Involvement of mitochondrial unfolded protein response and activating transcription factor 4 in the mitochondrial damage pathway of BEAS-2B cells induced by cigarette smoke extracts

Introduction

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease caused by multiple factors and characterised by chronic inflammation of the airways, lung tissue and pulmonary vasculature (1,2). Smoking is an established risk factor for COPD, and cigarette smoke (CS) is a major causative factor for many chronic lung diseases such as COPD (2), and approximately 90% of patients with COPD have a history of smoking (3). The reactive oxygen species (ROS) produced by the body under normal conditions can maintain a balance between production and scavenging under the action of oxidative and antioxidant systems in the body, and are involved in cell signaling and metabolism (4).

As the main site of ROS production in the body during oxidative phosphorylation, mitochondria play a very critical role in regulating superoxide radical production (5). It has been demonstrated that airway reactive oxygen radical production is significantly higher in patients with COPD compared to normal subjects, and when airway epithelial cells are exposed to cigarette smoke extract (CSE), this can lead to mitochondrial dysfunction through a variety of pathways, such as changes in mitochondrial membrane potential, increasing ROS production (6). In vitro and in vivo studies have shown that the degree and duration of cigarette smoke (CS) exposure are critical to the mitochondrial damage response (7). Increasing evidence indicates that CS-induced mitochondrial dysfunction promotes chronic inflammation in COPD patients, representing a key event in the pathogenesis (8,9). This suggests that mitochondrial-targeted interventions hold great promise as a research direction in COPD management. For example, current research indicates that CS exposure alters the abundance of several key mitochondrial proteins, such as PTEN-induced kinase 1 (PINK1), PARKIN, Ras homolog oncogene family member T1 (RHOT1), and dynamin-related protein 1 (DRP1), which are crucial for mitochondrial dynamics and quality control mechanisms (10); CS induces upregulation of DRP1 and mitochondrial fission factor (MFF), while downregulating mitochondrial fusion protein 2 (MFN2) and optic atrophy-related protein 1 (OPA1), thereby disrupting mitochondrial homeostasis and generating ROS (11); Maremanda et al. (10) found that the long OPA1 isoform, along with SLP2 and prohibitions, plays a crucial role in CS-induced lung damage, causing mitophagy/mitochondrial dysfunction in COPD. Therefore, investigating CSE-induced mitochondrial dysfunction may emerge as a potential therapeutic target for COPD, offering broad research prospects.

Mitochondrial dysfunction is associated with increased ROS production and aggregation of unfolded proteins (12). For this reason, mitochondria have renewal mechanisms, such as mitochondrial autophagy or the unfolded protein response (UPR). The mitochondrial UPR (UPR^mt) regulates mitochondrial protein homeostasis, originally defined as a process consisting of a transcriptional response triggered by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) (13). This transcriptional response increases mitochondrial chaperone expression in response to mitochondrial stress generated by the accumulation of unfolded or misfolded proteins within the mitochondria and repairs and rescues dysfunctional mitochondria (14). Regulation of the mitochondrial protein import pathway is an important mechanism for regulating mitochondrial protein homeostasis and function during stress. Mitochondrial protein import complexes, such as translocase of the outer membrane (TOM) and translocase of the inner membrane 23 (Tim23), are responsible for the post-translational import of 99% of the mitochondrial proteins encoded by the nuclear genome (15). Tim23 is the transporter enzyme responsible for the import of two-thirds of the mitochondrial proteome into the mitochondrial matrix through the inner mitochondrial membrane (16). It was found that knocking down Tim23 disrupts the protein import pathway and leads to mitotic nucleus imbalance, which stimulates ROS emission and selectively activates the C/EBP homologous protein (CHOP) branch of the UPR (17).

The UPR is regulated by the mitochondrial import efficiency of the transcription factor ATFS-1 in nematodes and by the potential homologous transcription factors activating transcription factor (ATF) 4 (ATF-4), ATF-5, and CHOP in mammals (18). Accumulation of mislocalised mitochondrial proteins in the cytoplasm activates the UPR^mt pathway, which is regulated by competing organelles that target sequences in the transcription factor ATF-4 (19). Under normal conditions, ATF-4 can be efficiently imported into healthy mitochondria (20). When OXPHOS or mitochondrial protein homeostasis is disturbed, its import efficiency is reduced, further exacerbating mitochondrial dysfunction. If ATF-4 fails to enter the mitochondria, it is transported to the nucleus where it induces transcription of mitochondrial protective genes, including mitochondrial chaperones and proteases, antioxidants, etc. (19). In turn, the mitochondrial import of protective gene products promotes organelle stabilization and recovery (21). In previous studies, the UPR branch of eIF2-α/ATF-4/CHOP was activated in trabecular meshwork cells upon exposure to tert-butyl hydroperoxide (TBHP), and inhibition of ATF-4 improved apoptosis and inflammatory cytokine production and reduced ROS production (22).

Therefore, we hypothesised that CSE-induced mitochondrial dysfunction in BEAS-2B cells might be related to the UPR^mt pathway. To validate this hypothesis, we detected the changes in cell membrane potential and ROS after CSE exposure. Concurrently, we assessed ATF-4 expression via RNA sequencing (RNA-seq) and signaling pathway analysis, and conducted measurements of Tim23 expression. We present this article in accordance with the MDAR reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-460/rc).

Methods

Reagents and antibodies

BEAS-2B cells were purchased from Pricella (Wuhan, China), CSE was obtained from Murty Pharmaceuticals (USA), Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were bought from Gibco (Shanghai, China), anti-ATF-4 (10835), anti-Tim23 (67535), anti-Alex-Fluor 488 were obtained from Proteintech (Wuhan, China), Mito TrackerTM RED CMXRos was purchased from Thermo (Shanghai, China).

CSE preparation

The initial concentration of CSE purchased from Murty Pharmaceuticals was 40 mg/mL. A total of 0.5 µL of CSE stock solution was added to 2 mL of cell culture medium to make 10 µg/mL of CSE working solution; 1 µL of CSE stock solution was added to 2 mL of cell culture medium to make 20 µg/mL of CSE working solution. Configure in equal proportions according to the amount of liquid required.

Cell culture and passaging

A DMEM solution containing 10% FBS was prepared and the recovered BEAS-2B cells were cultured in a cell culture incubator with 5% CO₂ at 37 ℃ and the cells were passaged when they reached approximately 80–90% growth. When the cells have reached more than 80% growth in the six-well plates, the medium is discarded and different concentrations of CSE are diluted with DMEM are added and a control group is set up.

Detection of mitochondrial ROS

Prepare 5 mM of MitoSox Red Reagent Storage Solution and 100 nM of MitoSox Red Reagent Working Solution (keep away from light). When the cells grew to a suitable concentration, the prepared medium containing CSE was added, placed in a cell culture incubator, incubated for different times, and 1 mL of MitoSox Red working solution at a concentration of 100 nM was added to each well to fully cover the cells, and the cells were incubated for 10 min at 37 ℃ and then observed under a confocal microscope.

Western blot (WB)

Cells were lysed using a wave cell grinder, RIPA buffer and harvested proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were sealed with 20% skimmed milk for 1 hour at room temperature. After incubation with primary and secondary antibodies, immunoblot detection was performed using ECL blotting reagents and proteins were quantified using ImageJ software.

Immunofluorescence staining and laser confocal microscopy

Treated cells grown on round coverslip were washed three times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 minutes at room temperature. At the end of fixation, the cells were washed three times with PBS solution and permeabilised by adding 0.25% Triton X-100 solution for 10 min. After permeabilisation, the samples were washed with PBS and incubated with the fluorescent secondary antibody for 1 h at room temperature in the dark, followed by 4',6-diamidino-2-phenylindole (DAPI) staining for 15 min. All samples were observed and photographed using a confocal microscope (LEICA TCS SP8, Germany).

Statistical analysis

All data were plotted and processed using GraphPad Prism 9.0 software. A t-test was used to compare the differences between the two groups and if P<0.05, the difference was considered statistically significant.

Results

CSE causes a decrease in mitochondrial membrane potential

BEAS-2B cells were treated with CSE and the changes in mitochondrial membrane potential were detected by JC-1 staining (Figure 1). The results showed that the mitochondrial membrane potential was reduced in the CSE-treated group of BEAS-2B cells compared to the control group.

Figure 1 Detection of mitochondrial membrane potential by JC-1 staining (magnification, 40×). The membrane potential was determined by the ratio of red to green. Compared with the control group, the ratio of red decreased and the ratio of green increased in the CSE-treated BEAS-2B cells, indicating that the mitochondrial membrane potential decreased after CSE treatment, and the difference was statistically significant (*, P<0.05). CSE, cigarette smoke extract.

CSE causes increased mitochondrial ROS production

To investigate the effect of CSE on mitochondrial ROS production, we treated BEAS-2B cells with different concentrations of CSE (1–10%), treated cells with the same concentration of CSE for different times (10–60 min), and added 100 nM MitoSox red to the medium for 10 min before observing ROS production under confocal microscopy. The results showed that the mitochondrial ROS production correlated with the concentration and duration of action of CSE (Figure 2).

Figure 2 CSE induced increased mitochondrial ROS production in cells (MitoSOX Red staining; magnification, 40×). (A) Mitochondrial ROS production in BEAS-2B cells after exposure to CSE at 1%, 5% and 10% for the same time. (B) Mitochondrial ROS production in BEAS-2B cells after exposure to the same concentration of CSE for 10, 30 and 60 min (***, P<0.001). CSE, cigarette smoke extract; ROS, reactive oxygen species.

RNA-seq to detect gene expression after CSE action

To further understand the changes in cellular transcription induced by CSE, we performed RNA-seq and signaling pathway analysis on normal and CSE-treated cells.

The results showed that the effect of CSE caused changes in messenger RNA (mRNA) expression of 773 genes, including 518 genes with increased expression and 255 genes with decreased expression. Analysis of the signaling pathways of these genes revealed that the genes altered by CSE were mainly in the pathways of UPR^mt, telomere regulation genes, anti-aging genes and mitochondrial membrane transfer protein genes. Functional analysis of these genes revealed that they are involved in cellular senescence, repair, and stress to the outside world, and are also closely associated with the development of COPD. We selected the ATF-4 gene with the most significant up-regulation of expression for subsequent experiments (Figure 3).

Figure 3 Analysis of RNA-seq and signaling pathways in cells after CSE treatment. (A) RNA-seq results show that CSE induced changes in multiple signaling pathways in cells. (B) CSE induced changes in genes involved in the UPR^mt pathway. Con, control; CSE, cigarette smoke extract; log₂FC, log₂ fold change; RNA-seq, RNA sequencing; UPR^mt, mitochondrial unfolded protein response.

Cells were treated with CSE, total RNA was extracted with TRIzol, then purified with RNeasy Mini kit, 500 ng of RNA was taken for polyA RNA enrichment, and libraries were constructed using Lexogen Mrna Sense kit for mRNA with sequencing primers, and libraries were amplified by PCR for 13 cycles. A 200–350 bp fragment from each library was purified by Pippin Prep gel and sequenced on an Ion Torrent Proton sequencer at 30× coverage depth. The sequencing results were aligned in the Human Genome (GRCh37) database after removal of sequencing primers. Differentially expressed genes were statistically analysed using by R software. The final 773 differentially expressed genes were identified. The differentially expressed genes were analysed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and the fold change was taken as −log₂. Red represents increased expression and green represents decreased expression.

Activation of the Tim23 during CSE stimulation of BEAS-2B cells

Next, given that Tim23 functions as a protein import channel in mitochondria, its upregulation is likely a downstream effect of UPR^mt activation. We thus suggest that the changes in Tim23 expression and localization are potentially linked to the UPR^mt. We collected BEAS-2B cell lysates after treatment with CSE at concentrations of 10, 20 µg/mL for 2, 4, 8 and 12 h, extracted cellular proteins, divided them into two groups according to their concentrations, and detected them by WB and immunofluorescence assays (Figure 4).

Figure 4 Altered expression of Tim23 after CSE treatment. (A,B) After treatment of BEAS-2B cells with 10 and 20 μg/mL CSE, the expression of Tim23 was upregulated as detected by the WB method and the difference was statistically significant after 12 h of stimulation (**, P<0.01). (C,D) Immunofluorescence staining (magnification: ×100) followed by laser confocal detection of Tim23 expression. The results showed that the expression of Tim23 was at an increased level after CSE treatment of the cells, with a significant increase in the expression of the cells treated for 12 hours compared to the control group. The difference was statistically significant after 10 μg/mL CSE treatment (**, P<0.01); the difference was statistically significant after 20 μg/mL CSE treatment (*, P<0.05). CSE, cigarette smoke extract; Tim23, translocase of inner membrane 23; WB, Western blot.

Altered ATF-4 expression after CSE stimulation of BEAS-2B cells

We have demonstrated at the genetic level that ATF-4 expression is up-regulated after CSE stimulation of cells, and we then verified the altered expression of ATF-4 at the protein level. We treated BEAS-2B cells with different concentrations of CSE and examined ATF-4 expression by WB assay and found that its expression was upregulated, suggesting involvement in the CSE-induced mitochondrial damage pathway. Immunofluorescence staining was performed to observe the changes in ATF-4 expression and localization in the cells using laser confocal microscopy, and the results showed that the expression of ATF-4 increased after different concentrations of CSE-treated cells for different times, accompanied by changes in nuclear expression (Figure 5).

Figure 5 Expression of ATF-4 after CSE treatment of cells. (A,B) ATF-4 expression was up-regulated after stimulation of BEAS-2B cells at different concentrations of CSE for different times, where the expression of ATF-4 was up-regulated after 10 μg/mL CSE stimulation and the difference was statistically significant after 8 and 12 h of stimulation; the expression of ATF-4 was up-regulated after 20 μg/mL CSE stimulation and the difference was statistically significant after 4, 8 and 12 hours after stimulation were statistically significant. (C,D) Altered ATF-4 expression after immunofluorescence staining (magnification: ×100), in which the expression of ATF-4 was statistically different after 4, 8 and 12 h of 10 μg/mL CSE compared to the control group; the expression of ATF-4 was statistically different after 4, 8 and 12 h of stimulation of BEAS-2B cells by 20 μg/mL CSE compared to the control group. *, P<0.05; **, P<0.01; ***, P<0.001. ATF-4, activating transcription factor 4; con, control; CSE, cigarette smoke extract; DAPI, 4',6-diamidino-2-phenylindole.

Discussion

COPD is a growing global health problem. Approximately 10% of people over the age of 45 years currently suffer from COPD, but this rises to 50% among heavy smokers (4). Chronic obstructive lung disease is characterised by persistent respiratory symptoms caused by abnormalities of the airways and/or alveoli, usually due to exposure to large amounts of harmful particles or gases (23). Despite the fact that slow-onset lung is one of the few non-communicable diseases with increasing morbidity and mortality worldwide, there is currently limited ability to identify patients at risk of slow-onset lung progression at an early stage (24). Therefore, early diagnosis and treatment of slow-onset lung disease are increasingly important.

Cigarette smoking is a major risk factor for COPD. ROS in CS can directly damage airway epithelial cells (25). Oxidative stress is caused by an excess of ROS resulting in impaired endogenous antioxidant defense mechanisms and has been demonstrated to be increased in COPD patients (26). The onset of uncontrolled chronic inflammation and oxidative stress resulting from exposure to cigarette smoke is a major driver of chronic obstructive pulmonary pathogenesis in airway epithelial cells and is involved in multiple forms of cell death (including apoptosis, necrosis, and autophagy) (27). Current studies demonstrate that the high oxidative burden caused by mitochondrial dysfunction is the main cause of refractory inflammation and abnormal responses in the lungs due to exposure to cigarette smoke (28).

Mitochondria are a major source of oxidative stress in vivo, and mitochondrial damage plays an important role in the pathogenesis of slow-onset lung (29). Previous studies have reported that CS-induced mitochondrial dysfunction and loss of mitochondrial phagocytosis can induce cellular senescence and impair lung function (30); a more recent study has shown that CSE leads to the accumulation of damaged mitochondria and severe mitochondrial damage through mitochondrial autophagy (31). Reduced levels of the proteins PHB1 and PHB2, which maintain mitochondrial homeostasis and have antioxidant effects, have been observed in lung tissue from both patients with and without LBP smokers (32). This suggests that CS alters mitochondrial structure and function, leading to increased levels of ROS and cellular damage. The lipophilic component of CSE was found to interfere with mitochondrial function; mitochondrial membrane potential, ATP levels were dose-dependently reduced in BEAS-2B cells exposed to CSE; and CSE induced ROS production in human alveolar epithelial cells (33). Ballweg et al. (34) observed an increase in mitochondrial potential after treatment of MLE12 cells with CSE, which may suggest that mitochondrial function is disrupted at the inner or outer membrane (32). Carbonylated proteins are the main products of ROS-mediated oxidation reactions, and Giordano et al. (35) found that the levels of carbonylated proteins in CSE-induced BEAS-2B increased with increasing CSE concentrations, suggesting that CSE exposure induces mitochondrial membrane depolarisation and increases superoxide production, leading to protein oxidation. Previous studies have similarly confirmed that CSE treatment of primary lung epithelial cells for short (24 h) and long (15 days) periods resulted in an increase in cellular mitochondrial ROS and a decrease in mitochondrial membrane potential as detected by immunofluorescence, suggesting that CSE causes mitochondrial dysfunction (36). In this study, the results showed that CSE can cause impaired mitochondrial membrane potential and mitochondrial ROS production. Although it was confirmed that CSE caused impaired mitochondrial function in BEAS-2B cells, the specific mechanism of the injury remains unclear, which may include mitochondrial autophagy (37), altered mitochondrial morphology (28), altered telomeres (38), and activation of the UPR^mt pathway (39), etc. Therefore, to further understand the transcriptional changes induced by CSE, we performed RNA-seq and signaling pathway analysis on normal and CSE-treated cells, and the results showed that the mRNA of several genes was altered. The analysis of these genes revealed that they were mainly concentrated in pathways such as UPR^mt and cellular telomere regulatory genes. It should be emphasized that our study has limitations. The assessment of mitochondrial damage is multidimensional. To fully elucidate the comprehensive picture of CSE-induced mitochondrial damage, more comprehensive assessments are required. These should include measuring cellular ATP levels, evaluating the activity of electron transport chain complexes, and examining mitochondrial cristae architecture and swelling via electron microscopy. These critical studies represent core directions for our future work, aimed at further elucidating the mechanisms by which CSE impairs mitochondrial health.

The UPR can alter the expression of various genes involved in antioxidant defense, inflammation, energy metabolism, and protein synthesis (21). The UPR consists of three distinct but interconnected arms that act through PERK, IRE1a, and ATF-6, respectively (40). In the ER, to reduce unfolded proteins, PERK is activated to phosphorylate eukaryotic initiation factor 2a (eIF2a), thereby transiently attenuating overall translation of mRNA (41); in mitochondria, GCN2 kinase activated for mitochondrial dysfunction phosphorylates eIF2a, mediating translation attenuation during mitochondrial dysfunction (42). However, eIF2a phosphorylation also upregulates the translation of many genes, including ATF4 and ATF5, to increase the ability to transport proteins (41). At the same time, ATF-4 upregulates apoptosis-inducing factors such as CHOP (43). Under normal conditions, ATF-4 transmits adaptive signals and upregulates genes that promote ER homeostasis and survival (43). In Cryptobacterium showyeri, the transcription factor ATFS-1 is thought to be a key regulator of UPR^mt, due to its elevated expression of mitochondrial chaperones and proteases that promote mitochondrial homeostasis (44). Recent evidence suggests that ATF-4, ATF-5 and CHOP may be homologous transcription factors of ATFS-1 in mammals involved in the regulation of UPR^mt (14). Tang et al. (45) detected activation of the UPR in an animal model of CS-induced COPD and identified proteins associated with unfolded proteins in the lung tissue of COPD rats. The expression levels of CHOP, ATF-4, and ATF-6 increased with the duration of CSE exposure (46). Jiang et al. (47) identified UPR^mt in alveolar epithelial cells (AEC) and suggested that ATF-4 mediates the activation of UPR^mt, thereby promoting in vitro and in vivo ER stress-induced mitochondrial dysfunction; furthermore, they identified ATF-4 as a key regulator of UPR^mt that mediates ER stress-induced mitochondrial dysfunction in alveolar epithelial cells. Also, in epilepsy-induced apoptosis experiments, it was confirmed that ATF-4 could regulate UPR^mt levels, attenuate mitochondrial oxidative stress and inhibit apoptotic pathway activation (48).

We detected the involvement of the UPR^mt and ATF-4 pathways in transcriptional changes in cells following CSE treatment through RNA-seq analysis. In the present study, we found that the expression of Tim23 and ATF-4 was up-regulated after CSE. These findings are consistent with the model that CSE may impair mitochondrial homeostasis, potentially through mechanisms involving. We acknowledge the limitations of this study, which failed to further test classical markers of the UPR^mt pathway to demonstrate its activation. This will be a key focus of our subsequent work. Mitochondrial stress responses and ATF-4 signaling suggest that the UPR^mt pathway may be involved. Although our research has certain limitations, it provides a potential mechanism for mitochondrial damage in CSE-mediated bronchial epithelial cell damage. Our research also offers a new theoretical framework for the development of mitochondrial-targeted therapies for lung injury caused by CS. Our data suggest that protecting mitochondrial integrity by targeting these specific nodes may be a feasible strategy to alleviate bronchial epithelial damage induced by CSE. Our future research will focus on verifying these targets in in vitro and in vivo CS-induced lung injury models using gene (knockout/overexpression) and pharmacological methods to assess their true therapeutic potential.

Conclusions

The effect of CSE on BEAS-2B cells leads to a decrease in mitochondrial membrane potential and an increase in ROS generation. The expression of Tim23 and ATF-4 changed after the action of CSE.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-460/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-460/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-460/prf

Funding: This study was supported by the Ningbo Key Research and Development Plan (No. 2023Z179) and the Natural Science Foundation Key Project of Ningbo (No. 202003N4023).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-460/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.

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. Kaur M, Chandel J, Malik J, et al. Particulate matter in COPD pathogenesis: an overview. Inflamm Res 2022;71:797-815. [Crossref] [PubMed]
2. Song Q, Chen P, Liu XM. The role of cigarette smoke-induced pulmonary vascular endothelial cell apoptosis in COPD. Respir Res 2021;22:39. [Crossref] [PubMed]
3. Kim GD, Lim EY, Shin HS. Macrophage Polarization and Functions in Pathogenesis of Chronic Obstructive Pulmonary Disease. Int J Mol Sci 2024;25:5631. [Crossref] [PubMed]
4. Barnes PJ. Oxidative stress-based therapeutics in COPD. Redox Biol 2020;33:101544. [Crossref] [PubMed]
5. Larson-Casey JL, He C, Carter AB. Mitochondrial quality control in pulmonary fibrosis. Redox Biol 2020;33:101426. [Crossref] [PubMed]
6. Quan Y, Xin Y, Tian G, et al. Mitochondrial ROS-Modulated mtDNA: A Potential Target for Cardiac Aging. Oxid Med Cell Longev 2020;2020:9423593. [Crossref] [PubMed]
7. Wang S, Song X, Wei L, et al. Role of Mitophagy in Cigarette Smoke-induced Lung Epithelial Cell Injury In Vitro. Curr Mol Med 2023;23:1130-40. [Crossref] [PubMed]
8. Shen Y, Chen L, Chen J, et al. Mitochondrial damage-associated molecular patterns in chronic obstructive pulmonary disease: Pathogenetic mechanism and therapeutic target. J Transl Int Med 2023;11:330-40. [Crossref] [PubMed]
9. Zhang MY, Jiang YX, Yang YC, et al. Cigarette smoke extract induces pyroptosis in human bronchial epithelial cells through the ROS/NLRP3/caspase-1 pathway. Life Sci 2021;269:119090. [Crossref] [PubMed]
10. Maremanda KP, Sundar IK, Rahman I. Role of inner mitochondrial protein OPA1 in mitochondrial dysfunction by tobacco smoking and in the pathogenesis of COPD. Redox Biol 2021;45:102055. [Crossref] [PubMed]
11. Zeng T, Liu L, Xu D, et al. The Mitochondrial Fusion Promoter M1 Mitigates Cigarette Smoke-Induced Airway Inflammation and Oxidative Stress via the PI3K-AKT Signaling Pathway. Lung 2024;203:12. [Crossref] [PubMed]
12. Keerthiga R, Pei DS, Fu A. Mitochondrial dysfunction, UPR(mt) signaling, and targeted therapy in metastasis tumor. Cell Biosci 2021;11:186. [Crossref] [PubMed]
13. Cilleros-Holgado P, Gómez-Fernández D, Piñero-Pérez R, et al. Mitochondrial Quality Control via Mitochondrial Unfolded Protein Response (mtUPR) in Ageing and Neurodegenerative Diseases. Biomolecules 2023;13:1789. [Crossref] [PubMed]
14. Cilleros-Holgado P, Gómez-Fernández D, Piñero-Pérez R, et al. mtUPR Modulation as a Therapeutic Target for Primary and Secondary Mitochondrial Diseases. Int J Mol Sci 2023;24:1482. [Crossref] [PubMed]
15. Jishi A, Qi X. Altered Mitochondrial Protein Homeostasis and Proteinopathies. Front Mol Neurosci 2022;15:867935. [Crossref] [PubMed]
16. Jain N, Gomkale R, Rehling P. TOM-TIM23 supercomplex formation. Methods Enzymol 2024;707:3-22. [Crossref] [PubMed]
17. Oliveira AN, Hood DA. Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPR(mt) in muscle. Am J Physiol Cell Physiol 2018;315:C516-26. [Crossref] [PubMed]
18. Wu Y, Yang Y, Qin X, et al. Unfolded proteins in the mitochondria activate HRI and inhibit mitochondrial protein translation. Cell Signal 2024;123:111353. [Crossref] [PubMed]
19. Kim H, Chen Q, Ju D, et al. ER-tethered stress sensor CREBH regulates mitochondrial unfolded protein response to maintain energy homeostasis. Proc Natl Acad Sci U S A 2024;121:e2410486121. [Crossref] [PubMed]
20. Lu H, Wang X, Li M, et al. Mitochondrial Unfolded Protein Response and Integrated Stress Response as Promising Therapeutic Targets for Mitochondrial Diseases. Cells 2022;12:20. [Crossref] [PubMed]
21. Zhou Z, Fan Y, Zong R, et al. The mitochondrial unfolded protein response: A multitasking giant in the fight against human diseases. Ageing Res Rev 2022;81:101702. [Crossref] [PubMed]
22. Ying Y, Xue R, Yang Y, et al. Activation of ATF4 triggers trabecular meshwork cell dysfunction and apoptosis in POAG. Aging (Albany NY) 2021;13:8628-42. [Crossref] [PubMed]
23. Song Y, Fu W, Zhang Y, et al. Azithromycin ameliorated cigarette smoke-induced airway epithelial barrier dysfunction by activating Nrf2/GCL/GSH signaling pathway. Respir Res 2023;24:69. [Crossref] [PubMed]
24. Xu J, Zeng Q, Li S, et al. Inflammation mechanism and research progress of COPD. Front Immunol 2024;15:1404615. [Crossref] [PubMed]
25. Chiaradia E, Sansone A, Ferreri C, et al. Phospholipid fatty acid remodeling and carbonylated protein increase in extracellular vesicles released by airway epithelial cells exposed to cigarette smoke extract. Eur J Cell Biol 2023;102:151285. [Crossref] [PubMed]
26. Barnes PJ. Oxidative Stress in Chronic Obstructive Pulmonary Disease. Antioxidants (Basel) 2022;11:965. [Crossref] [PubMed]
27. Wu X, Zhang G, Du X. Cigarette Smoke Extract Induces MUC5AC Expression Through the ROS/ IP3R/Ca(2+) Pathway in Calu-3 Cells. Int J Chron Obstruct Pulmon Dis 2024;19:1635-47. [Crossref] [PubMed]
28. Thapa R, Marianesan AB, Rekha A, et al. Hypoxia-inducible factor and cellular senescence in pulmonary aging and disease. Biogerontology 2025;26:64. [Crossref] [PubMed]
29. Caldeira DAF, Weiss DJ, Rocco PRM, et al. Mitochondria in Focus: From Function to Therapeutic Strategies in Chronic Lung Diseases. Front Immunol 2021;12:782074. [Crossref] [PubMed]
30. Zhao X, Zhang Q, Zheng R. The interplay between oxidative stress and autophagy in chronic obstructive pulmonary disease. Front Physiol 2022;13:1004275. [Crossref] [PubMed]
31. Araya J, Tsubouchi K, Sato N, et al. PRKN-regulated mitophagy and cellular senescence during COPD pathogenesis. Autophagy 2019;15:510-26. [Crossref] [PubMed]
32. Kosmider B, Lin CR, Karim L, et al. Mitochondrial dysfunction in human primary alveolar type II cells in emphysema. EBioMedicine 2019;46:305-16. [Crossref] [PubMed]
33. van der Toorn M, Rezayat D, Kauffman HF, et al. Lipid-soluble components in cigarette smoke induce mitochondrial production of reactive oxygen species in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2009;297:L109-14. [Crossref] [PubMed]
34. Ballweg K, Mutze K, Königshoff M, et al. Cigarette smoke extract affects mitochondrial function in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 2014;307:L895-907. [Crossref] [PubMed]
35. Giordano L, Gregory AD, Pérez Verdaguer M, et al. Extracellular Release of Mitochondrial DNA: Triggered by Cigarette Smoke and Detected in COPD. Cells 2022;11:369. [Crossref] [PubMed]
36. Sundar IK, Maremanda KP, Rahman I. Mitochondrial dysfunction is associated with Miro1 reduction in lung epithelial cells by cigarette smoke. Toxicol Lett 2019;317:92-101. [Crossref] [PubMed]
37. Picca A, Mankowski RT, Burman JL, et al. Mitochondrial quality control mechanisms as molecular targets in cardiac ageing. Nat Rev Cardiol 2018;15:543-54. [Crossref] [PubMed]
38. Gao X, Yu X, Zhang C, et al. Telomeres and Mitochondrial Metabolism: Implications for Cellular Senescence and Age-related Diseases. Stem Cell Rev Rep 2022;18:2315-27. [Crossref] [PubMed]
39. Guo J, Chiang WC. Mitophagy in aging and longevity. IUBMB Life 2022;74:296-316. [Crossref] [PubMed]
40. Anderson NS, Haynes CM. Folding the Mitochondrial UPR into the Integrated Stress Response. Trends Cell Biol 2020;30:428-39. [Crossref] [PubMed]
41. Wang M, Kaufman RJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 2016;529:326-35. [Crossref] [PubMed]
42. Wu Z, Senchuk MM, Dues DJ, et al. Mitochondrial unfolded protein response transcription factor ATFS-1 promotes longevity in a long-lived mitochondrial mutant through activation of stress response pathways. BMC Biol 2018;16:147. [Crossref] [PubMed]
43. Jeon JH, Im S, Kim HS, et al. Chemical Chaperones to Inhibit Endoplasmic Reticulum Stress: Implications in Diseases. Drug Des Devel Ther 2022;16:4385-97. [Crossref] [PubMed]
44. Melber A, Haynes CM. UPR(mt) regulation and output: a stress response mediated by mitochondrial-nuclear communication. Cell Res 2018;28:281-95. [Crossref] [PubMed]
45. Tang F, Ling C. Curcumin ameliorates chronic obstructive pulmonary disease by modulating autophagy and endoplasmic reticulum stress through regulation of SIRT1 in a rat model. J Int Med Res 2019;47:4764-74. [Crossref] [PubMed]
46. Emanuelli G, Nassehzadeh-Tabriz N, Morrell NW, et al. The integrated stress response in pulmonary disease. Eur Respir Rev 2020;29:200184. [Crossref] [PubMed]
47. Jiang D, Cui H, Xie N, et al. ATF4 Mediates Mitochondrial Unfolded Protein Response in Alveolar Epithelial Cells. Am J Respir Cell Mol Biol 2020;63:478-89. [Crossref] [PubMed]
48. Yu X, Wang X, Xie Y, et al. Activating Transcription Factor 4-mediated Mitochondrial Unfolded Protein Response Alleviates Hippocampal Neuronal Damage in an In Vitro Model of Epileptiform Discharges. Neurochem Res 2023;48:2253-64. [Crossref] [PubMed]