Novel therapeutic strategy: Nrf2 activation in targeting senescence-related changes in chronic obstructive pulmonary disease

Introduction

Chronic obstructive pulmonary disease (COPD) stands as a global public health concern characterized by incompletely reversible airflow obstruction, frequently accompanied by airway and/or alveolar inflammation (1-3). According to the World Health Organization (WHO), COPD ranks as the third leading cause of death worldwide, with its incidence and mortality expected to rise continuously over the coming decades (4,5). The development of COPD is associated with various environmental and genetic factors, though its precise mechanisms remain incompletely understood (6). In recent years, research has progressively unveiled the critical role of cell senescence in COPD progression, particularly highlighting the close correlation between epithelial cell senescence and disease advancement (7,8). Cellular senescence constitutes a complex biological process involving permanent cell cycle arrest, where cells in this state release a significant amount of pro-inflammatory and tissue remodeling factors known as senescence-associated secretory phenotype (SASP), a pivotal process in COPD (9-11).

Epithelial cell senescence holds a central position in the pathogenesis of COPD (9,11,12). Studies indicate that as COPD progresses, pulmonary epithelial cells undergo accelerated senescence, leading to the extensive release of SASP factors that further drive airway inflammation and fibrosis, exacerbating airflow limitation (9,11). SASP factors such as interleukin (IL)-6, tumor necrosis factor α (TNF-α), and other inflammatory mediators not only exacerbate local inflammation but also impact systemic health through the bloodstream, increasing the risk of cardiovascular diseases (13-15). Furthermore, SASP contributes to promoting neighboring non-senescent cell senescence by influencing intercellular signaling, creating a vicious cycle that propels COPD progression (16-18).

Nuclear factor erythroid 2-related factor 2 (Nrf2) represents a crucial antioxidant response transcription factor that protects cells from oxidative stress damage by upregulating various antioxidant enzymes and corresponding defense mechanisms (19,20). Activation of Nrf2 can diminish oxidative stress, inhibit inflammatory responses, and prevent cell senescence, making Nrf2 activators extensively studied as potential therapeutic agents for various diseases, including COPD (21). However, there is relatively limited research on the specific effects of Nrf2 activators on COPD bronchial epithelial cell senescence and SASP and their role in inhibiting COPD progression through these mechanisms (22-24). Understanding the role of Nrf2 in regulating cell senescence and inflammatory responses holds significant importance in developing new strategies for treating COPD.

Despite recent advancements in understanding the pathophysiology of COPD, current treatment methods still fail to completely reverse the disease progression, partly due to existing treatment strategies not adequately targeting the fundamental pathological processes of COPD, such as cell senescence and airway inflammation (25,26). In this context, exploring new treatment targets, especially those directly intervening in the pathophysiological processes, becomes notably important (27-29). Nrf2 activators have garnered attention for their potential role in regulating oxidative stress and cell senescence (30,31). However, despite showing some therapeutic potential in other disease models, their effects and mechanisms in COPD models are not fully understood, particularly their ability to prevent or reverse epithelial cell senescence and SASP (29,32,33).

Given this research gap, this study aims to thoroughly investigate the mechanisms of action of Nrf2 activators in COPD development, specifically their impact on bronchial epithelial cell SASP. By integrating single-cell transcriptome sequencing data and RNA sequencing chip data from public databases, this research first identifies senescent cells related to COPD and key molecular signaling pathways. Subsequently, through in vitro cell experiments and in vivo animal models, the study systematically assesses the effects of Nrf2 activators on inhibiting cell senescence, regulating inflammatory factor expression, improving respiratory function, and alleviating lung tissue pathology. These experimental designs aim to comprehensively elucidate, from the molecular to cellular to systemic levels, the potential mechanisms by which Nrf2 activators inhibit COPD progression. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-710/rc).

Methods

Public data download

COPD-related single-cell RNA sequencing (scRNA-seq) data of whole lung tissue were obtained through Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/). The dataset GSE167295 comprises 3 COPD patients (GSM5100998, GSM5100999, GSM5101000), and the data were analyzed using the R software package “Seurat” (34). Data quality control was performed based on the criteria of 200< nFeature_RNA <5,000 & percent.mt <20, and highly variable genes with the top 2,000 variances were selected. The COPD-related RNA sequencing chip dataset GSE162154 from the GEO database includes 3 COPD patients (GSM4943618, GSM4943619, GSM4943620) and 3 never-smoked healthy individuals (GSM4943621, GSM4943622, GSM4943623). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Since these data were obtained from public databases, ethical approval or informed consent was not required (35).

Clustering analysis

To reduce the dimensionality of the scRNA-Seq dataset, principal component analysis (PCA) was conducted based on the top 2,000 highly variable genes by variance. The top 20 principal components were selected for downstream analysis using the Elbowplot function provided by the Seurat software package. The FindClusters function in Seurat was utilized to identify main cell subgroups, with the resolution set to the default (res =1). Subsequently, the uniform manifold approximation and projection (UMAP) algorithm was applied to reduce the nonlinear dimensionality of the scRNA-seq sequencing data. Various cell subgroup marker genes were filtered using the Seurat software package. Cell annotation was performed by combining known cell lineage-specific marker genes with the online platform CellMarker and the “SingleR” package for annotation (36).

Pseudotime analysis

Pseudotime analysis was performed on the epithelial cell subgroups to explore dynamic changes in gene expression. A pseudotime axis was constructed, and dynamic gene sets were identified using Monocle (version 2.14.0).

Differential gene

Expression selection of differentially expressed genes (DEGs) in the scRNA-Seq dataset were selected using the “limma” package in R software. DEGs between COPD and normal samples were filtered based on criteria of |log fold change (FC)| >1 & P.adjust <0.05 (37).

Cell culture and treatment

The mouse lung epithelial type II cell line MLE-12 (CRL-2110, American Type Culture Collection, Manassas, VA, USA) was cultured in Dulbecco’s modified eagle medium (DMEM)/F-12 medium (11320033, Gibco, Gaithersburg, MD, USA) supplemented with 2% fetal bovine serum (FBS) (10099141, Gibco), 2 mM L-glutamine (25030149, Gibco), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (11344041, Gibco), 1:100 insulin/transferrin/selenium supplement (41400045, Gibco), and 1% penicillin-streptomycin (15140148, Gibco). Primary mouse bronchial smooth muscle cells (CP-M158, Procell) were cultured in a specific complete culture medium (CM-M158, Procell) for bronchial smooth muscle cells.

Cell groups were as follows: control (normal cultured MLE-12 or primary mouse bronchial smooth muscle cells), model group [cigarette smoke extract (CSE)-stimulated MLE-12 cells or primary mouse bronchial smooth muscle cells], model + sulforaphane group (CSE-stimulated and sulforaphane-treated MLE-12 cells or primary mouse bronchial smooth muscle cells). Cell stimulation with CSE (5%) was conducted for 48 hours. Treatment with sulforaphane (S4441, Sigma-Aldrich, Darmstadt, Germany) was administered at concentrations of 20 µmol/L for 48 hours (38-40).

Preparation of CSE

CSE was prepared to mimic cigarette smoke effects in vitro. A peristaltic pump burned an unfiltered cigarette, and the smoke was slowly bubbled into 10 mL of medium for 5 minutes. The pH of the medium containing the smoke extract was adjusted to 7.4, and it was then sterile-filtered through a 0.22 µm syringe filter to remove bacteria and large particles. The formulation was considered to have a 100% CSE concentration and was applied within 30 minutes for experiments (39,41).

Senescence-associated β-galactosidase (SA-β-Gal) staining experiment

Cellular senescence was determined using the SA-β-Gal staining kit (C0602, Beyotime, Shanghai, China). In brief, cells were seeded in 24-well plates at a density of 7×10⁴ cells per well. After cell treatment, fixation was carried out at room temperature for 15 minutes, followed by three washes with cold phosphate-buffered saline (PBS). Subsequently, 500 µL of SA-β-Gal solution was added to each well, and cells were washed again before overnight incubation at 37 ℃. Finally, cell images were captured using an electron microscope. The SA-β-Gal-positive rate was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) (42).

Cell counting kit-8 (CCK-8) assay

MLE-12 cells or primary mouse bronchial smooth muscle cells were seeded at a density of 5,000 cells per well in 96-well plates. Cell viability was assessed using the CCK-8 assay kit (C0038, Beyotime). Briefly, after 48 hours of experimental treatment, CCK-8 solution was added to the culture medium and incubated at 37 ℃ in a humidified atmosphere of 95% air and 5% CO₂ for 1 hour. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Berkeley, CA, USA) (43).

Establishment of COPD mouse model

Thirty male C57BL/6 mice (18–22 g) were purchased from Charles River (strain 219, Wilmington, MA, USA) and housed under specific pathogen-free (SPF) conditions with ad libitum access to water and food. A protocol was prepared before the study without registration. All animal experiments were approved by the Animal Ethics Committee of The Second Affiliated Hospital of Wenzhou Medical University (No. 20240108b0241701[007]). All procedures involving animals were conducted in accordance with the national guidelines for the care and use of animals. Except for the normal group, all mice were anesthetized with 100 mg/kg pentobarbital sodium (P3761, Sigma-Aldrich) and intratracheally exposed to 200 µL of lipopolysaccharide (LPS, L2630, Sigma-Aldrich) at a concentration of 1 mg/mL. Subsequently, mice were placed in a chamber containing 50 g of sawdust and 0.682 g of cigarette (tar 13.5 mg/g, nicotine 0.48 mg/g) for smoke exposure for 30 minutes per day for 28 days to establish a mouse model of COPD. Sulforaphane treatment involved intraperitoneal injection of 200 µL of sulforaphane (10 mg/kg) 1 hour before each LPS exposure (44,45).

Animal groups comprised ten mice each: normal (untreated), COPD (model group), and COPD + sulforaphane (COPD with sulforaphane treatment). Upon completion of in vivo experiments, 5 mL of blood was collected via the tail vein, centrifuged at 2,000 rpm, 4 ℃ for 15 minutes, and the serum was stored at −80 ℃ for cytokine analysis. Mice were euthanized, and lung tissues were harvested for further analysis (46).

Pulmonary function evaluation

On the 30th day post-treatment in the mouse models, pulmonary function tests were conducted using a small-animal pulmonary function testing apparatus (PFT, BUXCO, Chapel Hill, NC, USA). Parameters monitored included respiratory rate, peak expiratory flow (PEF), and forced vital capacity (FVC). Forced expiratory volume at 0.3 seconds (FEV_0.3) was calculated as FEV_0.3/FVC×100% using a specific formula (46).

Enzyme-linked immunosorbent assay (ELISA)

Mouse serum or cell culture supernatant was collected for analysis of TNF-α (ab208348, Abcam, Cambridge, UK) and IL-6 (ab222503, Abcam) using ELISA kits as per the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader (BioRad, CA, USA). The concentrations of TNF-α and IL-6 were calculated by constructing standard curves (47).

The determination of catalase (CAT), superoxide dismutase (SOD), and reactive oxygen species (ROS) levels involved collecting mouse lung tissue or processed primary cells and MLE-12 cells. Following the manufacturer’s instructions, commercially available assay kits were used to measure CAT (S0051, Beyotime), SOD (S0101S, Beyotime), and ROS (S0033S, Beyotime) levels. Enzyme activity of CAT and SOD was determined at 520 and 450 nm absorbance, respectively, using a microplate reader (BioRad). ROS levels were assessed using the Varioskan LUX microplate reader (Thermo Fisher Scientific, Rockford, IL, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm (48,49).

Histological studies

The left lung was isolated and fixed by intratracheal instillation of 2 mL 4% formaldehyde, followed by PBS washing and immersion in the fixative for at least 24 hours. Subsequently, after formaldehyde fixation and paraffin embedding, 3–4 µm sections were obtained and stained with hematoxylin and eosin (H&E) and Masson trichrome. Lung injury was semiquantitatively analyzed morphologically using a specific histological scoring system with the following criteria: 0, normal lung; 1, increased septal thickness; 2, epithelial thickening; 3, nasal septum inflammatory infiltration; 4, alveolar hemorrhage and/or hyaline membrane; 5, severe disruption of lung structure. Additionally, in three randomly chosen microscopic fields at a magnification of 100×, changes in wall thickness and area were observed in bronchioles with diameters <100 µm (ratio of shortest distance to lumen diameter, ≥0.7), calculating the wall area/total bronchiole area [mucosal area percent (MA%)] and wall thickness/bronchiole diameter [mucosal thickness percent (MT%)]. Furthermore, at a magnification of 200×, muscular arteries with diameters of 50–100 µm were selected for evaluating microvascular remodeling (50).

Western blot

Total protein was extracted from tissues or cells using RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF, P0013B, Beyotime). Nuclear proteins were isolated using a nuclear protein extraction kit (P0028, Beyotime) and quantified using a bicinchoninic acid (BCA) protein analysis kit (23225, Thermo Fisher Scientific). The protein samples (50 µg) were dissolved in 2× sodium dodecyl sulfate (SDS) sample buffer, boiled at 100 ℃ for 5 minutes, and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane using a wet transfer system. The membrane was blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation at 4 ℃ with primary antibodies against IL-1β (ab315084, 1:1,000, Abcam), matrix metalloproteinase 1 (MMP1, ab215715, 1:1,000, Abcam), transforming growth factor beta (TGF-β, ab315254, 1:1,000, Abcam), Nrf2 (16396-1-AP, 1:1,000, PROTEINTECH), p16 (ab151303, 1:1,000, Abcam), p21 (ab109520, 1:1,000, Abcam), p53 (ab26, 1:1,000, Abcam), HDAC1 (ab109411, 1:1,000, Abcam), and GAPDH (ab9485, 1:1,000, Abcam). The membrane was then washed with Tris-buffered saline with Tween 20 (TBST) three times for 10 minutes each and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (ab97051, 1:2,000, Abcam) for 1 hour at room temperature. After washing with TBST, the membrane was placed on a clean glass plate. Pierce^TM enhanced chemiluminescence (ECL) detection reagents (32209, Thermo Fisher Scientific) A and B were mixed in the dark, applied to the membrane, and imaged using the Bio-Rad image analysis system (ChemiDoc^TM XRS+, Bio-Rad) (39).

Statistical analysis

Data were derived from at least three independent experiments and were presented as mean ± standard deviation (SD). Independent samples t-tests were used for comparisons between the two groups. One-way analysis of variance (ANOVA) was utilized for comparisons involving three or more groups, followed by Tukey’s HSD post-hoc test if ANOVA results indicated significant differences to compare group differences. For non-normally distributed or unequal variance data, the Mann-Whitney U test or Kruskal-Wallis H test was applied. Statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA) and R. The significance level for all tests was set at 0.05, with a two-tailed P value <0.05 considered statistically significant.

Results

Isolation of ciliated epithelial cells in COPD using single-cell transcriptome sequencing

Preprocessed COPD-related single-cell transcriptome data GSE167295, encompassing lung tissues from 3 COPD patients, revealed transcriptome data from a total of 12,073 single cells. Utilizing Seurat for dimensionality reduction clustering analysis, 27 cell clusters were successfully identified (Figure S1A,S1B). Subsequently, cell annotation was performed using the “Single R” package in conjunction with marker genes, identifying 7 cell types, including epithelial cells (KRT19), natural killer (NK)/T cells (CD3E), B cells (CD79A), macrophages/monocytes (CD68, CXCL13), fibroblasts (COL1A1), mast cells (MAOB), and endothelial cells (PECAM1) (Figure S1C; Figure 1A). Correlation heatmaps for the top three cell types confirmed the reliability of the annotations (Figure 1B). To further pinpoint key genes regulating epithelial cells, we selectively annotated the epithelial cell cluster, revealing 11 cell subtypes (Figure S1D). The marker genes for various cells annotated epithelial cells as 6 cell types (Figure S1E), including type 1 (AGER) and type 2 (SFTPD) alveolar epithelial cells, basal cells (KRT5, FGFBP1), ciliated epithelial cells (FOXJ1, CFAP43), club cells (SCGB1A1), and goblet cells (MUC5AC) (Figure 1C). Correlation heatmaps for the top three cell types were presented (Figure 1D). Analysis of the proportion of the six cell types per sample revealed a higher prevalence of ciliated epithelial cells, distinctly identified by specific marker genes and visualized in two-dimensional (2D) space through t-distributed stochastic neighbor embedding (t-SNE)/UMAP (Figure 1A-1D). Secondary clustering of epithelial cells revealed further subdivision into multiple subclusters, illustrating the heterogeneity of epithelial cells in the COPD environment (Figure 1E). A higher abundance of goblet cells and a lower proportion of ciliated cells suggest that goblet cell proliferation and ciliary abnormalities are characteristic features of COPD. Subsequent analyses involved conducting pseudotime analysis of the epithelial cell subtypes using the “Monocle2” package. The cell transition trajectory within pseudotime exhibited distinct clusters, with the origin tissue used for color-coding in the pseudotime plot to identify cell composition, revealing ciliated epithelial cells positioned at the end of the timeline (Figure 1F). In COPD patients, ciliated epithelial cells may undergo morphological and functional alterations, such as abnormal cell differentiation and compromised epithelial barrier function (51). These changes may render the airways more susceptible to pathogen invasion and the effects of inflammatory factors (52-54), warranting the focus on ciliated epithelial cells.

Figure 1 Heterogeneity and subgroup classification of lung tissues in COPD. (A) Visualization of cell annotation results based on UMAP clustering; (B) correlation heat map of expression levels of the top 3 genes with 7 cell types; (C) visualization of cell annotation results based on UMAP clustering after extracting epithelial cell groups; (D) correlation heat map of expression levels of the top 3 genes with 6 cell types; (E) relative abundance of different cell subtypes in each sample represented by different colors; (F) trajectory skeleton map color-coded in pseudotime, with each point representing a cell type (right). The darker the color, the higher the pseudotime value, indicating that the cells are at a later stage of development or differentiation. The numbers on the figure are used to label different branch points or clusters, representing key bifurcations in the developmental trajectory. The arrows indicate the dynamic pathways of cells transitioning from one state to another during development. COPD, chronic obstructive pulmonary disease; NK, natural killer; UMAP, uniform manifold approximation and projection.

Nrf2 as a key regulator of COPD epithelial cell aging

Ciliated epithelial cells play an essential role in the respiratory tract by clearing mucus and foreign particles through the movement of their cilia, integral to the respiratory self-cleaning mechanism (55,56). Aging ciliated epithelial cells may alter their secretion function, leading to excessive mucus production (57). The accumulation of mucus further obstructs the airways, exacerbating breathing difficulties, a common symptom among COPD patients (58,59).

Subsequently, we further characterized aging ciliated epithelial cells within the epithelial cell population, identifying P21 (CDKN1A) and H2AFX as key molecular markers in the aging pathway. These genes were found to be highly expressed in cluster3 and cluster10 (Figure 2A), leading to the classification of cluster3 as aging ciliated epithelial cells and cluster10 as aging basal cells (Figure 2B).

Figure 2 Identification of key genes in aging ciliated epithelial cells in COPD. (A) Dot plot of CDKN1A and H2AFX in the epithelial cell group based on clustering; (B) visualization of cell annotation results based on UMAP clustering after annotation of senescent cells; (C) trajectory skeleton map color-coded in pseudotime, with each point representing a cell type. The darker the color, the higher the pseudotime value, indicating that the cells are at a later stage of development or differentiation. The numbers on the figure are used to label different branch points or clusters, representing key bifurcations in the developmental trajectory. The circle marks the senescent ciliated epithelial cells at the terminal end of the developmental trajectory; (D) volcano plot of differential analysis in the GSE162154 dataset, highlighting significantly upregulated (red) and downregulated (blue) genes; (E) Venn diagram of genes related to COPD downloaded from GSE162154, GSE167295, and GeneCards; (F) box plot of expression levels of 5 intersecting genes. COPD, chronic obstructive pulmonary disease; UMAP, uniform manifold approximation and projection.

Temporal analysis using the “Monocle2” package located aging ciliated epithelial cells at the end of the timeline (Figure 2C). The “FindAllMarkers” function was then utilized to analyze differential characteristic genes of specific aging ciliated epithelial cells, identifying a total of 1,697 genes.

Furthermore, COPD-related transcriptomic data was acquired from the GEO database, and differential gene expression analysis was conducted using the LIMMA package. A total of 461 genes with significant differences between the COPD and control groups were identified. Among these genes, 239 were upregulated in the COPD group, while 222 genes were downregulated, reflecting notable changes in cellular functionality under COPD conditions (Figure 2D).

From the GeneCards website, 3,505 genes related to COPD were retrieved based on a relevance score exceeding 10, and an intersection was performed with 461 differential genes and characteristic genes of aging ciliated epithelial cells, resulting in the identification of five intersecting genes: GFPT1, NFE2L2, GTF2IRD2, TRPV4, and ALMS1 (Figure 2E). Expression analysis of these genes in the GSE162154 dataset revealed relatively high expression levels of NFE2L2 with significant differences (Figure 2F).

Nuclear factor erythroid 2-related factor 2 (NFE2L2), also known as Nrf2, plays a crucial role in COPD (60,61). Nrf2, acting as a transcription factor, is pivotal in maintaining cellular defense mechanisms against oxidative stress and inflammation (62-64).

Therefore, this study identifies Nrf2 as a key gene influencing the aging of COPD epithelial cells.

Nrf2 activators SASP in COPD epithelial cells

Based on the bioinformatics analysis mentioned above, we hypothesize that Nrf2 serves as a crucial gene influencing cellular senescence in COPD. To further confirm the role of Nrf2 in COPD, we treated mouse lung epithelial type II cells (MLE-12 cells) with CSE to establish a COPD model. Subsequently, we co-treated the COPD model cells with an Nrf2 activator. The SASP refers to a series of inflammatory factors, chemokines, growth factors, and proteases secreted by senescent cells, representing a hallmark of aging (65,66).

To assess SASP, we analyzed the expression of IL-1β, MMP1, and TGF-β in MLE-12 cells. Western blot experiments were conducted to examine the expression levels of Nrf2 and IL-1β, MMP1, and TGF-β in the different MLE-12 cell groups. The results revealed that compared to the Control group, the Model group displayed significantly decreased levels of nuclear and total Nrf2 expression in MLE-12 cells, while IL-1β, MMP1, and TGF-β proteins were significantly elevated. Conversely, compared to the Model group, the Model + Sulforaphane group showed significantly increased levels of nuclear and total Nrf2 expression and decreased levels of IL-1β, MMP1, and TGF-β proteins (Figure 3A,3B). Furthermore, compared to the control group, the model group exhibited significantly increased expression of p53, p21, and p16 in MLE-12 cells. In contrast, the model + sulforaphane group showed significantly decreased expression levels of p53, p21, and p16 compared to the Model group (Figure 3C).

Figure 3 Influence of Nrf2 activator on the SASP of COPD epithelial cells. (A) Western blot experiment detecting the protein expression of nuclear Nrf2 in each group of cells; (B) Western blot experiment assessing the expression of Nrf2, IL-1β, MMP1, and TGF-β proteins in MLE-12 cells of each group; (C) Western blot experiment measuring the expression levels of p53, p21, and p16 proteins in MLE-12 cells of each group; (D) CCK-8 assay evaluating the proliferative capacity of MLE-12 cells in each group; (E) ELISA experiment determining the levels of TNF-α and IL-6 in the supernatant of MLE-12 cell cultures of each group; (F) SA-β-Gal staining experiment assessing the proportion of SA-β-Gal-positive cells in MLE-12 cells of each group; (G-I) measurement of enzyme activities of CAT (G), SOD (H) and ROS levels (I) in MLE-12 cells of each group. ANOVA was used to compare differences between groups: *, P<0.05 compared to the control group; ^#, P<0.05 compared to the model group. Experiments were performed in triplicate. ANOVA, analysis of variance; CAT, catalase; CCK-8, cell counting kit-8; COPD, chronic obstructive pulmonary disease; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MMP1, matrix metalloproteinase 1; Nrf2, nuclear factor erythroid 2-related factor 2; prot, protein; ROS, reactive oxygen species; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype; SOD, superoxide dismutase; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor-alpha.

The CCK-8 experiment was conducted to assess the proliferation capability of MLE-12 cells in different groups. The results revealed a significant decrease in the proliferation ability of MLE-12 cells in the model group compared to the control group. Following treatment with an Nrf2 activator, the proliferation capability of MLE-12 cells in the COPD cell model significantly increased (Figure 3D).

In addition, the ELISA experimental results demonstrate that compared to the control group, the levels of TNF-α and IL-6 in the supernatant of MLE-12 cell culture medium in the model group significantly increased. Following treatment with an Nrf2 activator, there was a significant decrease in the levels of TNF-α and IL-6 in the supernatant of the MLE-12 cell culture medium in the COPD cell model, further confirming the inhibitory effect of the Nrf2 activator on inflammatory response in the COPD cell model (Figure 3E).

The SA-β-Gal staining experiment revealed that compared to the control group, the proportion of SA-β-Gal positive cells in the MLE-12 cells significantly increased in the model group; after treatment with the Nrf2 activator, the proportion of SA-β-Gal positive cells in the COPD cell model significantly decreased (Figure 3F).

We also observed the levels of oxidative stress in different groups, indicating that in the Model group, the enzyme activities of CAT and SOD significantly decreased while the ROS levels significantly increased compared to the control group; however, after treatment with the Nrf2 activator, the enzyme activities of CAT and SOD in the COPD cell model significantly increased, and the ROS levels significantly decreased (Figures 3G-3I).

Finally, to corroborate our research findings, we conducted experiments using primary bronchial epithelial cells and observed results consistent with those of MLE-12 cells, including the Nrf2 activator delaying cell senescence and reducing oxidative stress responses (Figure S2).

These findings indicate that Nrf2 activators effectively suppress the SASP in COPD bronchial epithelial cells, potentially serving as a promising therapeutic strategy to inhibit COPD progression.

Nrf2 activators inhibit disease progression in COPD mice

Following the confirmation in vitro cell experiments that Nrf2 activators effectively suppress the SASP in COPD bronchial epithelial cells, to further explore the impact of Nrf2 activators on the progression of COPD in mice, our constructed COPD mouse model was treated with Nrf2 activators.

Pulmonary function tests revealed that compared to the normal group, mice in the COPD group exhibited a significant increase in respiratory rate, a marked reduction in the FEV_0.3/FVC ratio, and a significant decrease in PEF, indicating obstructive lung ventilation dysfunction. Post-treatment with Nrf2 activator (sulforaphane), the pulmonary function of mice in the COPD model group significantly improved, with a notable decrease in respiratory rate, a significant increase in the FEV_0.3/FVC ratio, and a significant increase in PEF (Figure 4A).

Figure 4 Evaluation of the therapeutic effects of Nrf2 activators in a COPD mouse model. (A) Lung function tests for each group; (B) ELISA analysis of IL-6 and TNF-α levels in mouse serum for each group; (C) H&E staining and scoring analysis of mouse lung tissue for each group; (D) statistical analysis of the wall area of bronchiole, the wall thickness of the bronchiole, the wall area/total bronchiole area (MA%), the wall thickness/bronchiole diameter (MT%) for each group; (E) Masson staining to detect collagen expression in mouse lung tissue for each group; (F) Western blot analysis of Nrf2 protein expression in mouse lung tissue; (G) Western blot analysis of Nrf2 and IL-1β, MMP1, and TGF-β protein expression in mouse lung tissue for each group; (H-J) enzyme activity of CAT (H) and SOD (I), and ROS (J) levels in mouse lung tissue for each group were measured. ANOVA was used to compare differences between groups: *, P<0.05 compared to the normal group; ^#, P<0.05 compared to the COPD group. Each group consisted of 10 mice. ANOVA, analysis of variance; CAT, catalase; COPD, chronic obstructive pulmonary disease; ELISA, enzyme-linked immunosorbent assay; FEV_0.3, forced expiratory volume at 0.3 seconds; FVC, forced vital capacity; H&E, hematoxylin and eosin; IL, interleukin; MA%, mucosal area percent; MMP1, matrix metalloproteinase 1; MT%, mucosal thickness percent; Nrf2, nuclear factor erythroid 2-related factor 2; PEF, peak expiratory flow; prot, protein; ROS, reactive oxygen species; SOD, superoxide dismutase; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor-alpha.

ELISA experiments assessing the levels of inflammatory factors in mouse serum showed that in the COPD model group, the levels of inflammatory factors TNF-α and IL-6 were significantly elevated, reflecting inflammatory responses under COPD conditions. Treatment with Nrf2 activator (sulforaphane) resulted in a significant decrease in the levels of TNF-α and IL-6 inflammatory factors in the serum of COPD mice (Figure 4B).

H&E staining was conducted to observe the morphological features of airway epithelium, alveolar septa, and inflammatory cell infiltration in the lungs of COPD mice. The results revealed that the COPD group exhibited larger alveolar spaces, increased inflammatory cell infiltration, and higher tissue scores compared to the normal group. In contrast, the COPD + sulforaphane group of mice showed reduced inflammatory cell infiltration, decreased alveolar septa destruction, and lower tissue scores compared to the COPD group (Figure 4C). Additionally, relative to the normal group, the COPD group showed a significant increase in the area of terminal bronchial walls, wall thickness, wall area/total area (MA%), and wall thickness/diameter of terminal bronchi (MT%). Conversely, the COPD + sulforaphane group of mice demonstrated significant decreases in these parameters compared to the COPD group (Figure 4D).

Masson staining was utilized to assess collagen expression in the lung tissues of mice in various groups. The results indicated a significant increase in the volume fraction of collagen protein in the lung tissues of the COPD group compared to the normal group. In contrast, the COPD + sulforaphane group of mice showed a significant reduction in collagen protein volume fraction compared to the COPD group (Figure 4E).

The Western blot experiment was conducted to examine the expression of Nrf2, IL-1β, MMP1 and TGF-β proteins in the lung tissues of each group of mice. The results showed that compared to the normal group, the expression levels of Nrf2 in both nuclear and whole-cell proteins of lung tissues in the COPD group mice were significantly decreased, while IL-1β, MMP1, and TGF-β proteins were significantly increased. In comparison to the COPD group, the COPD + sulforaphane group of mice exhibited a significant increase in the expression levels of Nrf2 in both nuclear and whole-cell proteins of lung tissues, while IL-1β, MMP1, and TGF-β proteins were significantly reduced (Figure 4F,4G).

Oxidative stress levels in the tissues were also measured. The results indicated that compared to the normal group, the activities of CAT and SOD enzymes in lung tissues of mice in the COPD group were significantly decreased, while the ROS levels were significantly elevated. In contrast to the COPD group, the COPD + sulforaphane group of mice showed a significant increase in CAT and SOD enzyme activities in lung tissues, alongside a significant reduction in ROS levels (Figure 4H-4J).

Overall, these results demonstrate that Nrf2 activators can alleviate obstructive lung ventilation dysfunction and inhibit inflammation and non-fibrotic processes, thereby mitigating disease progression in COPD mice.

Discussion

The Nrf2 activators demonstrated in this study the potential to effectively inhibit COPD progression by targeting the SASP in COPD bronchial epithelial cells, complementing existing research findings. While previous studies have shown that Nrf2 activators can enhance the expression of antioxidant enzymes and alleviate oxidative stress and inflammation, their specific impact on the cellular aging process in COPD progression has yet to be thoroughly explored (67-69). In comparison, this study not only elucidated the molecular mechanisms of Nrf2 activators but also confirmed their effectiveness in mitigating COPD-related pathological changes through in vitro and in vivo experiments, providing novel scientific evidence for the application of Nrf2 activators in COPD treatment.

The utilization of single-cell transcriptome analysis in this study represents a significant advancement, enabling precise identification of aging ciliated epithelial cells in lung tissues of COPD patients and the discovery of the key gene Nrf2. This high-resolution analytical method offers unprecedented detail in understanding the complex pathology of COPD, contrasting starkly with traditional whole-tissue RNA sequencing or proteomics studies (70-72). By unveiling cell-specific pathological processes and key regulatory factors, single-cell technology not only enhances our comprehension of COPD pathogenesis but also hints at possible future therapeutic directions (73).

In in vitro cell experiments, Nrf2 activators significantly reduced the proportion of SA-β-Gal-positive cells, suggesting their potential role in inhibiting cellular aging. Furthermore, the experimental results indicated that Nrf2 activators could enhance cell proliferation and decrease the expression of inflammatory factors, consistent with previous findings on the anti-aging and anti-inflammatory effects of the Nrf2 pathway (74). However, this study further comprehensively evaluated these effects through multiple experimental methods, providing a comprehensive profile of actions that delves deeper into the potential therapeutic mechanisms of Nrf2 activators in COPD.

This study further confirmed the therapeutic effects of Nrf2 activators using a COPD mouse model, including improving respiratory function, reducing levels of inflammatory factors, and alleviating lung tissue pathological damage. These animal experiment results not only corroborated the in vitro findings but also aligned with previous studies using Nrf2 activators in other disease models, demonstrating the broad involvement of the Nrf2 pathway in regulating inflammation and cellular protection mechanisms (75-77). However, compared to prior research, this study more specifically elucidated the ability of Nrf2 activators to slow down lung aging and enhance lung function within the pathological context of COPD, offering new avenues for COPD treatment. Moreover, by assessing the treatment effects at different time points, this study also provided initial insights into the optimal treatment timing and dosage, laying the foundation for subsequent clinical investigations.

Upon integrating the experimental results of this study, we further investigated the potential molecular mechanisms underlying the inhibitory effect of Nrf2 activators on COPD progression. Activating the Nrf2 pathway, these activators not only directly suppress oxidative stress and inflammation but may also slow down cellular aging processes by regulating the expression of SASP factors. This multifaceted intervention is crucial within the complex pathological background of COPD as it not only alleviates the inflammatory burden but also interrupts a significant driving force of COPD progression through the retardation of cellular aging. While these mechanisms have been mentioned in various studies, our research provides more direct evidence for this mechanism by integrating multiple experimental methods and models, reinforcing the theoretical foundation for the application of Nrf2 activators in COPD treatment. In summary, through a comprehensive approach involving single-cell transcriptome sequencing, in vitro cell models, and in vivo animal models, this study elucidates the mechanism of Nrf2 activators in inhibiting the SASP in COPD bronchial epithelial cells (Figure 5).

Figure 5 Molecular mechanisms of Nrf2 activators in suppressing aging and inflammation in COPD bronchial epithelial cells. COPD, chronic obstructive pulmonary disease; Nrf2, nuclear factor erythroid 2-related factor 2; SA-β-Gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory phenotype.

Despite significant progress in exploring the potential of Nrf2 activators for COPD treatment, there are limitations in this study. Firstly, while utilizing in vitro cell and animal models, these models may not entirely replicate the complex pathology of human COPD. Secondly, the primary focus of this study lies in activating the Nrf2 pathway without delving deeply into its interaction with other key signaling pathways in COPD. Furthermore, additional research is needed to validate the long-term safety and efficacy of Nrf2 activators and to assess their potential application in human COPD patients.

In conclusion, this study confirms that Nrf2 activators effectively inhibit the progression of COPD by targeting the SASP, shedding light on new mechanistic insights and potential therapeutic strategies for COPD treatment. These findings not only enhance our scientific understanding of the pathological mechanisms underlying COPD but also provide significant guidance for exploring novel treatment approaches clinically. Particularly, the application of Nrf2 activators may offer a novel therapeutic option for COPD patients with suboptimal responses to traditional treatments, thereby potentially improving their conditions and quality of life. Nevertheless, translating these laboratory findings into clinical practice entails addressing various challenges, including assessing the safety of long-term treatments, determining optimal drug dosages and administration timing, and validating the therapeutic effects in humans.

Looking ahead, further research should focus on several key areas. Firstly, clinical trials are needed to validate the therapeutic efficacy and safety of Nrf2 activators in a broader population over extended treatment periods. Secondly, exploring the potential synergistic effects of Nrf2 activators with existing COPD treatment strategies may introduce new combination therapies. Additionally, delving into the interactions between the Nrf2 pathway and other key signaling pathways in the pathological processes of COPD could provide a comprehensive understanding of the complex disease mechanisms, potentially revealing additional therapeutic targets. Lastly, considering the diversity in the etiology and pathological progression of COPD, future studies should investigate the varying responses of different COPD subtypes to Nrf2 activator treatment to develop more personalized therapeutic strategies.

Conclusions

This study underscores the therapeutic potential of Nrf2 activation in mitigating the progression of COPD by targeting SASP in bronchial epithelial cells. Our findings demonstrate that Nrf2 activators significantly reduce cellular senescence, decrease inflammation, and improve lung function in COPD models, both in vitro and in vivo. By effectively modulating oxidative stress and inflammatory responses, Nrf2 activators present a promising avenue for novel therapeutic strategies aimed at slowing COPD progression. These results not only advance our understanding of the molecular mechanisms underlying COPD but also pave the way for future clinical applications of Nrf2 activators in COPD treatment.

Further research is required to validate these findings in human clinical trials and to explore the long-term safety and efficacy of Nrf2 activators. Additionally, investigating the interaction between Nrf2 and other signaling pathways could provide deeper insights into the comprehensive management of COPD.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-710/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-710/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-710/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-710/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). All animal experiments were approved by the Animal Ethics Committee of The Second Affiliated Hospital of Wenzhou Medical University (No. 20240108b0241701[007]). All procedures involving animals were conducted in accordance with the national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Yang IA, Jenkins CR, Salvi SS. Chronic obstructive pulmonary disease in never-smokers: risk factors, pathogenesis, and implications for prevention and treatment. Lancet Respir Med 2022;10:497-511. [Crossref] [PubMed]
2. Guo P, Li R, Piao TH, et al. Pathological Mechanism and Targeted Drugs of COPD. Int J Chron Obstruct Pulmon Dis 2022;17:1565-75. [Crossref] [PubMed]
3. Agarwal AK, Raja A, Brown BD. Chronic Obstructive Pulmonary Disease. In: StatPearls. Treasure Island (FL): StatPearls Publishing; August 7, 2023.
4. Global burden of chronic respiratory diseases and risk factors, 1990-2019: an update from the Global Burden of Disease Study 2019. EClinicalMedicine 2023;59:101936. [Crossref] [PubMed]
5. Mei F, Dalmartello M, Bonifazi M, et al. Chronic obstructive pulmonary disease (COPD) mortality trends worldwide: An update to 2019. Respirology 2022;27:941-50. [Crossref] [PubMed]
6. Hou ZF, Yuan ZH, Chang K, et al. NLRP3 rs1539019 is significantly associated with chronic obstructive pulmonary disease in a Chinese Han population: a case-control study. Eur Rev Med Pharmacol Sci 2022;26:5821-8. [Crossref] [PubMed]
7. Bateman G, Guo-Parke H, Rodgers AM, et al. Airway Epithelium Senescence as a Driving Mechanism in COPD Pathogenesis. Biomedicines 2023;11:2072. [Crossref] [PubMed]
8. Liu L, Wei Y, Giunta S, et al. Potential role of cellular senescence in pulmonary arterial hypertension. Clin Exp Pharmacol Physiol 2022;49:1042-9. [Crossref] [PubMed]
9. Chin C, Ravichandran R, Sanborn K, et al. Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis. Cell Rep Med 2023;4:100945. [Crossref] [PubMed]
10. Lagoumtzi SM, Chondrogianni N. Senolytics and senomorphics: Natural and synthetic therapeutics in the treatment of aging and chronic diseases. Free Radic Biol Med 2021;171:169-90. [Crossref] [PubMed]
11. Wu H, Ma H, Wang L, et al. Regulation of lung epithelial cell senescence in smoking-induced COPD/emphysema by microR-125a-5p via Sp1 mediation of SIRT1/HIF-1a. Int J Biol Sci 2022;18:661-74. [Crossref] [PubMed]
12. Burgoyne RA, Fisher AJ, Borthwick LA. The Role of Epithelial Damage in the Pulmonary Immune Response. Cells 2021;10:2763. [Crossref] [PubMed]
13. Haston S, Gonzalez-Gualda E, Morsli S, et al. Clearance of senescent macrophages ameliorates tumorigenesis in KRAS-driven lung cancer. Cancer Cell 2023;41:1242-1260.e6. [Crossref] [PubMed]
14. Su L, Dong Y, Wang Y, et al. Potential role of senescent macrophages in radiation-induced pulmonary fibrosis. Cell Death Dis 2021;12:527. [Crossref] [PubMed]
15. Suda M, Paul KH, Minamino T, et al. Senescent Cells: A Therapeutic Target in Cardiovascular Diseases. Cells 2023;12:1296. [Crossref] [PubMed]
16. Saul D, Kosinsky RL, Atkinson EJ, et al. A new gene set identifies senescent cells and predicts senescence-associated pathways across tissues. Nat Commun 2022;13:4827. [Crossref] [PubMed]
17. Fraile M, Eiro N, Costa LA, et al. Aging and Mesenchymal Stem Cells: Basic Concepts, Challenges and Strategies. Biology (Basel) 2022;11:1678. [Crossref] [PubMed]
18. Zhao B, Wu B, Feng N, et al. Aging microenvironment and antitumor immunity for geriatric oncology: the landscape and future implications. J Hematol Oncol 2023;16:28. [Crossref] [PubMed]
19. Guo SP, Chang HC, Lu LS, et al. Activation of kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2/antioxidant response element pathway by curcumin enhances the anti-oxidative capacity of corneal endothelial cells. Biomed Pharmacother 2021;141:111834. [Crossref] [PubMed]
20. Qi DH, Ma H, Chen YY, et al. Research progress in mechanism of puerarin in treating vascular dementia. Zhongguo Zhong Yao Za Zhi 2023;48:5993-6002. [Crossref] [PubMed]
21. Bewley MA, Budd RC, Ryan E, et al. Opsonic Phagocytosis in Chronic Obstructive Pulmonary Disease Is Enhanced by Nrf2 Agonists. Am J Respir Crit Care Med 2018;198:739-50. [Crossref] [PubMed]
22. Wan Y, Shen K, Yu H, et al. Baicalein limits osteoarthritis development by inhibiting chondrocyte ferroptosis. Free Radic Biol Med 2023;196:108-20. [Crossref] [PubMed]
23. Boo YC. Arbutin as a Skin Depigmenting Agent with Antimelanogenic and Antioxidant Properties. Antioxidants (Basel) 2021;10:1129. [Crossref] [PubMed]
24. Tang YC, Hsiao JR, Jiang SS, et al. c-MYC-directed NRF2 drives malignant progression of head and neck cancer via glucose-6-phosphate dehydrogenase and transketolase activation. Theranostics 2021;11:5232-47. [Crossref] [PubMed]
25. Zeng M, Zhang X, Xing W, et al. Cigarette smoke extract mediates cell premature senescence in chronic obstructive pulmonary disease patients by up-regulating USP7 to activate p300-p53/p21 pathway. Toxicol Lett 2022;359:31-45. [Crossref] [PubMed]
26. Delgado-Eckert E, James A, Meier-Girard D, et al. Lung function fluctuation patterns unveil asthma and COPD phenotypes unrelated to type 2 inflammation. J Allergy Clin Immunol 2021;148:407-19. [Crossref] [PubMed]
27. MacLeod M, Papi A, Contoli M, et al. Chronic obstructive pulmonary disease exacerbation fundamentals: Diagnosis, treatment, prevention and disease impact. Respirology 2021;26:532-51. [Crossref] [PubMed]
28. Forman HJ, Zhang H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat Rev Drug Discov. 2021;20:689-709. Erratum in: Nat Rev Drug Discov 2021;20:652. [Crossref] [PubMed]
29. Uwagboe I, Adcock IM, Lo Bello F, et al. New drugs under development for COPD. Minerva Med 2022;113:471-96. [Crossref] [PubMed]
30. Barnes PJ. Oxidative Stress in Chronic Obstructive Pulmonary Disease. Antioxidants (Basel) 2022;11:965. [Crossref] [PubMed]
31. Li MY, Qin YQ, Tian YG, et al. Effective-component compatibility of Bufei Yishen formula III ameliorated COPD by improving airway epithelial cell senescence by promoting mitophagy via the NRF2/PINK1 pathway. BMC Pulm Med 2022;22:434. [Crossref] [PubMed]
32. Agustí A, Celli BR, Criner GJ, et al. Global Initiative for Chronic Obstructive Lung Disease 2023 Report: GOLD Executive Summary. Eur Respir J 2023;61:2300239. [Crossref] [PubMed]
33. Singh D, Mathioudakis AG, Higham A. Chronic obstructive pulmonary disease and COVID-19: interrelationships. Curr Opin Pulm Med 2022;28:76-83. [Crossref] [PubMed]
34. Hao Y, Hao S, Andersen-Nissen E, et al. Integrated analysis of multimodal single-cell data. Cell 2021;184:3573-3587.e29. [Crossref] [PubMed]
35. Lin S, Wu J, Chen B, et al. Identification of a Potential MiRNA-mRNA Regulatory Network for Osteoporosis by Using Bioinformatics Methods: A Retrospective Study Based on the Gene Expression Omnibus Database. Front Endocrinol (Lausanne) 2022;13:844218. [Crossref] [PubMed]
36. Ma L, Hernandez MO, Zhao Y, et al. Tumor Cell Biodiversity Drives Microenvironmental Reprogramming in Liver Cancer. Cancer Cell 2019;36:418-430.e6. [Crossref] [PubMed]
37. Ritchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 2015;43:e47. [Crossref] [PubMed]
38. Zago M, Sheridan JA, Traboulsi H, et al. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability. PLoS One 2017;12:e0180881. [Crossref] [PubMed]
39. Li L, Bao H, Wu J, et al. Baicalin is anti-inflammatory in cigarette smoke-induced inflammatory models in vivo and in vitro: A possible role for HDAC2 activity. Int Immunopharmacol 2012;13:15-22. [Crossref] [PubMed]
40. Zeng X, Liu X, Bao H. Sulforaphane suppresses lipopolysaccharide- and Pam3CysSerLys4-mediated inflammation in chronic obstructive pulmonary disease via toll-like receptors. FEBS Open Bio 2021;11:1313-21. [Crossref] [PubMed]
41. Panayiotidis MI, Stabler SP, Allen RH, et al. Cigarette smoke extract increases S-adenosylmethionine and cystathionine in human lung epithelial-like (A549) cells. Chem Biol Interact 2004;147:87-97. [Crossref] [PubMed]
42. Zhang Q, Qi J, Luo Q, et al. Yishen Xiezhuo formula ameliorates the development of cisplatin-induced acute kidney injury by attenuating renal tubular epithelial cell senescence. Ann Transl Med 2022;10:1392. [Crossref] [PubMed]
43. Guo F, Lin SC, Zhao MS, et al. microRNA-142-3p inhibits apoptosis and inflammation induced by bleomycin through down-regulation of Cox-2 in MLE-12 cells. Braz J Med Biol Res 2017;50:e5974. [Crossref] [PubMed]
44. Li W, Li Y, Wang Q, et al. Therapeutic effect of phycocyanin on chronic obstructive pulmonary disease in mice. J Adv Res 2024;66:285-301. [Crossref] [PubMed]
45. Yao H, Hwang JW, Moscat J, et al. Protein kinase C zeta mediates cigarette smoke/aldehyde- and lipopolysaccharide-induced lung inflammation and histone modifications. J Biol Chem 2010;285:5405-16. [Crossref] [PubMed]
46. Wang C, Ding H, Tang X, et al. Effect of Liuweibuqi Capsules in Pulmonary Alveolar Epithelial Cells and COPD Through JAK/STAT Pathway. Cell Physiol Biochem 2017;43:743-56. [Crossref] [PubMed]
47. Shin NR, Ko JW, Park SH, et al. Protective effect of HwangRyunHaeDok-Tang water extract against chronic obstructive pulmonary disease induced by cigarette smoke and lipopolysaccharide in a mouse model. J Ethnopharmacol 2017;200:60-5. [Crossref] [PubMed]
48. Park J, Jang J, Cha SR, et al. L-carnosine Attenuates Bleomycin-Induced Oxidative Stress via NFκB Pathway in the Pathogenesis of Pulmonary Fibrosis. Antioxidants (Basel) 2022;11:2462. [Crossref] [PubMed]
49. Chen M, Zheng J, Liu G, et al. Ceruloplasmin and hephaestin jointly protect the exocrine pancreas against oxidative damage by facilitating iron efflux. Redox Biol 2018;17:432-9. [Crossref] [PubMed]
50. Wang L, Xu Z, Chen B, et al. The Role of Vascular Endothelial Growth Factor in Small-airway Remodelling in a Rat Model of Chronic Obstructive Pulmonary Disease. Sci Rep 2017;7:41202. [Crossref] [PubMed]
51. Raby KL, Michaeloudes C, Tonkin J, et al. Mechanisms of airway epithelial injury and abnormal repair in asthma and COPD. Front Immunol 2023;14:1201658. [Crossref] [PubMed]
52. Qin S, Xiao W, Zhou C, et al. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther 2022;7:199. [Crossref] [PubMed]
53. Zoulikha M, Xiao Q, Boafo GF, et al. Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome. Acta Pharm Sin B 2022;12:600-20. [Crossref] [PubMed]
54. Lee JW, Chun W, Lee HJ, et al. The Role of Macrophages in the Development of Acute and Chronic Inflammatory Lung Diseases. Cells 2021;10:897. [Crossref] [PubMed]
55. Petit LMG, Belgacemi R, Ancel J, et al. Airway ciliated cells in adult lung homeostasis and COPD. Eur Respir Rev 2023;32:230106. [Crossref] [PubMed]
56. McMahon DB, Kuek LE, Johnson ME, et al. The bitter end: T2R bitter receptor agonists elevate nuclear calcium and induce apoptosis in non-ciliated airway epithelial cells. Cell Calcium 2022;101:102499. [Crossref] [PubMed]
57. Sachs N, Papaspyropoulos A, Zomer-van Ommen DD, et al. Long-term expanding human airway organoids for disease modeling. EMBO J 2019;38:e100300. [Crossref] [PubMed]
58. Rahmawati SF, Te Velde M, Kerstjens HAM, et al. Pharmacological Rationale for Targeting IL-17 in Asthma. Front Allergy 2021;2:694514. [Crossref] [PubMed]
59. Yang C, Zeng HH, Du YJ, et al. Correlation of Luminal Mucus Score in Large Airways with Lung Function and Quality of Life in Severe Acute Exacerbation of COPD: A Cross-Sectional Study. Int J Chron Obstruct Pulmon Dis 2021;16:1449-59. [Crossref] [PubMed]
60. Hu J, Wang J, Li C, et al. Fructose-1,6-bisphosphatase aggravates oxidative stress-induced apoptosis in asthma by suppressing the Nrf2 pathway. J Cell Mol Med 2021;25:5001-14. [Crossref] [PubMed]
61. Huang J, Chen X, Xie A. Formononetin ameliorates IL-13-induced inflammation and mucus formation in human nasal epithelial cells by activating the SIRT1/Nrf2 signaling pathway. Mol Med Rep 2021;24:832. [Crossref] [PubMed]
62. Ulasov AV, Rosenkranz AA, Georgiev GP, et al. Nrf2/Keap1/ARE signaling: Towards specific regulation. Life Sci 2022;291:120111. [Crossref] [PubMed]
63. Chen QM. Nrf2 for protection against oxidant generation and mitochondrial damage in cardiac injury. Free Radic Biol Med 2022;179:133-43. [Crossref] [PubMed]
64. Sánchez-Ortega M, Carrera AC, Garrido A. Role of NRF2 in Lung Cancer. Cells 2021;10:1879. [Crossref] [PubMed]
65. Ovadya Y, Krizhanovsky V. Senescent cells: SASPected drivers of age-related pathologies. Biogerontology 2014;15:627-42. [Crossref] [PubMed]
66. Chen Q, Gu P, Liu X, et al. Gold Nanoparticles Encapsulated Resveratrol as an Anti-Aging Agent to Delay Cataract Development. Pharmaceuticals (Basel) 2022;16:26. [Crossref] [PubMed]
67. Wang L, He C. Nrf2-mediated anti-inflammatory polarization of macrophages as therapeutic targets for osteoarthritis. Front Immunol 2022;13:967193. [Crossref] [PubMed]
68. Gęgotek A, Skrzydlewska E. Ascorbic acid as antioxidant. Vitam Horm 2023;121:247-70. [Crossref] [PubMed]
69. Lee JJ, Ng SC, Hsu JY, et al. Galangin Reverses H(2)O(2)-Induced Dermal Fibroblast Senescence via SIRT1-PGC-1α/Nrf2 Signaling. Int J Mol Sci 2022;23:1387. [Crossref] [PubMed]
70. Sikkema L, Ramírez-Suástegui C, Strobl DC, et al. An integrated cell atlas of the lung in health and disease. Nat Med 2023;29:1563-77. [Crossref] [PubMed]
71. Lai HC, Lin TL, Chen TW, et al. Gut microbiota modulates COPD pathogenesis: role of anti-inflammatory Parabacteroides goldsteinii lipopolysaccharide. Gut 2022;71:309-21. [Crossref] [PubMed]
72. Van Eeckhoutte HP, Donovan C, Kim RY, et al. RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD. Eur Respir J 2023;61:2201506. [Crossref] [PubMed]
73. Wei D, Xu M, Wang Z, et al. The Development of Single-Cell Metabolism and Its Role in Studying Cancer Emergent Properties. Front Oncol 2021;11:814085. [Crossref] [PubMed]
74. Yang H, Song P, Li B, et al. Design, synthesis and biological evaluation of Nrf2 modulators for the treatment of glioblastoma multiforme. Bioorg Med Chem 2024;103:117684. [Crossref] [PubMed]
75. Li Z, Liu T, Feng Y, et al. PPARγ Alleviates Sepsis-Induced Liver Injury by Inhibiting Hepatocyte Pyroptosis via Inhibition of the ROS/TXNIP/NLRP3 Signaling Pathway. Oxid Med Cell Longev 2022;2022:1269747. [Crossref] [PubMed]
76. Wang Y, Liu Z, Ma J, et al. Lycopene attenuates the inflammation and apoptosis in aristolochic acid nephropathy by targeting the Nrf2 antioxidant system. Redox Biol 2022;57:102494. [Crossref] [PubMed]
77. Song C, Zhang A, Zhang M, et al. Nrf2/PINK1-mediated mitophagy induction alleviates sodium fluoride-induced hepatic injury by improving mitochondrial function, oxidative stress, and inflammation. Ecotoxicol Environ Saf 2023;252:114646. [Crossref] [PubMed]