FASN regulates CSE-induced apoptosis, oxidative stress and mitochondrial damage in type 2 alveolar epithelial cells by regulating NRF2 expression and nuclear translocation

ABSTRACT

Smoking is a major etiological factor in numerous chronic lung diseases. However, the precise underlying mechanisms remain incompletely elucidated. In this study, we investigated the effects of cigarette smoke extract (CSE) on mitochondrial oxidative phosphorylation (OXPHOS), mitochondrial structure, and the antioxidant regulator Nuclear factor erythroid 2-related factor 2 (NRF2) in a rat lung epithelial-T-antigen negative cell line (RLE-6TN), focusing on the associated molecular pathways. CSE exposure significantly reduced cell viability, induced oxidative-antioxidant imbalance, and disrupted OXPHOS complex subunit expression and mitochondrial ultrastructure. Furthermore, an increased BCL2-Associated X (BAX) / B-cell lymphoma/leukemia 2 (BCL2) ratio activated the intrinsic apoptosis pathway. NRF2 knockdown exacerbated CSE-induced mitochondrial damage and apoptosis. Co-immunoprecipitation (co-IP) analysis revealed a direct interaction between NRF2 and Fatty Acid Synthase (FASN). CSE treatment significantly reduced NRF2-FASN binding. Notably, FASN knockout amplified oxidative stress, exacerbated damage to OXPHOS and mitochondrial structure, and diminished NRF2 expression and nuclear translocation. Collectively, our findings demonstrate that CSE exposure impairs NRF2 expression and nuclear translocation by disrupting FASN expression and its interaction with FASN. This impairment leads to mitochondrial OXPHOS dysfunction, structural damage, and ultimately apoptosis. Our findings identify FASN as a potential therapeutic target for mitigating smoking-associated lung injury.

Introduction

Smoking is a major etiological factor and significant risk factor for chronic lung diseases, including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), contributing substantially to mortality [1]. Cigarette smoke (CS) is a complex toxic mixture comprising over 7,000 identified chemicals. Its particulate and gaseous phases contain numerous oxidants, including phenols, semiquinones, superoxide, epoxides, peroxide, nitric oxide, nitrogen dioxide, peroxynitrite, and so on [2,3]. These components induce systemic oxidative stress, with pronounced effects in the lungs due to direct exposure. While reactive oxygen species (ROS) physiologically regulate processes like cell proliferation, metabolism, and differentiation, CS-derived ROS overload disrupts lipids, proteins, and DNA, causing cellular damage [4,5]. The alveoli are the terminal gas-exchange units of the respiratory system. Type II alveolar epithelial cells (AEC2) produce and secrete pulmonary surfactant proteins (SPs) and convert to AEC1 when the alveolar structure is impaired, which is critical for maintaining distal lung homeostasis after injury [6]. Multiple studies have suggested that an important cause of emphysema and pulmonary fibrosis is alveolar epithelial injury [7–9]. CS exposure drives AEC dysfunction via direct and indirect pathways, promoting apoptosis, oxidative stress, inflammation, mitochondrial respiratory chain blockade, and impaired DNA repair, ultimately exacerbating lung tissue destruction [10–12]. It was confirmed that AEC2 and SP activities were decreased in patients with COPD, and these lesions further aggravated oxidative stress to destroy AEC2 [13,14]. Lower SP-A and SP-D levels in bronchoalveolar lavage fluid correlate with poorer lung function, increased complications, and higher ventilator dependence [15]. Consequently, AEC2 dysfunction is a pivotal contributor to lung pathology. However, little is known about the specific mechanism of AEC2 in CS-induced injury, so further studies are necessary.

Nuclear factor erythroid-like 2-related factor 2 (NRF2) is a key transcription factor regulating cellular defense against oxidative stress [16]. It normally resides in the cytoplasm and is degraded by proteasomes after binding to Kelch Like ECH Associated Protein 1 (KEAP1). Various endogenous and exogenous oxidants can disrupt their binding, leading to nuclear translocation of NRF2 and transcriptional activation of downstream antioxidant response elements (ARE), such as heme oxygenase-1(HO-1) and NAD(P)H: Quinone Oxidoreductase 1 (NQO1), to mitigate oxidative damage [17]. Studies indicate dysregulation of the NRF2 signaling pathway within AEC2s across multiple lung pathologies. Pharmacological NRF2 activators demonstrate efficacy in enhancing downstream ARE activation [18–20]. Several NRF2 pharmacological activators have been approved for treatment; for example, dimethyl fumarate, diroximel fumarate, and monomethyl fumarate. Their application in multiple sclerosis suggests that the NRF2/ARE pathway is a promising target for disease therapy [21–23].

AEC2 is highly proliferative and metabolically active and therefore have a high energy requirement [24]. Cellular energy in the form of ATP is produced through mitochondrial respiration, a process comprising the electron transport chain (ETC) and oxidative phosphorylation (OXPHOS) [25]. The ETC comprises a series of protein complexes that sequentially transfer electrons while pumping protons across the inner mitochondrial membrane. This establishes a proton gradient that drives ATP synthesis [26]. ROS are natural by-products of OXPHOS, whose resulting oxidative damage is an important molecular basis for various pathological states [27]. On the other hand, oxidative stress impairs mitochondrial function, leading to increased ROS production and further deterioration of oxidative stress. Studies further indicate that NRF2 regulates critical mitochondrial processes, including biogenesis and quality control [28,29].

AEC2 have a complex lipid metabolic network, which can regulate the synthesis, secretion, and circulation of SPs under different pathophysiological conditions. Dysregulated AEC2 lipid metabolism is implicated in multiple lung pathologies [30,31]. Fatty Acid Synthase (FASN), a key enzyme of lipid metabolism, is highly expressed in AEC2 (Supplementary Fig. 1B). FASN inhibition impairs mitochondrial function and increases ROS production [32]. The expression of FASN in the lung tissue of COPD patients was significantly decreased. FASN deficiency results in impaired mitochondrial glycolysis and anaerobic metastasis [33]. CS-exposed mice with targeted deletion of FASN (Fasn^iΔAEC2) in AEC2 exhibited more severe alveolar destruction compared to wild-type controls [34]. FASN knockdown significantly enhanced Bleomycin (BLM)-induced AEC2 death, whereas overexpression of FASN alleviated mitochondrial membrane potential (MMP) loss, decreased ROS production, and rescued cell death [35]. Despite this evidence linking FASN to AEC2 protection, the mechanistic role of FASN in chronic lung disease progression remains poorly defined, warranting further investigation.

Methods and materials

Cell lines and cultures

In vitro, Rat type II alveolar epithelial cell line (RLE-6TN) was purchased from Fuheng Biological Company (Shanghai), and cells were grown in 1640 culture medium supplemented with 10% fetal bovine serum (FBS, GIBCO), 1% penicillin/streptomycin(P/S), and the cells were cultured at 37°C, 5% CO2.

Preparation of CSE

CSE was prepared as described previously [36]. Briefly, one unfiltered cigarette (Da Qian Men, 0.8 mg of nicotine, 11 mg of tar, and 13 mg of carbon monoxide per cigarette) was burned, and the smoke was passed through 10 ml of phosphate-buffered saline (PBS). The extract was used when adjusting the pH to between 7.00 and 7.40 after filtering through a filter with 0.22-μM pores to remove particles and bacteria. Fresh CSE-PBS solution was prepared before each use.

Cell proliferation assays

The viable cell number was estimated using a Cell Counting Kit-8 (Dojindo, Japan) assay. To measure the proliferative activity of cells in 96-well microplates, CCK-8 was added (10 µl/well) and incubated for 2 h. Absorbance was measured at 450 nm using an enzyme-labeled instrument (BioTek uQuant, USA) with a reference wavelength of 650 nm.

Analysis of intracellular ROS

Cells were seeded in 6-well plates. Depending on the experimental group, cells were pretreated with or without mito-Tempo (MCE, HY-112879, 100 μM) at concentrations previously described [37], followed by treatment with 10% CSE. Cells were harvested and incubated with dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, China). Each sample was examined by fluorescence microscopy (Olympus IX73, Japan).

Mitochondrial membrane potential (MMP)

Cells were cultured on six-well plates and the medium was replaced with 10 μg/mL JC-1 (dissolved in DMSO) containing a 1×Assay buffer (JC-1 Mitochondrial Membrane Potential Detection Kit, Beyotime, China). The cells were placed back into the incubator (37 °C, 5% CO2) for 20 min, followed by washing with PBS to remove unbound dye. Monomeric JC-1 was detected via excitation at 488 nm and emission at 527 nm. Aggregated JC-1 was detected by excitation at 543 nm and emission at 570 nm using a fluorescent microscope (Olympus IX73, Japan). Fluorescence intensity was measured using Image J software (USA).

Western blotting (WB)

Cells were harvested and total cell proteins were extracted using RIPA lysis buffer, The extraction of cytoplasmic and nuclear protein with kit (Beyotime, China) was used to obtain the cytoplasmic and nuclear proteins, According to the manufacturer's instruction. Subsequently, centrifugation was carried out, and the samples were denatured using a loading buffer. The proteins were separated by 7.5–15% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with milk for 1 h at room temperature. Then, incubated with corresponding primary antibodies at 4°C overnight. The following primary antibodies were used: Nrf2 (Proteintech, Cat No:16396-1-AP, 1:1000), KEAP1 (Proteintech, Cat No:10503-2-AP, 1:1000), HO-1 (Proteintech, Cat No:81281-1-RR, 1:1500), NQO1 (ABclonal, Cat No: A23486, 1:1000), BAX (Proteintech, Cat No:50599-2-Ig, 1:1000), BCL2 (Proteintech, Cat No:26593-1-AP, 1:1000), Caspase 3 (Proteintech, Cat No:66470-2-Ig, 1:1000), NDUFB8 (Abcam, Cat No: ab192878, 1:1000), SDHB (Abcam, Cat No: ab175225, 1:1000), UQCRC2 (Abcam, Cat No: ab203832, 1:1000), MTCO2 (Proteintech, Cat No:55070-1-AP, 1:1000), ATP5A (Abcam, Cat No:ab176569, 1:1000), FASN (Proteintech, Cat No:10624-2-AP, 1:1000) and β-actin (Proteintech, Cat No:66009-1-Ig, 1:1000). On the following day, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (H + L) (Abbkin, Cat No: A21020-1, 1:1000) or HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Cat No: A0216, 1:1000) for 1 h. The protein bands were visualized using an enhanced chemiluminescence reagent, and the relative protein expression was quantified using the Image Lab software (Bio-Rad; USA).

RNA extraction and qPCR

Total RNA was extracted from RLE-6TN cells with TRIzol reagent (Invitrogen) following the manufacturer’ s instructions. For qPCR, 0.2 mg total RNA was reverse transcribed with SuperScript III reagents (Invitrogen) with oligo-d(T) (Takara Bio, Shiga, Japan) and random hexamer (Takara Bio) primers. Real-time qPCR was performed using SYBR Green Supermix (Applied Biosystems, Foster City, CA) with the QuantStudio 3 Real-Time PCR System (Roche Diagnostics, ThermoFisher). PCR conditions were 95°C for 5 min, 40 cycles of 95°C for 10 s, 60°C for 30 s. The relative expression levels of each gene of interest were normalized using the level of β-actin in the same sample. Fold changes in target-gene expression were calculated using the 2^-ΔΔCt method. The primers used for PCR were: β-actin (F: 5′-TGGGTCAGAAGGACTCCTACG-3′, R: 5′-CAGGCAGCTCATAGCTCTTCT-3′); NRF2(F: 5′-CCCAGCACATCCAGACAGAC-3′, R: 5′-TATCCAGGGCAAGCGACTC-3′); HO-1(F: 5′-CCCTTCCTGTGTCTTCCTTTG-3′, R: 5′-ACAGCCGCCTCTACCGACCACA-3′); NQO-1(F: 5′-ATGTATGACAAAGGACCCTTCC-3′, R: 5′-TCCCTTGCAGAGAGTACATGG-3′).

Flow cytometry

The levels of apoptosis in RLE-6TN cells from each treatment group were assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, China), and cells were labeled according to manufacturer’ s instructions, the samples were then examined using a CytoFLEX S Flow Cytometry (BECKMAN COULTER).

Cell immunofluorescence microscopy

RLE-6TN were cultured on coverslips placed in 12-well plates. Coverslips were rinsed with PBS and immobilized using a 4% paraformaldehyde (Solarbio, China) for 20 min, Subsequently, Immunostaining Permeabilization Solution with Triton X-100 (Beyotime, China) was applied to permeabilize the cells for 20 min. After washing with PBS, coverslips were incubated in a blocking buffer (Beyotime, China) for 20 min. Then the cells were incubated with the antibodies against NRF2 (Proteintech, Cat No:16396-1-AP, 1:50) or FASN (Proteintech, Cat No:10624-2-AP, 1:50) at 4 °C overnight. After primary antibody incubation, cells were washed 3 times and incubated with secondary antibodies coupled with FITC/Cy3 at room temperature for 1 h (servicebio, China, 1:100). DAPI (Beyotime, China) was used to label nuclei (50 µl/well). Images were captured by fluorescent microscope (Olympus IX73, Japan). Fluorescence intensity determination and manders’ correlation coefficient analysis were then performed using Image J software (USA).

Immunoprecipitation followed by mass spectrometry (IP-MS) and co-immunoprecipitation (co-IP)

The Protein A/G beads (Beyotime, China, 20 µl/sample) were mixed with an anti-NRF2 antibody (Proteintech, Cat No:16396-1-AP, 1.6 µg/sample) for 1 h at room temperature. The cells were subjected to extraction using an IP lysis buffer on ice (Beyotime, China). The whole-cell lysates were mixed with beads-antibody complex overnight at 4 °C. The utilization of anti-IgG (Beyotime, China, 2 µg/sample) was employed as a negative control in the experiment. Following washing, the complex was eluted and submitted to denaturation. The samples were loaded onto a polyacrylamide gel. Subsequently, the gel was stained with coomassie brilliant blue. The proteins were analyzed utilizing Thermo Fisher LTQ Obitrap ETD (Thermo, USA). The Co-Immunoprecipitation (co-IP) test was employed to ascertain the interacting protein. Initially, the IP procedure, as previously delineated, was executed. Subsequently, the immunoprecipitates utilizing anti-NRF2 and anti-FASN antibodies were employed for Western blot validation.

Transmission electron microscope (TEM) assay

The morphological changes of mitochondria in cells were observed by TEM. After CSE treatment, cells were collected and fixed in 4% glutaraldehyde overnight at 4 °C, and then postfixed in 1% osmium tetroxide (OsO4) at 4 °C. Afterward, samples were dehydrated with a series of alcohol concentrations and embedded in Epon-Araldite resin. Ultrathin sections were obtained by an ultramicrotome and stained with uranyl acetate and lead citrate. Subsequently, ultra-structures were observed using TEM.

Transfection

The siRNA oligonucleotides targeting NRF2 and FASN were supplied by Gene Pharma(Shanghai, China). The transfection of siNrf2 or siFASN was carried out using lipofectamine 3000 (Thermo Fisher Scientific; USA) following the manufacturer's procedure. The silencing efficacy of the target genes was confirmed using Western blot analysis. Scrambled siRNA was used as a negative control. The target sequences for NRF2 siRNA #1 were sense 5′-GCCUUGUACUUUGAAGACUTT-3′ and antisense 5′-AGUCUUCAAAGUACAAGGCTT-3′, for NRF2 siRNA #2 were sense 5′-GUAGUCCACAUUUCCUUCATT-3′ and antisense 5′-UGAAGGAAAUGUGGACUACTT-3′, for NRF2 siRNA #3 were sense 5′-GAACACAGAUUUCGGUGAUTT-3′ and antisense 5′-AUCACCGAAAUCUGUGUUCTT-3′, for NRF2 siRNA #4 were sense 5′-GUGACUCGGAAAUGGAAGATT-3′ and antisense 5′-UCUUCCAUUUCCGAGUCACTT-3′, for FASN siRNA #1 were sense 5′-CGGAGUCUCUUGAAUAUAUTT-3′and antisense 5′-AUAUAUUCAAGAGACUCGGTT-3′, FASN siRNA #2 were sense 5′-GCUCCACCAAAUCCAACAUTT-3′and antisense 5′-AUGUUGGAUUUGGUGGAGCTT-3′, FASN siRNA #3 were sense 5′-GACCCUGACUCCAAGUUAUTT-3′and antisense 5′-AUAACUUGGAGUCAGGGUCTT-3′, FASN siRNA #4 were sense 5′-GCCCAGAGCAUAAGAGUUATT-3′and antisense 5′-UAACUCUUAUGCUCUGGGCTT-3′. After 48 h of transfection, cells were harvested.

Molecular docking

The protein models used for docking are FASN (Uniprot ID: P12785) and Nrf2 (Uniprot ID: O54968). The HDOCK SERVER (http://HDOCK.phys.hust.edu.cn/) is used for protein–protein molecular docking. The protein was pretreated with Pymol 2.4(removing water molecule and redundant ligand, adding hydrogen atom). Docking Score, Confidence Score, and Ligand RMSD were used as evaluation criteria for Docking, and the results were set to output 10 optimal Docking positions. Select the model with the highest score as the best docking model. Finally, we used Pymol 2.4 software to visualize the docking results. In this way, we can visually observe the binding between the proteins.

Statistical analysis

The experiment in this study was conducted with a minimum of three repetitions and the data were presented in the form of mean ± standard error of the mean (SEM). Data normality was determined using the Shapiro–Wilk test. An unpaired two-tailed Student's t-test was employed to compare the means of the two groups. For comparison between three or more groups, One-way analysis of variance (ANOVA) was used. A p-value less than 0.05 was considered to be statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Statistical analyses were performed using GraphPad Prism 8.0 Software (GraphPad Software, USA).

Results

CSE causes mitochondrial damage and apoptosis in AEC2

To explore the effect of CSE on AEC2 proliferation, we treated AEC2 with different concentrations of CSE. The results showed that CSE could inhibit the proliferation of AEC2 (Figure 1(A)), but with the further increase of treatment concentration, apoptosis increased significantly (Figure 1(F)). So the following experiments mainly treated the cells with 10% CSE.

Figure 1.

CSE causes mitochondrial damage and apoptosis in RLE-6TNs. (A) Cell viability was measured by CCK-8 after RLE-6TNs were exposed to different concentrations of CSE for 24 h. (B) ROS content in RLE-6TNs after being treated with 10% CSE for 24 h was determined by DCFH-DA fluorescence staining and visualized using a Fluorescence microscope (scale bar 100 μm). Fluorescence intensity was analyzed using Image J, and then graphics and analysis were done using GraphPad Prism8.0. (C) MMP was measured by JC-1 staining and visualized using a Fluorescence microscope (scale bar 200 μm): Red fluorescence represents normal MMP, while green fluorescence suggests a decrease in MMP and early apoptosis. Fluorescence intensity was analyzed using Image J, and then graphics analysis was done using GraphPad Prism8.0. (D) The expression of NDUFB8, SDHB, UQCRC2, MTCO2, ATP5A, and β-actin were analyzed by Western blot. (E) The expression of BAX, BCL2, Caspase-3, Cleaved caspase-3, and β-actin were analyzed by Western blot. (F) Apoptosis was detected by flow cytometry. (G) Mitochondrial ultrastructure was observed under TEM (scale bar 2μm and 500 nm). Images were analyzed using Image J, followed by data analysis using GraphPad Prism8.0. Values were shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Studies suggest that CSE affects cell proliferation by affecting ROS levels, and therefore, we used DCFH dye to assess intracellular oxidative stress levels after CSE intervention, and the results suggest that CSE treatment increased intracellular ROS production, and this effect was attenuated with the use of antioxidant mito-TEMPO (Figure 1(B)).

ROS induces irreversible apoptosis by inhibiting Mitochondrial membrane potential (MMP) and increasing Mitochondrial membrane permeability. So we can indirectly reflect early apoptosis by monitoring MMP. The results suggested that 10% CSE treatment caused a significant decrease in AEC2 MMP and a consequent change in mitochondrial membrane permeability (Figure 1(C)). This suggests that CSE inhibits MMP by increasing ROS levels, increasing mitochondrial membrane permeability and ultimately leading to mitochondrial damage.

OXPHOS is an important component of mitochondrial function. Therefore, we further examined the changes of mitochondrial OXPHOS-related complex proteins NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A after the cell was treated with CSE. The results showed that NDUFB8, UQCRC2, and MTCO2 decreased significantly after intervention, while SDHB and ATP5A did not change significantly (Figure 1(D)). These results suggest that CSE may cause mitochondrial dysfunction by inhibiting OXPHOS-related proteins NDUFB8, UQCRC2, and MTCO2. We further examined the effect of CSE on mitochondrial morphology using TEM. Mitochondrial cristae analysis was described previously [38]. Compared with the control group, the mitochondria swelled and mitochondrial cristae area and volume decreased after CSE treatment (Figure 1(G)). These results suggest that CSE leads to abnormalities in mitochondrial function and morphology.

Many studies have shown that abnormal mitochondrial function and morphology can promote apoptosis. So we examined the expression of apoptosis-related proteins in mitochondria after CSE treatment. The results showed that the expression of BAX and Cleaved Caspase-3 increased in a concentration-dependent manner, while the expression of BCL2 decreased in a concentration-dependent manner. The ratio of BAX/BCL2 gradually increased with the increase of the concentration of CSE (Figure 1(E)). The results of flow cytometry also showed that the degree of apoptosis was aggravated with the increase of CSE concentration (Figure 1(F)). These results suggest that mitochondrial dysfunction induced by CSE can promote apoptosis.

CSE causes oxidative stress and NRF2 activation in RLE-6TN

It has been reported that NRF2 is a major transcription factor for ROS-regulated mitochondrial OXPHOS [39]. We further examined the changes in NRF2 expression after CSE treatment and found that after CSE stimulation, total NRF2 protein levels first increased and then decreased, whereas nuclear NRF2 protein continued to increase and began to decrease after 24 h, RNA levels of NRF2 and its downstream ARE also showed similar results (Figure 2(A), Figure 2(B)). This is consistent with our previous results of MMP reduction and apoptosis after 24 h, suggesting that CSE regulates MMP-induced apoptosis by promoting NRF2 nuclear translocation. The level of ROS gradually increased with the increase of CSE intervention time (Figure 2(C)). The changes of NRF2 were further confirmed by immunofluorescence after different intervention times of CSE (Figure 2(D)). Interestingly, NRF2 expression decreased significantly after the peak and fell below the baseline level after 48 h of intervention. This indicates the depletion of the cellular antioxidant system.

Figure 2.

CSE causes NRF2 activation in RLE-6TNs. RLE-6TNs were exposed to CSE (10%) for 0, 6, 24, 48 h. (A) Western blot analysis showed that the expression level of Total NRF2, Cytoplasm-NRF2 and Nucleus-NRF2 was time-dependent. (B) PCR showed that the expression level of NRF2, HO-1 and NQO-1 was time-dependent. (C) ROS content in RLE-6TNs after being treated with 10% CSE for 0, 6, 24, 48 h was determined by DCFH-DA fluorescence staining and visualized using a Fluorescence microscope (scale bar 100 μm). (D) NRF2 and DAPI were detected by immunofluorescence (scale bar 100μm). Values were shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Effects of NRF2 gene knockout on oxidative stress, apoptosis, mitochondrial function, and structure with or without CSE intervention in RLE-6TN

These results suggest that CSE induces cell apoptosis by regulating NRF2. So we knocked out Nrf2 in RLE-6TN to further validate the effect of CSE on oxidative stress (Figure 3(A), Figure 3(B)). We treated NRF2 siRNA-inhibited RLE-6TN with CSE and detected ROS levels. The results showed that inhibition of NRF2 by siRNA increased intracellular ROS levels, indicating that NRF2 knockdown aggravates oxidative-antioxidant imbalance. The use of the antioxidant mito-TEMPO can partially alleviate ROS production. However, compared with the control group, the increase of ROS in siNrf2 RLE-6TN decreased after CSE treatment (Figure 3(C)). This suggests that CSE affects ROS levels by regulating NRF2 and that other pathways also play a role.

Figure 3.

Effects of Nrf2 gene knockout on oxidative stress, apoptosis, mitochondrial function, and structure with or without CSE intervention. RLE-6TN were transfected with control siRNA or NRF2 siRNA and then exposed to CSE (10%). (A)Western blot analysis showed the expression level of NRF2 after RLE-6TN was knocked down by the siRNA1-4 sequence. (B) Western blot analysis showed the expression level of NRF2 and its downstream HO-1 and NQO-1 after Nrf2 was knocked down. (C) ROS content in RLE-6TN treated with 10% CSE was determined by DCFH-DA fluorescence staining and visualized using a Fluorescence microscope (scale bar 100 μm). (D) MMP was measured by JC-1 staining and visualized using a Fluorescence microscope (scale bar 200 μm), Red fluorescence represents normal MMP, while green fluorescence suggests a decrease in MMP and early apoptosis. Fluorescence intensity was analyzed using Image J, and then graphics analysis was done using GraphPad Prism8.0. (E) The expression of BAX, BCL2, Caspase-3, Cleaved Caspase-3, and β-actin were analyzed by Western blot. (F) Apoptosis was detected by flow cytometry. (G) The expression of NDUFB8, SDHB, UQCRC2, MTCO2, ATP5A, and β-actin were analyzed by Western blot. (H) Mitochondrial ultrastructure was observed under TEM (scale bar 2μm and 500 nm). Values were shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

We further validated the effects of CSE treatment on apoptosis and mitochondrial damage in siNrf2 RLE-6TN. The results showed that NRF2 knockdown aggravated mitochondrial apoptosis. The results of JC-1 suggest that inhibition of NRF2 by siRNA aggravated the imbalance of MMP. However, the degree of reduction in MMP in siNrf2 RLE-6TN after CSE treatment was reduced compared with the control group (Figure 3(D)). These results suggest that CSE regulates mitochondrial function by regulating Nrf2. Compared with the control group, The changes of BAX, BCL2 and Cleaved caspase-3 in siNrf2 RLE-6TN decreased after CSE treatment (Figure 3(E)). The results of flow cytometry showed that NRF2 knockdown aggravated the apoptosis of RLE-6TN cells. However, compared with the control group, the increase of apoptosis of RLE-6TN cells after NRF2 knockdown was decreased after CSE treatment (Figure 3(F)). These results suggest that CSE can affect apoptosis of RLE-6TN by regulating NRF2. Changes in representative proteins of OXPHOS-associated complexes can reflect mitochondrial damage, so we examined changes in the expression of these proteins in siNrf2 RLE-6TN after CSE treatment. We found that the degree of reduction of NDUFB8, SDHB, UQCRC2, and MTCO2 was lower than that of the control group in siNrf2 RLE-6TN after CSE treatment. The expression of ATP5A did not change after NRF2 knockdown (Figure 3(G)). These results suggest that CSE may affect the expression of OXPHOS-related protein NDUFB8, SDHB, UQCRC2, and MTCO2 in mitochondria. Studies have found that mitochondrial OXPHOS abnormalities are usually accompanied by mitochondrial structural disorders [40], so we next examined mitochondria using TEM. We found that the RLE-6TN mitochondrial structure after NRF2 knockdown was significantly damaged compared with the control group, including mitochondrial cristae reduction, mitochondrial swelling, and vacuolation. However, compared with the wild-type cells after CSE treatment, the degree of mitochondrial structural abnormality in siNrf2 RLE-6TN treated was alleviated (Figure 3(H)). This suggests that CSE inhibits mitochondrial structure and function by affecting Nrf2, which may be the reason why CSE exposure affects RLE-6TN.

The interaction between NRF2 and FASN in RLE-6TN is abrogated upon CSE stimulation

There is no evidence that CSE directly regulates NRF2 expression or nuclear translocation. Therefore, to further investigate possible ways in which CSE regulates NRF2, we used Immunoprecipitation (IP)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins that might interact with NRF2 under CSE. A total of 1387 interacting proteins were detected (supplementary table 1). Next, we projected rat genes into the R-pack ‘Biomart' on human genes (supplementary table 2). We took the top 10 confidence-ranking genes and hybridized them with the mitochondrial gene set Mitocarta 3.0 (supplementary table 3) to obtain FASN (Figure 4(A)). We then downloaded three-dimensional structural models of the proteins FASN (P12785) and NRF2 (O54968) from the UniProt database. The HDOCK tool (http://HDOCK.phys.hust.edu.cn/) is utilized for the computation of protein–protein docking. In this instance, a model has been selected wherein the FASN and the NRF2 are linked with a binding affinity score of −238.00, and the confidence score is 0.8532. PYMOL software is used for 3D visualization of complexes (Figure 4(C)). The protein–protein docking results have yielded a structural foundation for the possible function of FASN in its interaction with NRF2.

Figure 4.

An interaction exists between NRF2 and FASN in RLE-6TNs under the stimulation of CSE. (A)Proteins interacting with Nrf2 were identified using immunoprecipitation-based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The top 10 genes were crossed with the mitochondrial gene set (Mitocarta 3.0) to obtain FASN. (B) The direct binding of NRF2 to FASN was analyzed by western blotting. (C) The Molecular docking model of NRF2 and FASN. (D) The direct binding of NRF2 to KEAP1 and NRF2 to FASN was quantitatively analyzed by western blot. (E) The expression of FASN under different CSE concentrations and time gradient intervention were analyzed by western blot. (F) FASN and DAPI were detected by immunofluorescence (scale bar 100μm). Values were shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

We further verified the interaction between NRF2 and FASN through co-IP (Figure 4(B)). As a natural inhibitor of NRF2, we used KEAP1 as a positive control and found a reduction in binding to both after the CSE intervention. We then further explored the binding effect of NRF2 to FASN, and our study confirmed that the degree of binding of NRF2 to FASN decreased with CSE intervention (Figure 4(D)). CSE may regulate nuclear translocation of NRF2 by affecting FASN. Furthermore, we found that the expression of FASN gradually decreased as the CSE concentration gradient increased (Figure 4(E)), and the same result was shown by immunofluorescence (Figure 4(F)). At the selected 10% CSE concentrations, the expression of FASN decreased with the prolongation of the intervention time (Figure 4(E)). Considering the interaction between FASN and NRF2, we next further investigated the expression and nuclear translocation of NRF2 in siFASN RLE-6TN after CSE treatment.

CSE affects oxidative stress, NRF2 expression, and nuclear translocation by inhibiting FASN

To further investigate the specific roles of the combination of FASN and NRF2 in RLE-6TN after CSE intervention. We knocked down FASN using siRNA (Figure 5(A)), immunoblotting showed a reduced degree of inhibition of NRF2 expression in siFasn RLE-6TN after CSE treatment, consistent with changes in downstream ARE. and nuclear translocation of Nrf2 was suppressed after CSE treatment compared with the control group (Figure 5(C)). Immunofluorescence showed the same results (Figure 5(D)). This indicates that CSE affects the expression and nuclear translocation of NRF2 via FASN. Inhibition of FASN expression also inhibited the expression of Nrf2 and nuclear translocation and enhanced oxidative stress. We further quantified colocalization using Manders’ correlation coefficients (M1 represents the proportion of NRF2 colocalized with FASN to total NRF2 protein, and M2 represents the proportion of FASN and NRF2 colocalized to total FASN protein). As shown in Figure 5(E), extensive colocalization of NRF2 with FASN (represented by M1) was observed in the CON group. This colocalization is significantly reduced after CSE alone, siFasn alone, or siFasn and CSE combined intervention. This suggests that these interventions significantly affect NRF2 expression and the interaction between NRF2 and FASN proteins. ROS staining further confirmed this conclusion. Knockdown of FASN further increased intracellular ROS, The use of the antioxidant mito-TEMPO can partially alleviate ROS production. Furthermore, Compared with the wild-type cells treated with CSE, the increased level of ROS in siFasn RLE-6TN decreased after CSE treatment (Figure 5(B)). These results suggest that CSE decreases the expression and nuclear translocation of NRF2 by inhibiting FASN, which results in the exacerbation of oxidative stress.

Figure 5.

CSE affects oxidative stress, NRF2 expression, and nuclear translocation by inhibiting FASN. (A) Western blot analysis showed the expression level of FASN after RLE-6TN was knocked down by the siRNA1-4 sequence. (B) ROS content in RLE-6TN treated with 10% CSE was determined by DCFH-DA fluorescence staining and visualized using Fluorescence (scale bar 100μm). (C) Western blot analysis showed the expression level of total NRF2 and nuclear NRF2, HO-1 and NQO-1 in RLE-6TN after siFasn knockdown. (D) NRF2 (red), FASN (green), and DAPI (blue) were detected by immunofluorescence (scale bar 100μm and 25μm). (E) Quantitative analysis of protein colocalization of NRF2 and FASN using Manders’ Colocalization Coefficients by Image J. Values were shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Effects of FASN gene knockout on apoptosis, mitochondrial function, and structure with or without CSE intervention

It has been found that FASN plays an important role in the production of ROS in lung homeostasis [35]. Therefore, we further investigated the effects of CSE on the mitochondrial function and structure of siFASN RLE-6TN. We found that the degree of increase in BAX and cleaved-Caspase3 and the reduction of BCL2 in siFasn RLE-6TN after CSE treatment were lower than in the control group (Figure 6(A)). Flow cytometry results showed that FASN knockdown after CSE treatment aggravated apoptosis in RLE-6TN cells compared with the control group, but the degree of apoptosis was reduced compared to control (Figure 6(B)). These results suggest that CSE can affect the expression of apoptosis-related proteins by regulating FASN. The reduction degree of NDUFB8, UQCRC2 and MTCO2 in siFasn RLE-6TN after CSE treatment was lower than that in the control group (Figure 6(C)). TEM showed that the mitochondrial structure of siFasn RLE-6TN was impaired, but the degree of structural abnormality of siFasn RLE-6TN mitochondria was alleviated compared with CSE-treated wild-type RLE-6TN (Figure 6(D)). These results suggest that CSE-treated AEC2 inhibits NRF2 expression and nuclear translocation by decreasing FASN, thereby disrupting OXPHOS and structural normality of mitochondria, ultimately leading to apoptosis.

Figure 6.

Effects of Fasn gene knockout on apoptosis, mitochondrial function, and structure with or without CSE intervention. RLE-6TN were transfected with control siRNA or siFasn and then exposed to CSE (10%). (A) The expression of BAX, BCL2, Caspase-3, Cleaved caspase-3, and β-actin were analyzed by western blot. (B) Apoptosis was detected by flow cytometry. (C) The expression of NDUFB8, SDHB, UQCRC2, MTCO2, ATP5A, and β-actin were analyzed by western blot. (D) Mitochondrial ultrastructure was observed under TEM (scale bar 2μm and 500 nm). Values were shown as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Discussion

As a rich source of oxidants, CS drives disease progression through oxidative stress and mitochondrial damage [41,42]. The central link is mitochondrial dysfunction. It has been shown that oxidative stress plays an important role in inducing mitochondrial dysfunction and disrupting redox balance. Our study investigated the mechanistic relationship between CS-induced redox imbalance and mitochondrial damage. We found that FASN is involved in regulating NRF2-mediated RLE-6TN protection under CSE. These results suggest that FASN is a potential therapeutic target for chronic lung disease.

CS contains numerous deleterious components, inhalation of which results in cell damage. Exposure to high concentrations of CSE can induce apoptosis of lung epithelial cells [43]. Our study confirms that the BAX/BCL2 ratio increases after CSE intervention to activate the mitochondrial-dependent apoptosis pathway. ROS serve as key mediators of CSE-induced apoptosis. Mitochondria are not only the source of ROS production but also the target of CSE-induced cell damage. ETC is an important structural foundation of cellular energy production, and the destruction of the ETC will lead to mitochondrial respiratory disorder, contributing to cellular injury. Consistent with prior reports, CSE exposure reduces mitochondrial complex subunit protein levels and compromises mitochondrial function in AEC [44]. The activities of mitochondrial complexes I and II were inhibited by CSE in a dose-dependent manner. At the same time, MMP, mitochondrial oxygen consumption, and ATP production decreased [10]. Our study also confirmed that CSE causes damage to respiratory chain-associated proteins.

NRF2 is a key regulator of the antioxidant system. It is ubiquitously expressed in the lung and is predominantly present in epithelial cells (supplementary Figure 1(A)). While studies in chondrocytes demonstrate IL-1β-induced NRF2 upregulation promotes antioxidant and anti-apoptotic effects. This suggests that increased NRF2 expression and nuclear translocation may represent an adaptive mechanism by which cells respond and survive in the oxidative stress microenvironment [45]. Following CSE treatment, total NRF2 expression exhibited transient upregulation followed by decline, whereas nuclear NRF2 accumulation progressively increased until 24 h post-exposure before decreasing. In combination with previous findings, MMP reduction and apoptosis were most pronounced after 24 h of CSE treatment, suggesting that CSE may regulate mitochondrial function by modulating NRF2 nuclear translocation, leading to apoptosis.

The study reported that NRF2 deficiency increases the production of oxidants, especially H2O2, which in turn exacerbates mitochondrial dysfunction. Further mechanistic studies confirmed that NRF2 regulates the production of NADPH (Isocitrate dehydrogenase) by affecting the shuttle activity of Nicotinamide adenine dinucleotide phosphate and pentose phosphate pathway, which in turn influences oxidants neutralization [46]. Our study confirms that the knockdown of NRF2 aggravates redox imbalance and increases cellular ROS levels. This suggests that CSE regulates ROS production in part by regulating NRF2.

While oxidative stress is established as a regulator of mitochondrial function, its precise mechanisms remain incompletely elucidated. Studies indicate that activation of NRF2 and its downstream targets (e.g. using Songorine) promotes mitochondrial biogenesis by regulating mitochondrial complex gene expression, rescuing LPS-induced myocardial dysfunction [47]. Our study confirmed that OXPHOS-related subunit protein expression was decreased after NRF2 knockdown, which indicated that NRF2 had a regulatory effect on them. Crucially, CSE-induced damage to OXPHOS subunits and mitochondrial structure was markedly exacerbated in NRF2-deficient cells compared to wild-type controls. This demonstrates that NRF2 exerts a protective regulatory effect on these components and that CSE-induced mitochondrial damage is, at least partially, mediated through NRF2-dependent pathways. While CSE may exert direct, acute effects on mitochondria, significant intracellular ROS accumulation and OXPHOS subunit alterations emerged predominantly at 24 h post-exposure. This temporal pattern aligns precisely with the peak and subsequent decline of NRF2 nuclear translocation observed in our study. Collectively, these findings indicate that CSE disrupts mitochondrial OXPHOS integrity primarily by impairing NRF2 nuclear translocation.

Co-IP assays confirmed a direct interaction between NRF2 and FASN. This interaction was significantly attenuated following CSE exposure. Furthermore, FASN expression exhibited dose- and time-dependent downregulation in response to CSE treatment. Studies reported that neonatal mice with AEC2-targeted deletion of Fasn (FasniΔAEC2) had significantly increased neutrophil and protein content in BALF compared with controls and more severe spatial expansion of the lungs after exposure to CS [34]. FASN expression was significantly reduced in IPF patients and mice treated with Bleomycin (BLM). After overexpression of FASN, BLM-induced death of AEC was alleviated and the ROS production and the loss of MMP was reduced [35]. These results suggest that FASN is involved in the maintenance of pulmonary structural homeostasis. While our data demonstrate that FASN knockdown exacerbates baseline apoptosis and mitochondrial damage, suggesting that FASN is essential for normal cell function. However, Li et al. had a different view. Their research showed that BLM treatment inhibited the Fatty acid synthesis of alveolar type 2 cells (AT2) and promoted cell proliferation. Further studies found that BLM-induced alveolar damage drives metabolic reediting of AT2 cells by affecting autophagy-regulated glucose and lipid metabolism, facilitating the transition from lipid metabolism to glucose metabolism; To ensure the energy supply to promote the regeneration of lung epithelium and maintain lung homeostasis [48]. This demonstrate the complex role of FASN in lung disease pathogenesis. Compared with the control group, the expression of Nrf2 and the degree of nuclear translocation in siFasn RLE-6TN decreased after CSE treatment. Furthermore, apoptosis, decrease in OXPHOS-associated subunit proteins, and the extent of mitochondrial structural damage were reduced, suggesting that FASN is involved in the regulation of these processes. Our study indicates that FASN is involved in the maintenance of mitochondrial function. FASN inhibition impairs NRF2 expression and nuclear translocation, aggravating oxidative stress and mitochondrial damage, leading to an imbalance of the antioxidant stress system and ultimately cell death.

There are several limitations to this study. First, the experiments were conducted exclusively in vitro using cell lines. Future research employing in vivo models, such as transgenic mice, is warranted to provide a more comprehensive understanding of pathophysiological mechanisms and toxicological effects of CSE. Furthermore, since FASN expression was reduced in our disease model, overexpressing FASN would be more valuable for elucidating its functional roles.

Funding Statement

This work was supported by the National Natural Science Foundation of China [grant number 82000039] (www.nsfc.gov.cn).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13510002.2025.2550412.