier. This impairment facilitates the translocation of bacteria and endotoxins into the bloodstream, triggering a systemic inflammatory response that can ultimately result in significant damage to distal organs [33]. To investigate the correlation between lung injury and ferroptosis in patients undergoing CPB, this study analyzed blood samples from patients 6 h post-CPB for linear correlation analysis. The characteristics of the patients were presented in Supplementary Table S2. The results indicated a positive correlation between SP-D and iFABP as well as DAO (Fig. 12A and B). Conversely, GSH exhibited a negative correlation with SP-D, TNF-α, IL-6, and IL-1β (Fig. 12C–F). These findings suggest that CPB-induced intestinal ischemia-reperfusion injury may be associated with ferroptosis.

Fig. 12.

Correlation analysis between pulmonary ferroptosis and intestinal injury in CPB patients. (A) Correlation between plasma SP-D and plasma iFABP. (B) Correlation between plasma SP-D and plasma DAO. (C) Correlation between plasma GSH and plasma SP-D. (D) Correlation between plasma GSH and plasma IL-1β. (E) Correlation between plasma GSH and plasma IL-6. (F) Correlation between plasma GSH and plasma TNF-α. Spearman's rank correlation, n = 13.

4. Discussion

Ferroptosis, a newly discovered form of cell death, is primarily characterized by lipid peroxidation and the accumulation of iron and ROS [5]. Numerous studies have demonstrated a strong link between ferroptosis and ischemia-reperfusion disease [[34], [35], [36]]. Moreover, research has shown that II/R and OGD/R play crucial roles in promoting ferroptosis [37]. To investigate the occurrence of ferroptosis in lung tissue after II/R and in MLE-12 cells after OGD/R, we examined the expression of ferroptosis-related markers (GSH/GSSG, MDA, Fe²⁺, ROS) and key proteins associated with ferroptosis. In the above model, our findings revealed a decrease in GSH/GSSG, and negative regulatory proteins (Nrf2, GPX4, SLC7A11), along with an increase in Fe²⁺, MDA and ROS. TEM analysis of lung type Ⅱ alveolar epithelial cells in ALI induced by II/R also showed characteristic changes of ferroptosis in mitochondria. To further investigate the impact of ferroptosis on II/R-induced ALI, we pretreated II/R mice with the ferroptosis inhibitor Fer-1. Fer-1 is a lipid peroxidation scavenger that directly quenches lipid radicals and reduces lipid hydroperoxides, thereby halting the execution phase of ferroptosis [38]. Crucially, Fer-1 does not directly inhibit upstream regulators like YAP, Nrf2, or SLC7A11. Instead, by blocking lipid peroxidation (a downstream event), Fer-1 mitigates oxidative stress and creates a permissive environment for cells to activate endogenous antioxidant pathways. In our data, Fer-1 treatment reduced ferroptosis execution markers including lipid peroxidation (MDA) and iron overload (Fe²⁺). Concurrently, it increased GSH/GSSG ratio and elevated protein levels of GPX4 and SLC7A11, indicating restoration of redox homeostasis through protective gene upregulation. The results demonstrated that Fer-1 effectively improved pulmonary edema, inhibited oxidative stress, and mitigated lung injury. In conclusion, our study confirms the occurrence of ferroptosis in II/R-induced ALI and OGD/R, exacerbating lung injury and cellular damage. Additionally, inhibiting ferroptosis proves to be an effective approach in reducing such damage.

YAP acts as a downstream transcriptional regulator of the Hippo signaling pathway, which is crucial in controlling organ development and cell proliferation [11]. Upon inhibition of the Hippo signaling pathway, YAP is transported to the nucleus where it interacts with downstream transcription factors, facilitating the transcription of target genes. Previous research has established a connection between YAP and ferroptosis [39,40]. Previous studies have primarily focused on the role of YAP in tumors. However, recent research has revealed its significance in ischemic disease as well [17]. The Hippo pathway operates as a conserved kinase cascade. Activation of the Hippo pathway triggers LATS1/2-mediated phosphorylation of YAP, leading to its cytoplasmic sequestration and degradation, thereby suppressing its transcriptional activity. Conversely, the Hippo pathway inactivation permits nuclear translocation of unphosphorylated YAP, enabling TEAD-mediated transcriptional activation. Our experiment demonstrate that OGD/R induces iron overload (Fe²⁺), ROS accumulation (DHE) and establishes an oxidative microenvironment. Previous studies indicate that oxidative stress can activate LATS1/2 kinases, promoting YAP phosphorylation and cytoplasmic degradation [41]. Concurrently, OGD/R-induced ATP depletion creates energy stress potentially activating AMPK, further suppressing YAP activity [42]. Actually, Fer-1 mitigates oxidative damage, leading to Hippo pathway inactivation, thereby alleviating inhibitory pressure on YAP to permit nuclear translocation and exerts anti-ferroptotic effects. Consequently, OGD/R-induced oxidative stress suppresses YAP function, facilitating ferroptosis through downregulation of antioxidant genes. Fer-1 disrupts this pathological cascade by inhibiting downstream peroxidation events. The reactivated YAP restores antioxidant defenses via upregulation of antioxidant genes, creating a positive feedback loop that reinforces cellular resistance to ferroptosis. Overall, we discovered that ALI induced by II/R inhibited YAP expression in lung tissue. Notably, overexpression of YAP can counteract the ferroptosis induced by II/R and OGD/R, thereby effectively reducing ALI.

Nrf2, a crucial transcription factor in oxidation reactions, plays a vital role in regulating ferroptosis by controlling the metabolism of glutathione, iron, lipids, and mitochondrial function [43]. Furthermore, the activation of Nrf2 has been observed to increase the expression of various ROS detoxification enzymes, such as heme oxygenase-1 (HO-1) [44]. YAP and Nrf2 are transcription regulators that are activated in response to sublethal oxidative stress to promote cell survival. However, intestinal I/R-induced excessive ferroptotic stress overwhelms these pathways, leading to their functional impairment through cytoplasmic sequestration or degradation. The Hippo pathway signaling is particularly sensitive to redox imbalance, with severe lipid peroxidation promoting YAP cytoplasmic retention and suppressing its transcriptional activity [41,45]. Similarly, extreme lipid peroxidation depletes reducing equivalents like GSH, impairing Nrf2 dissociation from Keap1 and subsequent nuclear translocation [46].

TAZ, the paralog of YAP, reveal direct functional interactions with Nrf2 in the context of mitigating inflammation and enhancing antioxidant capacity in microglia [47]. Specifically, (1) Nuclear TAZ interacts with TEAD transcription factors to bind the Nrf2 promoter and promote its transcription, and (2) TAZ can induce Nrf2 nuclear translocation. Based on this established role of Nrf2 in mediating the protective effects of Hippo pathway components like YAP/TAZ during I/R injury, the significant structural homology between YAP and TAZ, and the demonstrated roles of oxidative stress and ferroptosis in our intestinal I/R-ALI model, we hypothesized that YAP might similarly regulate the bioactivity of Nrf2, either at the transcriptional or protein level. To test this hypothesis, ChIP-PCR and dual-luciferase assays (Fig. 11C and D), designed to examine the interaction between YAP and the Nrf2 promoter, yielded negative results, suggesting that YAP does not transcriptionally regulate Nrf2 expression. Furthermore, to determine whether YAP can directly bind to Nrf2, we performed molecular docking analysis, which revealed potential binding sites between YAP and Nrf2 (Fig. 3A). The CoHave we correctly interpreted the following funding source(s) and country names you cited in your article: GSH, Australia?-IP experiments (Fig. 11E and F) further supported our hypothesis that YAP can regulate Nrf2 at the protein level. Moreover, we proceeded to investigate the functional role of the YAP-Nrf2 axis within our specific mouse model in vivo. We employed AAV to overexpress YAP in mice and used the Nrf2 inhibitor ML385 to inhibit Nrf2 expression. Interestingly, the Nrf2 inhibitor hindered the protective effect of YAP and the resistance to ferroptosis in mice. Consistently, in vitro experiments involving lentiviral overexpression of YAP and knockdown of Nrf2 yielded similar outcomes. Clinical correlation analysis revealed a positive correlation between lung injury markers and intestinal injury markers. Additionally, lung injury markers and inflammatory factors exhibited a negative correlation with GSH. These findings provide clinical evidence linking II/R-induced lung injury to ferroptosis. However, these clinical studies represent only preliminary results, and further clinical samples are necessary for comprehensive elucidation.

The findings of this study indicate that ferroptosis and YAP play significant roles in the pathophysiology of II/R-induced ALI. The increased expression of YAP in the nucleus contributes to resistance against ferroptosis and alleviates ALI by binding to Nrf2 to promote its nuclear translocation. Sustaining high levels of YAP expression in the nucleus could potentially serve as a novel therapeutic approach for ALIFig. 13. However, it is important to acknowledge certain limitations in this study. Firstly, ALI is influenced by various complex pathological mechanisms, and ferroptosis is just one of them. The study did not evaluate other potential mechanisms involved. Secondly, YAP affects ferroptosis in ALI and may be involved in a variety of pathways besides Nrf2 pathway. Ultimately, the interaction between YAP and Nrf2 may be indirect, suggesting the potential involvement of other mediating proteins.

Fig. 13.

YAP-mediated Nrf2 translocation to the nucleus ameliorates ferroptosis in II/R-Induced ALI. Overexpression of YAP (OE-YAP) promotes the translocation of Nrf2 into the nucleus, where it activates the HO-1 promoter, leading to increased HO-1 expression. HO-1 mitigates ferroptosis by reducing ROS and inhibiting lipid peroxidation, thereby alleviating ALI.

CRediT authorship contribution statement

Lu Tang: Writing – original draft, Methodology, Data curation, Conceptualization. Chengjie Yang: Writing – original draft, Methodology. Yanhua Peng: Methodology. Mudi Liu: Methodology. Na Wei: Methodology. Xin Fan: Methodology. Bo Yang: Methodology. Jing Jia: Methodology. Ye Chen: Methodology. Jianguo Feng: Writing – review & editing, Methodology. Jun Zhou: Writing – review & editing, Funding acquisition, Conceptualization.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 81873930), partly by grants from Sichuan Science and Technology Program (No.2025ZNSFSC0721), Luzhou Science and Technology Program (No.2023SYF099).

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Data availability

Data will be made available on request.