Hesperetin Alleviates Bleomycin‐Induced Pulmonary Fibrosis by Modulating Cellular Senescence and Promoting Impaired Autophagy in a CISD2‐Dependent Manner

ABSTRACT

Pulmonary fibrosis is an age‐related disease marked by progressive lung function decline and high mortality, with limited treatment options. Hesperetin, a citrus‐derived flavonoid with antioxidant and anti‐aging properties, has not been thoroughly investigated for its potential in pulmonary fibrosis. This study evaluated the prophylactic potential of hesperetin in pulmonary fibrosis in vivo and in vitro. In vivo experiments demonstrated that hesperetin can significantly reduce cellular senescence in bleomycin‐induced pulmonary fibrosis. Activation of Nrf2 signaling was involved in inhibiting cellular senescence and oxidative stress in A549 cells treated with hesperetin. We found that the deficiency of CISD2 contributed to bleomycin‐induced pulmonary fibrosis and was associated with the protective effects of hesperetin, which bound with high affinity to CISD2. Meanwhile, overexpression of CISD2 improved cellular senescence induced by bleomycin in vitro. This study further highlighted that the cellular senescence induced by bleomycin was associated with impaired autophagy, which might be related to the inhibition of the CISD2/BECN1 pathway through bioinformatics analysis. Additionally, hesperetin was found to restore bleomycin‐induced impaired autophagy by modulating the CISD2/BECN1 pathway. Notably, the protective effects of hesperetin against pulmonary fibrosis were diminished following CISD2 knockdown. Collectively, these findings suggest that hesperetin ameliorates bleomycin‐induced pulmonary fibrosis through inhibiting cellular senescence and attenuating impaired autophagy in a CISD2‐dependent manner.

In bleomycin‐induced pulmonary fibrosis, CISD2 deficiency was associated with cellular senescence, oxidative stress, and impaired autophagy. Hesperetin, as a novel anti‐aging strategy, counteracts senescence and oxidative stress by restoring autophagy in a CISD2‐dependent manner.

Abbreviations

AECs

Alveolar epithelial cells

ANOVA

One‐way analysis of variance

ARRIVE

Animal Research: Reporting of In Vivo Experiments

AT2

Alveolar type II

AV

Adenoviral

BCA

Bicinchoninic Acid

BCL2

B‐cell lymphoma‐2

BECN1

Beclin 1

BSA

Bovine serum albumin

CCK‐8

Cell counting Kit‐8

CDK4

Cyclin dependent kinase 4

CISD2

CDGSH Iron–Sulfur Domain 2

DEGs

Differentially expressed genes

ECM

Extracellular matrix

ELISA

Enzyme‐linked immunosorbent assay

EMT

Epithelial‐mesenchymal transition

ER

Endoplasmic reticulum

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde‐3‐phosphate dehydrogenase

GEO

Gene expression omnibus

H&E

Hematoxylin and eosin

HO‐1

Heme oxygenase 1

IHC

Immunohistochemistry

IL‐1β

Interleukin‐1β

IL6

Interleukin‐6

IPF

Idiopathic pulmonary fibrosis

LD50

Median lethal dose

Nrf2

Nuclear factor erythroid 2‐related factor 2

OD

Absorbance

oeCISD2

CISD2 overexpression

onCISD2 or snRNA

Empty vector controls

p21

Cyclin‐dependent kinase inhibitor 1A

p62

Sequestosome 1

PDB

Protein data bank

PMSF

Phenylmethylsulfonyl fluoride

qRT‐PCR

Quantitative real‐time polymerase chain reaction

RIPA

Radio immunoprecipitation assay

ROS

Reactive oxygen species

RPMI

Roswell Park Memorial Institute

SASP

Senescence‐associated secretory phenotype

SA‐β‐gal

Senescence‐associated β‐galactosidase

scRNA‐seq

Single‐cell RNA sequencing

SEM

Standard error of the mean

shCISD2

CISD2 silencing

shRNA

Short hairpin RNA

SMILES

Simplified molecular input line entry system

TGF‐β1

Transforming growth factor‐β1

WB

Western blot

α‐SMA

α‐smooth muscle actin

1. Background

Pulmonary fibrosis is a chronic, progressive, and interstitial fibrosing lung disease, with an irreversible decline in lung function and a median survival of 2 to 5 years after diagnosis [1, 2, 3]. With growing prevalence worldwide, pulmonary fibrosis represents a substantial public health threat that profoundly diminishes patients' quality of life [4]. Clinically, the disease manifests with progressive dyspnea, reduced lung compliance, and respiratory failure, ultimately leading to a dramatic reduction in both healthspan and lifespan [5, 6]. Histopathologically, pulmonary fibrosis is characterized by abnormal extracellular matrix (ECM) deposition, distortion of alveolar architecture, recruitment of inflammatory responses, and sustained fibroblast activation [7]. These processes are driven by repetitive stress injuries to alveolar epithelial cells (AECs), which disrupt the epithelial‐mesenchymal crosstalk [2, 8]. Recent studies have identified numerous pathogenic mechanisms contributing to the occurrence and development of pulmonary fibrosis [3, 4]. Cellular senescence, characterized by irreversible cell cycle arrest, resistance to apoptosis, secretion of the senescence‐associated secretory phenotype (SASP), and impaired autophagy, plays a pivotal role in the pathogenesis of pulmonary fibrosis [9, 10]. The components of SASP, such as interleukin‐6 (IL6) and interleukin‐1β (IL‐1β), appear to enhance the growth arrest in multiple cells through paracrine signaling, inducing senescent AECs that compromise their ability to resolve lung injury, thereby enhancing susceptibility to pulmonary fibrosis [6, 11]. Thus, targeting and reducing senescent cells holds promise as therapeutic strategies for pulmonary fibrosis.

As a bioactive citrus flavonoid, hesperetin is predominantly found in lemons and sweet oranges, possesses anti‐inflammatory and antioxidant properties with therapeutic potential for various diseases, such as cardiovascular diseases, neurological diseases, and respiratory diseases [12, 13, 14]. Hesperetin appears to attenuate D‐galactose‐induced brain aging by upregulating antioxidant enzymes and suppressing apoptosis [15]. Furthermore, hesperetin significantly elevated nuclear factor erythroid 2‐related factor 2 (Nrf2) and p‐Nrf2 levels, resulting in improved renal function and attenuation of structural damage in diabetic rats [16]. On the other hand, hesperetin could restore glycemic control in animal models of diabetes through senescent cell clearance [17]. These evidences suggested anti‐aging might represent a key pharmacological property of hesperetin [18]. However, studies on the regulated effect of hesperetin against pulmonary fibrosis are rare, and the regulated mechanism is unclear.

CDGSH Iron–Sulfur Domain 2 (CISD2), characterized as a pro‐longevity gene, is primarily localized to the outer mitochondrial membrane and the endoplasmic reticulum (ER). It plays a pivotal role in regulating several key cellular processes, including autophagy, cellular senescence, apoptosis, and ferroptosis [19, 20, 21]. Dysregulation of CISD2 has been implicated in a wide spectrum of pathological conditions, including obesity, diabetes, aging, and neurodegenerative diseases [12, 20]. Notably, upregulation of CISD2 has been associated with various aggressive malignancies [20], such as diffuse large B‐cell lymphoma [22], lung squamous carcinoma [23], and prostate cancer [24]. Inversely, the level of CISD2 tends to decline with age across multiple tissues. For instance, CISD2 expression is significantly reduced in naturally aged mice compared with young mice, and CISD2 deficiency leads to an aged cardiac phenotype characterized by perivascular fibrosis, disorganized myofibrils, and swollen, degenerated mitochondria [25]. Moreover, in human keratinocytes derived from elderly individuals, CISD2 expression is downregulated following chronic ultraviolet exposure [26]. Interestingly, Hesperetin has been shown to enhance mitochondrial function, inhibit inflammatory response, and protect against oxidative stress by upregulating CISD2 expression [26, 27]. These evidences suggest that CISD2 may be involved in the regulation of cellular senescence across various diseases. However, the functional role of CISD2 in pulmonary fibrosis remains unclear, underscoring the urgent need for further investigation.

Here, we investigated the therapeutic potential of hesperetin in pulmonary fibrosis through a comprehensive approach. Firstly, a bleomycin‐induced pulmonary fibrosis mouse model was employed. Secondly, a bleomycin‐exposed A549 cells were examined. Thirdly, the network pharmacology and bioinformatic analyses were conducted. Through these investigations, we elucidated the underlying mechanisms and biological pathways of hesperetin through CISD2 targeting, revealing novel therapeutic avenues for pulmonary fibrosis treatment.

2. Materials and Methods

2.1. Experimental Animal and Treatment

C57BL/6 mice (10–12 weeks old, 18–22 g) were housed under a 12/12‐h light/dark cycle at 25°C with access to food and water. The mice were randomly assigned to four groups. Following previous studies [28, 29], a pulmonary fibrosis model (BLM group) was established by administering a single intratracheal dose of bleomycin (3 mg/kg) on day 0. As established in previous studies [30, 31], a dose of 50 mg/kg/day hesperetin was selected based on its well‐documented efficacy and safety profile in preclinical models. Accordingly, the BLM group received hesperetin (50 mg/kg/day; MCE, USA) via oral gavage daily for 28 consecutive days (HEB group). Simultaneously, the mice treated with normal saline (Shuanghe Pharmacy, China) or hesperetin (50 mg/kg/day) alone were assigned to the CON and HES groups, respectively, as controls. After 28 days of exposure, the mice were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (1 mL/kg) and sacrificed. All animal experiments were conducted in strict accordance with the ARRIVE 2.0 guidelines (Animal Research: Reporting of In Vivo Experiments, https://arriveguidelines.org) [32] and were approved by the Ethics Committee of the Affiliated Hospital of Putian University (ID: 2025DW070).

2.2. Enzyme‐Linked Immunosorbent Assay (ELISA)

Blood samples were collected from mice after sacrifice, and serum was separated by centrifugation at 3000 rpm for 10 min at 4°C. The serum samples were stored at −80°C until analysis. The levels of IL‐6 and IL‐1β were quantified using ELISA kits (Multi Sciences, China) according to the manufacturer's instructions.

2.3. Histological Staining and Immunohistochemistry (IHC) Assay

First, the right lung tissues from mice were collected and fixed in 4% paraformaldehyde solution (Solarbio, China) overnight. After dehydration and embedding in paraffin, the tissues were sectioned into 4 μm slices for hematoxylin and eosin (H&E) and Masson staining to observe histomorphological changes. Second, IHC was performed. The slides were permeabilized with 0.3% Triton X‐100 (Solarbio, China) and blocked with bovine serum albumin (BSA, Beyotime, China) at room temperature. Subsequently, the slides were incubated with primary antibodies, including α‐smooth muscle actin (α‐SMA; cat. YT8040, Immunoway, USA), transforming growth factor‐β1 (TGF‐β1; cat. YM8257, Immunoway, USA), cyclin‐dependent kinase inhibitor 1A (p21; cat. YT3497, Immunoway, USA), and cyclin‐dependent kinase 4 (CDK4; cat. YT5198, Immunoway, USA), overnight at 4°C, followed by incubation with a secondary antibody (HRP‐labeled goat anti‐rabbit IgG (H + L), Beyotime, China). The images were visualized under a microscope (Eclipse C1, Nikon, Japan).

2.4. Cell Culture and Adenoviral Transfection

The human alveolar epithelial cell line (A549, Procell, China) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin, and streptomycin (Hyclone, USA). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. Adenoviral (AV) vectors encoding CISD2 (for overexpression and silencing) and a corresponding negative control were obtained from Zolgene (China), with vector sequences detailed in Table S1. A549 cells were seeded in 6‐well plates at a density of 1 × 10⁵ cells per well and cultured until they reached approximately 70%–80% confluence. Cells were transfected with the adenoviral vectors for 6 h following the manufacturer's instructions. After transfection, cells were treated with bleomycin (MCE, USA) for 24 h prior to the next experiments.

2.5. Cell Viability Assay

Cell viability was assessed using the Cell Counting Kit‐8 (CCK‐8, Biosharp, China) assay. Briefly, after treatment, cells were incubated with 10% CCK‐8 solution at 37°C in a CO₂ incubator for 2 h. The absorbance (OD) at 450 nm was then measured using a microplate reader (ThermoFisher, USA), and the cell viability was calculated using the following formula: Cell viability (%) = [OD (test) – OD (blank)]/[OD (Control) – OD (blank)] × 100%.

2.6. Senescence‐Associated β‐Galactosidase (SA‐β‐Gal) Activity

Cellular senescence in fresh lung tissue was assessed using a SA‐β‐gal Staining Kit (Solarbio, China). For in vitro analysis, bleomycin‐ and hesperetin‐treated cells were washed with phosphate‐buffered saline (PBS) and processed for SA‐β‐gal staining (Solarbio, China) according to the manufacturer's protocol. Senescent cells were identified by the presence of characteristic green staining. Stained samples were visualized and quantified using a Nikon Eclipse C1 microscope (Japan). The percentage of SA‐β‐gal positive cells was calculated based on the total number of cells.

2.7. Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR)

The cells were harvested after treatment, and the total RNA was isolated using TRIzol reagent (Invitrogen, USA), and then transcribed into cDNA using the HiScript Q RT SuperMix for qPCR (Vazyme, China). qRT‐PCR was conducted on the CFX Connect Real‐Time PCR Detection System (BioRad, USA) using HQ SYBR qPCR Mix (without ROX) (Zomanbio, China). SASP factors (IL6 and IL‐1β) levels were quantified using the 2^−ΔΔCt method, with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as a loading control. The primer sequences are provided in Table S2.

2.8. Reactive Oxygen Species (ROS) Assessment

The generation of ROS was performed using a DCFH‐DA kit (Zomanbio, China), respectively. After treatment, cells were incubated with 1 μM DCFH‐DA solution in the dark. Fluorescence signals were subsequently observed and imaged using a fluorescence microscope (Nikon Eclipse Ti, Japan).

2.9. Western Blot (WB) Assay

Total proteins were extracted from the lung tissue of experimental animals and treated cells. The samples were collected and lysed using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China), supplemented with 1% phenylmethylsulfonyl fluoride (PMSF; Solarbio, China). The total protein concentration was measured using a bicinchoninic acid (BCA) assay kit (Beyotime, China). The levels of the following proteins were subsequently detected: CISD2 (cat. 66082‐1‐IG, Proteintech, China), α‐SMA (cat. YT8040, Immunoway, USA), TGF‐β1 (cat. YM8257, Immunoway, USA), p21 (cat. YT3497, Immunoway, USA), CDK4 (cat. YT5198, Immunoway, USA), Beclin 1 (BECN1; cat. YM8333, Immunoway, USA), Nrf2 (cat. YM8624, Immunoway, USA), heme oxygenase 1 (HO‐1; cat. YM8337, Immunoway, USA), sequestosome 1 (p62; cat. YM8025, Immunoway, USA), and B‐cell lymphoma‐2 (BCL2; cat. YM8319, Immunoway, USA). β‐actin (cat. YM8343, Immunoway, USA) was used as a loading control. The blot signals were visualized using the JS‐M9P system (P&Q Science and Technology, China).

2.10. Molecular Docking Analysis

To obtain the simplified molecular input line entry system (SMILES) information of hesperetin from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and the PDB identifier for CISD2 from the UniProt database (https://www.uniprot.org). These compounds were imported into Discovery Studio 2019 Client to screen for dominant conformations, followed by molecular docking using the ‘Dock Ligands’ function. The docking results were then exported in PDB format and visualized as 3D structures using PyMOL (Version 3.1.3, Schrödinger).

2.11. Bioinformatics Analysis

The single‐cell transcriptomic dataset GSE227136 [33] was retrieved from the Gene Expression Omnibus (GEO) database, containing single‐cell RNA sequencing (scRNA‐seq) profiles of lung tissues from 33 pulmonary fibrosis patients and 27 non‐fibrotic controls. Raw data were preprocessed and analyzed using the Seurat package within the R programming environment (version 4.4.2) [34]. In addition, bulk RNA expression profiles and corresponding clinical data from GSE132607 [35] (n = 74) were also obtained from the GEO database. The correlation between CISD2 expression and that of related genes in pulmonary fibrosis cases was subsequently assessed.

2.12. Statistical Analysis

All experimental data analyses were performed using R programming (version 4.4.2). Data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments or biological replicates. Statistical comparisons were conducted using one‐way analysis of variance (ANOVA) for multiple groups and Student's t‐test for comparisons between two groups. A p‐value < 0.05 was considered statistically significant.

3. Results

3.1. Hesperetin Attenuated Bleomycin‐Induced Pulmonary Fibrosis in Mice

To investigate the effects of hesperetin on pulmonary fibrosis, mice were intratracheally administered Bleomycin to establish a pulmonary fibrosis model, followed by oral gavage of hesperetin (50 mg/kg) every other day for 28 days (Figure 1A). Throughout the experiment, mice in the BLM group exhibited slower body weight gain compared to the other groups, while hesperetin treatment resulted in increased body weight relative to the BLM group (Figure S1A). After sacrifice, lung injury induced by bleomycin was evident (Figure S1B). On day 28 after bleomycin treatment, the lungs of mice that were exposed to bleomycin were larger than those of mice that were treated with saline or hesperetin, accompanied by a significantly elevated lung coefficient (Figure 1B, p < 0.05), possibly as a consequence of the underlying tissue fibrosis. Bleomycin exposure induced extensive pulmonary fibrosis, characterized by the disruption of normal lung architecture and intense Masson's trichrome blue staining (Figure 1C,D). In contrast, hesperetin‐treated lungs showed significantly better preservation of pulmonary architecture, along with a marked reduction in the severity of alveolar inflammation and fibrosis. Fibrosis scoring analysis further supported these observations (Figure 1D, p < 0.05). The scores were elevated in the BLM group compared to the CON group (Figure 1D, p < 0.05), but hesperetin treatment significantly reduced them. Meanwhile, the levels of pro‐fibrotic SASP factors, including IL‐1β and IL‐6, were significantly elevated in the serum of the BLM group compared to the CON group (Figure 1E, p < 0.05). Moreover, hesperetin treatment effectively reversed the upregulation of IL‐1β and IL‐6 (Figure 1E, p < 0.05), confirming its anti‐inflammatory effects in bleomycin‐induced lung injury. The upregulated expression of α‐SMA and TGF‐β1 in bleomycin‐exposed lung tissues was effectively suppressed by hesperetin treatment (Figure 1F,G, p < 0.05). Furthermore, hesperetin treatment restored the upregulated expression of α‐SMA and TGF‐β1 observed in fibrotic regions of bleomycin‐treated lung tissues (Figure 1H,I). Together, these findings demonstrate that hesperetin attenuates bleomycin‐induced pulmonary fibrosis.

FIGURE 1.

Hesperetin attenuated bleomycin‐induced pulmonary fibrosis in mice. (A) Experimental flowchart; (B) Comparisons of lung coefficients among the different groups; (C) H&E staining results for the different groups, Scale bar: 50 μM; (D) Masson staining results for the different groups, scale bar: 50 μM; (E) Comparisons of fibrosis scoring among the different groups; (F) Representative WB images of lung tissue from different groups; (G) Relative protein levels in lung tissue from different groups, including α‐SMA and TGF‐β1, with β‐Actin used as the loading control. IHC staining observation (Scale bar: 50 μM; Up: Magnification, 20×; Down: Magnification, 40×): (H) α‐SMA expression; (I) TGF‐β1 expression. H&E, Hematoxylin and eosin; IHC, Immunohistochemistry; TGF‐β1, Transforming growth factor‐β1; WB, Western blot; α‐SMA, α‐smooth muscle Actin. Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. Hesperetin Ameliorates Cellular Senescence in Mice Lung Induced by Bleomycin Exposure

Cellular senescence is a pathological feature of pulmonary fibrosis in the lung following bleomycin‐induced injury [3]. The levels of pro‐fibrotic SASP factors, including IL‐1β and IL‐6, were significantly elevated in the serum of the BLM group compared to the CON group, as determined by ELISA. Hesperetin treatment effectively reversed the upregulation of IL‐1β and IL‐6 (Figure S1C, p < 0.05), confirming its anti‐inflammatory and anti‐aging role in the treatment of pulmonary fibrosis. To observe cellular senescence in bleomycin‐exposed lung tissue, the senescence marker SA‐β‐gal activity was quantified. Bleomycin exposure significantly increased the number of SA‐β‐gal‐positive cells compared to the CON group (Figure 2A,D, p < 0.05). In contrast, hesperetin treatment reduced the percentage of SA‐β‐gal‐positive cells relative to the BLM group (Figure 2A,D, p < 0.05). As previous studies [3, 36], p21, which is known as an activator of senescence‐associated growth arrest, was upregulated in senescent cells and contributes to the fibrotic process. Meanwhile, CDK4, which interacts with p21, plays a key role in promoting cellular senescence. The image of IHC showed significantly higher levels of p21 and CDK4 compared to the CON group (Figure 2B,C), and hesperetin treatment significantly reduced the elevated expression levels of p21 and CDK4 in the BLM group (Figure 2B,C). Semiquantitative analysis of lung tissue revealed that bleomycin‐induced upregulation of p21 and CDK4 was significantly suppressed by hesperetin treatment (Figure 2E,F, p < 0.05). These results demonstrate that hesperetin effectively attenuates bleomycin‐induced cellular senescence.

FIGURE 2.

Hesperetin ameliorates cellular senescence in mice lung induced by bleomycin exposure. (A) Representative images of SA‐β‐gal staining in fresh lung tissue following bleomycin exposure and hesperetin treatment, Scale bar: 50 μM; (D) Quantification of SA‐β‐gal positive cells in fresh lung tissue following bleomycin exposure and hesperetin treatment. IHC staining observation (Scale bar: 50 μM; Up: Magnification, 20×; Down: Magnification, 40×): (B) p21 expression; (C) CDK4 expression; (E) Relative protein levels in lung tissue from different groups, including p21 and CDK4, with β‐Actin used as the loading control; (F) Representative WB images of lung tissue from different groups. CDK4, Cyclin dependent kinase 4; IHC, Immunohistochemistry; p21, Cyclin‐dependent kinase inhibitor 1A; SA‐β‐gal, senescence‐associated β‐galactosidase; WB, Western blot. Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. Hesperetin Alleviated Bleomycin Induced Cellular Senescence and Oxidative Stress by Activating Nrf2 In Vitro

Cellular senescence contributes to impaired epithelial regeneration following bleomycin‐induced lung injury [37]. The A549 cell (Procell, China), characterized as human alveolar type II (AT2) cells, was selected for further analysis. Bleomycin exposed to A549 exhibited a dose‐dependent effect on cellular senescence in A549 cells (Figure S2A). Moreover, an increase in SA‐β‐gal positive cells was observed following 40 μM bleomycin treatment (Figure S2B,C), indicating that 40 μM bleomycin exposure effectively induces cellular senescence in vitro. The levels of SASP factors, including IL‐1β and IL‐6, were significantly elevated following bleomycin exposure, indicating an increase in inflammatory cytokine secretion (Figure S2D). Meanwhile, semiquantitative analysis confirmed that bleomycin exposure increased the expression of two cellular senescence markers, p21 and CDK4 (Figure S2E,F). These results suggested that bleomycin induces cellular senescence in epithelial cells. Next, a 10 μM hesperetin pretreatment (Figure S2G) significantly enhanced cell viability (Figure 5A, p < 0.05) and recovered the decreased cell viability induced by bleomycin exposure (Figure 3A, p < 0.05). Furthermore, the increased SA‐β‐gal positive cells were reduced by hesperetin treatment (Figure 3B,D, p < 0.05). In line with in vivo experiments, bleomycin exposure upregulated the levels of fibrotic markers (α‐SMA and TGF‐β1, Figure 3E,F, p < 0.05) and cellular senescence markers (p21 and CDK4, Figure 3E,F, p < 0.05). Cellular senescence is closely associated with oxidative stress, a key driver that both initiates and sustains the senescent phenotype [3, 6]. Hesperetin treatment could inhibit ROS production and alleviate the high ROS levels induced by bleomycin (Figure 3G,H, p < 0.05). Nrf2 serves as a crucial transcription factor that maintains cellular redox homeostasis and mitigates cellular senescence [38, 39]. Meanwhile, Nrf2 often interacts with HO‐1 through a complex regulatory network that modulates cellular senescence. In this study, bleomycin exposure significantly suppressed Nrf2 signaling activity (Figure 3G,H, p < 0.05). Conversely, hesperetin treatment effectively restored Nrf2 activation and mitigated bleomycin‐induced cellular senescence (Figure 3G,H, p < 0.05). Collectively, hesperetin could inhibit cellular senescence induced by bleomycin via activation of Nrf2 signaling.

FIGURE 5.

Overexpression of CISD2 improved cellular senescence induced by bleomycin in vitro. (A) Relative expression of CISD2 in A549 cells transfected with CISD2 overexpression, assessed by qRT‐PCR; (B) Cell viability in A549 cells transfected with CISD2 overexpression and exposed to bleomycin, evaluated using the CCK‐8 method; (C) Quantification of SA‐β‐gal‐positive cells in A549 cells transfected with CISD2 overexpression and exposed to bleomycin. (D) Relative levels of SASP factors (left: Il‐1β, right: IL‐6) detected by qRT‐PCR; (E) Representative images of SA‐β‐gal staining in A549 cells transfected with CISD2 overexpression and treated with bleomycin, scale bar: 50 μM; (F) Relative protein levels in A549 cells transfected with CISD2 overexpression and treated with bleomycin, including α‐SMA, TGF‐β1, p21, CDK4, and CISD2, with β‐Actin used as the loading control. (G) Representative WB images of A549 cells transfected with CISD2 overexpression and treated with bleomycin. CCK‐8, Cell Counting Kit‐8; CDK4, Cyclin‐dependent kinase 4; CISD2, CDGSH Iron–Sulfur Domain 2; IL‐1β, Interleukin‐1β; IL‐6, Interleukin‐6; p21, Cyclin‐dependent kinase inhibitor 1A; qRT‐PCR, Quantitative real‐time polymerase chain reaction; SASP, senescence‐associated secretory phenotype; SA‐β‐gal, senescence‐associated β‐galactosidase; TGF‐β1, Transforming growth factor‐β1; WB, Western blot; α‐SMA, α‐smooth muscle actin. Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Hesperetin alleviated bleomycin‐induced cellular senescence and oxidative stress by activating Nrf2 in A549 cells. (A) Cell viability in A549 cells treated with hesperetin and bleomycin, assessed by the CCK‐8 method. (B) Quantification of SA‐β‐gal‐positive cells in A549 cells treated with hesperetin and bleomycin. (C) Relative levels of SASP factors (left: Il‐1β, right: IL‐6) detected by qRT‐PCR. (D) Representative images of SA‐β‐gal staining in A549 cells after 40 μM bleomycin treatment, scale bar: 50 μM. (E) Representative Western blot images from different groups. (F) Relative protein levels from different groups, including α‐SMA, TGF‐β1, p21, and CDK4, with β‐Actin used as the loading control. (G) Representative images of DCFH‐DA staining in A549 cells, scale bar: 20 μM. (H) Quantification of mean fluorescence intensity of ROS in A549 cells treated with hesperetin and bleomycin. (I) Representative WB images from different groups. (J) Relative protein levels from different groups, including Nrf2 and HO‐1, with β‐Actin used as the loading control. CCK‐8, Cell Counting Kit‐8; CDK4, Cyclin‐dependent kinase 4; HO‐1, Heme oxygenase 1; IL‐1β, Interleukin‐1β; IL‐6, Interleukin‐6; Nrf2, Nuclear factor erythroid 2‐related factor 2; p21, Cyclin‐dependent kinase inhibitor 1A; qRT‐PCR, Quantitative real‐time polymerase chain reaction; ROS, Reactive oxygen species; SASP, senescence‐associated secretory phenotype; SA‐β‐gal, senescence‐associated β‐galactosidase; TGF‐β1, Transforming growth factor‐β1; WB, Western blot; α‐SMA, α‐smooth muscle actin. Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. CISD2 Was Involved in the Protective Effects of Hesperetin on Pulmonary Fibrosis

Numerous studies have demonstrated that CISD2 plays a pivotal role in regulating cellular senescence [12, 20, 21]. Hesperetin has been identified as a potential anti‐senescence compound through its ability to enhance CISD2 expression [12, 40]. Molecular docking analysis confirmed this mechanism, showing that hesperetin binds to CISD2 with high affinity (ΔG = −15.66 kcal/mol) through extensive hydrophobic interactions (Figure 4A), providing structural insights into its pharmacological activity. Our in vivo studies demonstrated that while bleomycin challenge markedly suppressed CISD2 expression (Figure 4B–D), therapeutic intervention with hesperetin not only restored but actually enhanced CISD2 levels beyond baseline, effectively reversing the bleomycin‐induced suppression (Figure 4B–D). Similarly, bleomycin exposure significantly suppressed CISD2 expression in A549 cells, while hesperetin treatment effectively attenuated this suppression (Figure 4E,F). Based on these findings, downregulation of CISD2 contributes to bleomycin‐induced pulmonary fibrosis. To analyze the expression pattern of CISD2 in pulmonary fibrosis, the GSE227136 dataset [33] was obtained from the GEO database. As shown in Figure 4G,H, this lung tissue array contains samples from 66 idiopathic pulmonary fibrosis cases and 48 control donors. The analysis of scRNA‐seq revealed differential expression of CISD2 across 20 cell types (Figure 4I), including alveolar macrophages, AT2 cells, monocytes, and NK cells.

FIGURE 4.

CISD2 was involved in the therapeutic effects of hesperetin on pulmonary fibrosis. (A) Hydrogen bonds formed between hesperetin and the CISD2 protein; (B) IHC staining observation on CISD2 expression (Scale bar: 50 μM; Up: Magnification, 20×; Down: Magnification, 40×); (C) Representative WB images of lung tissue from different groups in vivo; (D) Relative CISD2 levels in lung tissue from different groups, with β‐Actin used as the loading control; (E) Representative WB images of A549 cells treated with hesperetin and bleomycin in vitro; (F) Relative CISD2 levels in A549 cells treated with hesperetin and bleomycin, with β‐Actin used as the loading control; (G) uMAP plot of differential cell types in the GSE227136 dataset; (H) uMAP plot of CISD2 expression in the GSE227136 dataset; (I) Differential expression of CISD2 across different cell types in the GSE227136 dataset. CISD2, CDGSH Iron–Sulfur Domain 2; IHC, Immunohistochemistry; IPF, Idiopathic pulmonary fibrosis; WB, Western blot. Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. Overexpression of CISD2 Improved Cellular Senescence Induced by Bleomycin In Vitro

Notably, CISD2 may play a pivotal role in the cellular senescence process associated with pulmonary fibrosis. To investigate the impact of CISD2 on the process of pulmonary fibrosis, A549 cells were transfected with constructed AV plasmids for CISD2 overexpression (oeCISD2, Table S2). As shown in Figure 5A, oeCISD2 significantly enhanced A549 cell viability compared to empty vector controls (onCISD2). In vitro, overexpression of CISD2 inhibited the decrease in cell viability induced by bleomycin (Figure 5B, p < 0.05). SA‐β‐gal staining results revealed that oeCISD2 could reverse the bleomycin‐induced cellular senescence (Figure 5C,E, p < 0.05). Also, overexpression of CISD2 was able to reduce the elevated levels of IL‐1β and IL‐6 induced by bleomycin (Figure 5D, p < 0.05). Bleomycin treatment induced significant increases in both fibrotic markers (α‐SMA and TGF‐β1) and senescence markers (p21 and CDK4); these adverse effects were reduced by transfection of oeCISD2 (Figure 5F,G, p < 0.05). These results demonstrate that CISD2 attenuates bleomycin‐induced pulmonary fibrosis through inhibition of cellular senescence.

3.6. Hesperetin Restored Impaired Autophagy Induced by Bleomycin Through Modulating the CISD2/BECN1 Pathway

To assess the potential mechanisms of CISD2 in pulmonary fibrosis, the top 10 CISD2‐related genes (CANX, BECN1, NDUFA8, TIMM10, TIMM10B, CISD3, BCL2, TIMM9, GIMAP5, and WFS1) were identified using the STRING database (Figure 6A). The correlations between these genes and CISD2 were analyzed based on raw data from the GSE132607 dataset [35] (Figure 6B). The results showed a significant positive correlation between CISD2 and BECN1 expression (Figure 6C, R ² = 0.5008, p < 0.05). BECN1 is a key regulator of autophagy, essential for initiating and progressing autophagosome formation [21]. It acts as a scaffold protein that recruits and interacts with various autophagy‐related proteins to form functional autophagy complexes [41]. Previous studies have demonstrated that CISD2 interacts with BCL2, an anti‐apoptotic protein that suppresses autophagy by binding to BECN1 [41, 42]. As shown in Figure 6D,E, bleomycin exposure significantly downregulated BECN1 expression while concurrently upregulating BCL2 levels (p < 0.05), suggesting bleomycin exposure induced impaired autophagy in A549 cells [43]. The ability of hesperetin to simultaneously upregulate BECN1 and downregulate BCL2 suggested a dual mechanism for mitigating bleomycin‐induced impaired autophagy (Figure 6D,E, p < 0.05). These results indicated that hesperetin may play a beneficial effect on the activation of autophagy induced by bleomycin.

FIGURE 6.

Hesperetin restored impaired autophagy induced by bleomycin through modulating the CISD2/BECN1 pathway. (A) The PPI network of 10 genes related to CISD2, visualized using STRING (https://string‐db.org); (B) The correlation plot between CISD2 and its related genes, visualized based on the GSE132607 dataset; (C) The scatter plot showing the correlation between CISD2 and BECN1 based on the GSE132607 dataset; (D) Representative WB images from different groups; (E) Relative protein levels from different groups, including BECN1, p62, and BCL2, with β‐Actin used as the loading control. BECN1, Beclin 1; BCL2, B‐cell lymphoma‐2; p62, Sequestosome 1; PPI, Protein–protein interaction; WB, Western Blotting. Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

3.7. Inhibition of CISD2 Decreased the Prophylactic Effects of Hesperetin on Pulmonary Fibrosis

To further elucidate whether hesperetin attenuates pulmonary fibrosis by suppressing cellular senescence through CISD2 knockdown. CISD2 inhibition was achieved through AV transfection of short hairpin RNA (shRNA) targeting CISD2 (CISD2 silencing, shCISD2, Table S2). As shown in Figure S3A, transfection with shCISD2_01 (selected as shCISD2) significantly reduced both CISD2 expression levels and cell viability in A549 cells compared to empty vector controls (snCISD2). Silencing CISD2 exacerbated the reduction in cell viability induced by bleomycin exposure (Figure S3B), while shCISD2 increased the number of SA‐β‐gal positive cells upon bleomycin exposure (Figure S3C,E). The intervention of CISD2 appeared to elevate the levels of SASP factors, including IL‐1β and IL6 (Figure S3D, p < 0.05). Notably, both shCISD2 transfection and bleomycin exposure significantly increased protein expression of fibrotic and senescence markers while reducing CISD2 levels (Figure S3F,G, p < 0.05). Next, we treated shCISD2‐transfected A549 cells with hesperetin. Notably, hesperetin failed to rescue the shCISD2‐induced reduction in cell viability (Figure 7A, p > 0.05). Meanwhile, the increase of SA‐β‐gal positive cells (Figure 7B,D, p > 0.05) and upregulation of SASP factors (IL‐1β and IL6, Figure 7C, p > 0.05) caused by shCISD2 transfection could not be inhibited by hesperetin treatment, and fibrotic and senescence markers demonstrated consistent phenotypic changes (Figure 7E,F, p > 0.05). Our results establish that CISD2 knockdown is sufficient to induce fibrotic activation and cellular senescence, phenotypes that prove refractory to hesperetin treatment. In addition, shCISD2 transfection reduced BECN1 while increasing p62 and BCL2 levels, indicating autophagy impairment. However, hesperetin treatment failed to restore this dysregulated autophagy. Furthermore, in CISD2‐knockdown cells, hesperetin did not activate CISD2 expression (Figure 7E,F, p > 0.05). These results demonstrate that hesperetin cannot rescue the autophagy defects caused by CISD2 deficiency.

FIGURE 7.

Inhibition of CISD2 decreased the therapeutic effects of hesperetin on pulmonary fibrosis. (A) Cell viability in A549 cells transfected with silencing CISD2 and treated with hesperetin, assessed by the CCK‐8 method; (B) Quantification of SA‐β‐gal positive cells in A549 cells transfected with silencing CISD2 and treated with hesperetin; (C) Relative levels of SASP factors (left: Il‐1β, right: IL‐6) detected by qRT‐PCR; (D) Representative images of SA‐β‐gal staining in A549 cells transfected with silencing CISD2 and treated with hesperetin, scale bar: 50 μM; (E) Representative WB images from different groups; (F) Relative protein levels from different groups, including α‐SMA, TGF‐β1, p21, CDK4, BECN1, p62, BCL2, and CISD2, with β‐Actin used as the loading control. BECN1, Beclin 1; BCL2, B‐cell lymphoma‐2; CCK‐8, Cell Counting Kit‐8; CDK4, Cyclin dependent kinase 4; CISD2, CDGSH Iron–Sulfur Domain 2; IL‐1β, Interleukin‐1β; IL‐6, Interleukin‐6; p21, Cyclin‐dependent kinase inhibitor 1A; p62, Sequestosome 1; qRT‐PCR, Quantitative real‐time polymerase chain reaction; SASP, senescence‐associated secretory phenotype; SA‐β‐gal, senescence‐associated β‐galactosidase; shCISD2, CISD2 silencing; snCISD2, Empty vector controls; TGF‐β1, Transforming growth factor‐β1; WB, Western blot; α‐SMA, α‐smooth muscle Actin. Results were expressed as mean ± SE (n ≥ 3). Results were expressed as mean ± SEM (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001, NS, no significance.

4. Discussion

Currently, only two FDA‐approved anti‐fibrotic therapies, such as nintedanib and pirfenidone, have demonstrated efficacy in slowing disease progression in most pulmonary fibrosis patients [44, 45, 46]. Despite ongoing research, these treatments remain limited in their ability to improve survival rates. The growing evidence indicates that early initiation of anti‐fibrotic therapy reduces the risk of acute exacerbations in patients with pulmonary fibrosis and prolongs survival by slowing disease progression [47, 48]. However, pulmonary fibrosis is often diagnosed at an established stage, as its symptoms are frequently misattributed to more common respiratory diseases such as asthma or chronic obstructive pulmonary disease [47, 49], making early‐stage diagnosis particularly challenging. Therefore, targeting the early stage of disease not only provides a unique opportunity to disrupt the pathological cascade that drives fibrogenesis but also fundamentally alters the natural progression of pulmonary fibrosis. Several natural products, such as curcumin and quercetin, have emerged as promising candidates for inhibiting the progression of pulmonary fibrosis due to their multi‐target mechanisms and diverse biological activities [45, 50, 51]. Curcumin has been shown to activate the Nrf2 pathway, leading to the upregulation of antioxidant enzymes such as HO‐1, thereby protecting lung tissue from oxidative damage induced by excessive ROS during pulmonary fibrosis development [28, 50]. Similarly, quercetin has been reported to attenuate pulmonary fibrosis by suppressing TGF‐β/Smad signaling, inhibiting fibroblast proliferation, and reducing extracellular matrix (ECM) deposition induced by SiO₂ both in vitro and in vivo [51]. These results suggest that natural products may serve as a viable prophylactic strategy for pulmonary fibrosis.

Notably, flavonoids are a diverse family of naturally occurring phenolic compounds widely distributed in fruits, vegetables, and tea, exhibiting a broad spectrum of biological effects, including anti‐inflammatory, antioxidant, neuroprotective, and anti‐aging activities [52, 53]. Based on their carbon skeleton and degree of oxidation, flavonoids can be classified into six major subclasses: flavones, flavonols, flavanones, isoflavones, flavanols, and anthocyanins [52]. Myricetin, a naturally occurring flavonoid structurally related to quercetin, has been reported to protect against oxidative stress, inflammation, and fibrosis by modulating key signaling pathways, including NF‐κB and Nrf2, across multiple organ systems [54]. Meanwhile, Kaempferol, categorized as a senomorphic flavonoid, has also been demonstrated to exert anti‐fibrotic effects by inhibiting epithelial‐mesenchymal transition (EMT), protect mitochondrial function, enhance autophagy, and reduce apoptosis [55]. Given its well‐documented antioxidant and anti‐aging properties, hesperetin, the aglycone metabolite of hesperidin, has been reported to exert prophylactic effects and to attenuate the progression of various diseases [18, 56]. Recent studies indicate that hesperetin demonstrates anti‐inflammatory, antioxidant, antitumor, and anti‐aging potential [13, 18]. However, the potential therapeutic or protective role of hesperetin in pulmonary fibrosis remains unexplored. Therefore, investigating the impact of hesperetin on pulmonary fibrosis and elucidating its underlying mechanisms is of significant research interest.

Mechanically, oxidative stress is a hallmark condition in pulmonary fibrosis [4]. Numerous studies have demonstrated that bleomycin exposure increases ROS, leading to significant cellular damage [57, 58]. Nrf2, a key regulator of redox homeostasis and cellular integrity, plays a pivotal role in the pathogenesis of pulmonary fibrosis [59]. Our results demonstrated that hesperetin promoted the expression of Nrf2 and HO‐1, counteracts their depletion induced by bleomycin exposure, and improved the ability to combat oxidative stress and inflammation, consistent with previous reports [60, 61]. On the other hand, emerging evidence implicates cellular senescence as a key pathogenic mechanism in pulmonary fibrosis, with patient‐derived fibrotic tissue exhibiting hallmark senescence features [3]. The AT2 cells isolated from pulmonary fibrosis lungs display transcriptomic hallmarks of cellular senescence and serve as the primary drivers of tissue repair and regeneration following lung injury [11, 62]. The accumulation of senescent AT2 cells that is driven by SASP secretion activates pro‐fibrotic myofibroblasts through multiple biological pathways, and these processes are involved in cell proliferation, inflammation, and autophagy [62, 63], and are resulted from increased SA‐β‐gal cells and the upregulation of senescence markers, such as p16, p21, and CDK4 [64]. Furthermore, senescent AT2 cells exhibited the loss of alveolar barrier integrity and persistent cell cycle arrest, contributing to fibroblast activation and excessive collagen deposition [65]. On the other hand, the excessive generation of ROS is another critical inducer of cellular senescence [3, 66]. The dramatic activation of oxidative stress and the mitochondrial dysfunction signaling pathway has been observed in senescent AT2 cells [3, 62]. Our previous study showed that bleomycin induced AT2 cell senescence through increased mitochondrial ROS and decreased mitophagy [67]. Collectively, these triggering factors promote cellular senescence in the initiation and progression of pulmonary fibrosis. Nitazoxanide, a broad‐spectrum antiparasitic and antiviral agent, has demonstrated anti‐fibrotic properties. In the bleomycin and D‐galactose‐induced senescence model, nitazoxanide effectively attenuates senescence progression in pulmonary fibrosis, and this effect occurs through suppression of SASP factor release [68]. Therefore, targeted cellular senescence has shown potential to restore pulmonary function and tissue homeostasis [11].

Generally, pulmonary fibrosis is a complex and progressive condition characterized by irreversible scarring and architectural distortion of the lung. While some forms of fibrosis, such as those triggered by acute lung injury, drug toxicity, or connective tissue diseases, may exhibit partial reversibility following removal of the triggering factor, established and irreversible fibrosis remains largely refractory to treatment [69, 70]. Therefore, therapeutic strategies are primarily aimed at slowing disease progression, preserving residual lung function, and alleviating symptoms to improve quality of life. In this study, the prophylactic potential of hesperetin emerged as the primary focus of investigation. Numerous studies had shown that oral administration of hesperetin has been shown to extend both lifespan and healthspan in vivo [12, 26]. These benefits have been associated with improvements in systemic metabolic health, including reduced adiposity, enhanced glucose homeostasis, and inhibition of organ senescence [12]. No specific reports on the median lethal dose (LD50) of hesperetin are readily available. Nevertheless, previous studies have shown that chronic administration of hesperetin at doses up to 100 mg/kg per day for 6 months in mice resulted in no detectable toxicity [27, 56]. Based on this safety profile, a 50 mg/kg dose was selected for the present study. This dosage has been consistently reported in the literature to exert robust antioxidant, anti‐inflammatory, and neuroprotective effects in mice [30, 31]. Accordingly, 50 mg/kg represents a well‐established, effective, and safe dose for evaluating the prophylactic potential of hesperetin in the context of pulmonary fibrosis. As previous studies [71, 72] noted, once absorbed, hesperetin undergoes rapid phase II conjugation, primarily yielding glucuronide and sulfate derivatives. Pharmacokinetic analyses have systematically demonstrated hesperetin sulfonation and glucuronidation across 12 individual UGT and 12 individual SULT enzymes, highlighting organ‐specific differences in UGT and SULT expression [72]. As a result, the predominant circulating form of hesperetin in plasma is its conjugated metabolites rather than the free aglycone, which retain significant biological activities, particularly antioxidant and anti‐inflammatory effects.

Consequently, hesperetin exerts antioxidant, anti‐inflammatory, and anti‐aging effects by modulating multiple signaling [18, 26, 61]. It has also been reported to attenuate the pathological progression of cardiac aging, characterized by hypertrophy and fibrosis, by reducing oxidative stress and improving glucose homeostasis [73]. In addition, hesperetin enhances mitochondrial function in HEK001 human keratinocytes derived from elderly individuals, protects against ultraviolet B‐induced cellular damage, and suppresses matrix metalloproteinase‐1 expression. Furthermore, in vitro studies have identified hesperetin as a CISD2 activator of considerable scientific interest, with molecular docking analyses confirming its strong steric and electrostatic complementarity with the CISD2. Decrease of CISD2 was the first to be observed in lung tissues from both pulmonary fibrosis patients and bleomycin‐induced fibrotic mouse models, accompanied by oxidative stress and cellular senescence [11]. Previous studies have indicated that increasing CISD2 levels show promise in ameliorating age‐associated diseases [20, 74, 75]. Given these findings, targeting CISD2 may serve as a valuable therapeutic strategy for mitigating pulmonary fibrosis. The altered expression of CISD2 across different cell types may lead to distinct disease outcomes. Single‐cell sequencing analysis of lung tissues from IPF patients and non‐fibrotic donors revealed that CISD2 is enriched in various lung cell populations, including epithelial, immune, and mesenchymal cells [33]. Consistent with previous studies [12, 26], our results demonstrated that hesperetin treatment protected A549 cells from bleomycin exposure by reducing the generation of ROS and inhibiting cellular senescence, which this effect is attributed to the enhancement of CISD2 expression.

Specifically, the complex role of CISD2 makes it challenging to regulate the biological function in various diseases [26]. CISD2 is localized on the membranes of the ER and mitochondria, where it plays a critical role in maintaining mitochondrial integrity and function [76]. CISD2 exerts its regulatory effects in coordination with related genes, influencing essential cellular processes such as metabolism, cell survival, and senescence [20]. Here, we performed bioinformatic correlation analysis, which revealed a positive correlation between CISD2 and BECN1. BECN1 forms part of the class III PI3 kinase complex (PI3KC3), where it initiates autophagosome formation and interacts with anti‐apoptotic proteins of the Bcl‐2 family [41, 77]. Reduction of BECN1 expression has been observed in fibroblasts from pulmonary fibrosis patients [67, 77]. Recent studies have further demonstrated that BECN1 plays critical roles in mitochondrial quality control and the autophagic machinery, processes that progressively decline with aging and are frequently impaired in senescent cells [78]. CISD2 acts as a negative regulator of autophagy by interacting with BCL2 at the ER. This interaction strengthens the inhibitory effect of BCL2 on BECN1, thereby suppressing BECN1‐mediated initiation of autophagy [79]. This process is a fundamental quality control mechanism responsible for the lysosomal degradation and recycling of cellular components to preserve homeostasis [7, 79]. Indeed, autophagy deficiency has been associated with cellular senescence in pulmonary fibrosis [80]. Our data also demonstrated that both CISD2 and BECN1 are integral regulators of autophagy and were markedly downregulated in A549 cells following bleomycin exposure, which was accompanied by elevated ROS production and cellular senescence, leading to impaired autophagy. The disruption of autophagy may promote SASP secretion by increasing DNA damage, activating senescence‐related pathways, and further driving both senescence and inflammation [81]. Meanwhile, hesperetin treatment ameliorated impaired autophagy by upregulating the CISD2/BECN1 signaling pathway. Moreover, CISD2 knockdown could reverse hesperetin‐mediated protection against pulmonary fibrosis. Collectively, these findings indicate that hesperetin mitigates cellular senescence and defective autophagy in a CISD2‐dependent manner.

Additionally, several limitations should be addressed in future studies. First, although hesperetin demonstrates promising prophylactic potential for regulating CISD2 expression, further research is required to evaluate its therapeutic efficacy in established pulmonary fibrosis, as well as to comprehensively investigate its metabolic profile. Second, while CISD2 and BECN1 were shown to be positively correlated and both implicated in autophagy regulation, the precise molecular mechanisms underlying their interaction were not examined in depth. Third, although AT2 cells were the primary focus of this study, it remains possible that CISD2 also influences senescence in other cell populations, such as immune and mesenchymal cells, which warrants further exploration.

5. Conclusion

Collectively, bleomycin exposure induced both pulmonary fibrosis and cellular senescence. Hesperetin, a promising natural product, was demonstrated to have protective effects against pulmonary fibrosis by activating Nrf2 signaling and attenuating impaired autophagy in a CISD2‐dependent manner. These findings suggest that hesperetin is a potential novel therapeutic strategy for pulmonary fibrosis.

Author Contributions

Study concepts and design: Chaofeng Zhang and Qi Lin. Experimental studies: Qi Lin, Wenjie Fan, Ziyi Chen and Bin Chen. Data acquisition and analysis: Wenjie Fan and Ziyi Chen. Statistical analysis: Qi Lin. Manuscript writing and editing: Wenjie Fan and Qi Lin. All authors read and approved the final manuscript.

Ethics Statement

This study is performed in accordance with relevant guidelines and regulations. The animal experiments were conducted in accordance with ARRIVE 2.0 guidelines and were approved by the Ethics Committee of the Affiliated Hospital of Putian University (ID: 2025DW070).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: fsb271083‐sup‐0001‐supinfo.pdf.

Lin Q., Fan W., Chen Z., Chen B., and Zhang C., “Hesperetin Alleviates Bleomycin‐Induced Pulmonary Fibrosis by Modulating Cellular Senescence and Promoting Impaired Autophagy in a CISD2‐Dependent Manner,” The FASEB Journal 39, no. 19 (2025): e71083, 10.1096/fj.202502311R.

Data Availability Statement

The publicly available datasets GSE227136 and GSE132607 were analyzed in this study. The R analysis code has been uploaded to GitHub. Additional data supporting the findings are available from the corresponding author upon reasonable request.