N‑acetyl‑L‑cysteine protects rat lungs and RLE‑6TN cells from cigarette smoke‑induced oxidative stress

Chen,Jiameng; Cheng,Yuefeng; Cui,Huijuan; Li,Shuangyan; Duan,Lantian; Jiao,Zongxian

doi:10.3892/mmr.2025.13462

N‑acetyl‑L‑cysteine protects rat lungs and RLE‑6TN cells from cigarette smoke‑induced oxidative stress

Authors:
- Jiameng Chen
- Yuefeng Cheng
- Huijuan Cui
- Shuangyan Li
- Lantian Duan
- Zongxian Jiao
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Affiliations: Department of Pathology, Research Institute of Pathology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu 730000, P.R. China, Department of Pathology, Research Institute of Pathology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu 730000, P.R. China
Published online on: February 18, 2025 https://doi.org/10.3892/mmr.2025.13462
Article Number: 97
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Cigarette smoke (CS) is a key contributor of chronic obstructive pulmonary disease (COPD); however, its role in the pathogenesis of COPD has not been fully elucidated. N‑acetyl‑L‑cysteine (NAC), as an antioxidant, has been used in the treatment of COPD; however, the mechanisms of action of NAC are not fully understood. Alveolar epithelial type 2 (ATII) cells serve an essential role in the maintenance of alveolar integrity. The aim of the present study was to identify the effect of CS on rat lungs and ATII cells. A subacute lung injury model of Wistar rats was established using CS exposure for 4 weeks. Interalveolar septa widening, infiltration of inflammatory cells, edema fluid in airspaces and abnormal enlargement of airspaces were observed through H&E staining. ELISA revealed that NAC could protect against CS‑induced increases in serum levels of malondialdehyde and decreases in serum levels of superoxide dismutase. Additionally, 8‑hydroxy‑deoxyguanosine was detected using immunohistochemical staining, and this was also expressed at increased levels in the lung tissue of the CS‑exposed group. In addition, the expression levels of Bcl‑2, BAX and caspase‑3 p12 in lung tissue were detected by western blotting or immunohistochemical staining. The expression levels of Bcl‑2 decreased and those of caspase3 p12 were increased in response to CS exposure when compared with those in the control group. These effects were prevented by treatment with NAC. In vitro, the effect of CS extract (CSE) on rat lung epithelial‑6‑T‑antigen negative (RLE‑6TN) cells was observed, flow cytometry was used to detect intracellular reactive oxygen species (ROS) levels and the occurrence of apoptosis, and the content of glutathione (GSH) was detected using a colorimetric assay. Additionally, the expression levels of heme oxygenase‑1 (HO‑1), p53 and Bcl‑2 were examined by western blotting, and HO‑1 mRNA expression was also examined using reverse transcription‑quantitative PCR. The results of the present study revealed that CSE induced apoptosis of RLE‑6TN cells, accompanied by increased levels of intracellular ROS and exhaustion of GSH. Significantly increased protein levels of HO‑1 and p53, as well as decreased protein levels of Bcl‑2 were also observed. These effects were prevented by administration of NAC. Overall, these findings suggested that CS could promote apoptosis in rat lung tissues and alveolar epithelial cells by inducing intracellular oxidative injury, and NAC may serve an antioxidant role by replenishing the intracellular GSH content.

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1	Kahnert K, Jörres RA, Behr J and Welte T: The diagnosis and treatment of COPD and its comorbidities. Dtsch Arztebl Int. 120:434–444. 2023.PubMed/NCBI
2	Song Q, Chen P and Liu XM: The role of cigarette smoke-induced pulmonary vascular endothelial cell apoptosis in COPD. Respir Res. 22:392021. View Article : Google Scholar : PubMed/NCBI
3	Devulder JV: Unveiling mechanisms of lung aging in COPD: A promising target for therapeutics development. Chin Med J Pulm Crit Care Med. 2:133–141. 2024. View Article : Google Scholar : PubMed/NCBI
4	Kaur M, Chandel J, Malik J and Naura AS: Particulate matter in COPD pathogenesis: An overview. Inflamm Res. 71:797–815. 2022. View Article : Google Scholar : PubMed/NCBI
5	Chen L, Zhu D, Huang J, Zhang H, Zhou G and Zhong X: Identification of hub genes associated with COPD through integrated bioinformatics analysis. Int J Chron Obstruct Pulmon Dis. 17:439–456. 2022. View Article : Google Scholar : PubMed/NCBI
6	Prieux R, Eeman M, Rothen-Rutishauser B and Valacchi G: Mimicking cigarette smoke exposure to assess cutaneous toxicity. Toxicol In Vitro. 62:1046642020. View Article : Google Scholar : PubMed/NCBI
7	Fischer BM, Voynow JA and Ghio AJ: COPD: Balancing oxidants and antioxidants. Int J Chron Obstruct Pulmon Dis. 10:261–276. 2015. View Article : Google Scholar : PubMed/NCBI
8	Woźniak A, Górecki D, Szpinda M, Mila-Kierzenkowska C and Woźniak B: Oxidant-antioxidant balance in the blood of patients with chronic obstructive pulmonary disease after smoking cessation. Oxid Med Cell Longev. 2013:8970752013. View Article : Google Scholar : PubMed/NCBI
9	Sies H and Jones DP: Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol. 21:363–383. 2020. View Article : Google Scholar : PubMed/NCBI
10	Barnes PJ: Oxidative stress in chronic obstructive pulmonary disease. Antioxidants (Basel). 11:9652022. View Article : Google Scholar : PubMed/NCBI
11	Cha SR, Jang J, Park SM, Ryu SM, Cho SJ and Yang SR: Cigarette smoke-induced respiratory response: Insights into cellular processes and biomarkers. Antioxidants (Basel). 12:12102023. View Article : Google Scholar : PubMed/NCBI
12	Paintlia MK, Paintlia AS, Contreras MA, Singh I and Singh AK: Lipopolysaccharide-induced peroxisomal dysfunction exacerbates cerebral white matter injury: Attenuation by N-acetyl cysteine. Exp Neurol. 210:560–576. 2008. View Article : Google Scholar : PubMed/NCBI
13	Aldini G, Altomare A, Baron G, Vistoli G, Carini M, Borsani L and Sergio F: N-Acetylcysteine as an antioxidant and disulphide breaking agent: The reasons why. Free Radic Res. 52:751–762. 2018. View Article : Google Scholar : PubMed/NCBI
14	Pedre B, Barayeu U, Ezeriņa D and Dick TP: The mechanism of action of N-acetylcysteine (NAC): The emerging role of H(2)S and sulfane sulfur species. Pharmacol Ther. 228:1079162021. View Article : Google Scholar : PubMed/NCBI
15	Millea PJ: N-acetylcysteine: Multiple clinical applications. Am Fam Physician. 80:265–269. 2009.PubMed/NCBI
16	Sanguinetti CM: N-acetylcysteine in COPD: Why, how, and when? Multidiscip Respir Med. 11:82016. View Article : Google Scholar : PubMed/NCBI
17	Messier EM, Day BJ, Bahmed K, Kleeberger SR, Tuder RM, Bowler RP, Chu HW, Mason RJ and Kosmider B: N-Acetylcysteine protects murine alveolar type II cells from cigarette smoke injury in a nuclear erythroid 2-related factor-2-independent manner. Am J Respir Cell Mol Biol. 48:559–567. 2013. View Article : Google Scholar : PubMed/NCBI
18	Cazzola M, Page CP, Wedzicha JA, Celli BR, Anzueto A and Matera MG: Use of thiols and implications for the use of inhaled corticosteroids in the presence of oxidative stress in COPD. Respir Res. 24:1942023. View Article : Google Scholar : PubMed/NCBI
19	Burkhardt BR, Lyle R, Qian K, Arnold AS, Cheng H, Atkinson MA and Zhang YC: Efficient delivery of siRNA into cytokine-stimulated insulinoma cells silences Fas expression and inhibits Fas-mediated apoptosis. FEBS Lett. 580:553–560. 2006. View Article : Google Scholar : PubMed/NCBI
20	Murphy MP, Bayir H, Belousov V, Chang CJ, Davies KJA, Davies MJ, Dick TP, Finkel T, Forman HJ, Janssen-Heininger Y, et al: Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab. 4:651–662. 2022. View Article : Google Scholar : PubMed/NCBI
21	Dang X, He B, Ning Q, Liu Y, Guo J, Niu G and Chen M: Alantolactone suppresses inflammation, apoptosis and oxidative stress in cigarette smoke-induced human bronchial epithelial cells through activation of Nrf2/HO-1 and inhibition of the NF-κB pathways. Respir Res. 21:952020. View Article : Google Scholar : PubMed/NCBI
22	Vijayan VK: Chronic obstructive pulmonary disease. Indian J Med Res. 137:251–269. 2013.PubMed/NCBI
23	Salvi SS and Barnes PJ: Chronic obstructive pulmonary disease in non-smokers. Lancet. 374:733–743. 2009. View Article : Google Scholar : PubMed/NCBI
24	Martinez FJ, Donohue JF and Rennard SI: The future of chronic obstructive pulmonary disease treatment-difficulties of and barriers to drug development. Lancet. 378:1027–1037. 2011. View Article : Google Scholar : PubMed/NCBI
25	Upadhyay P, Wu CW, Pham A, Zeki AA, Royer CM, Kodavanti UP, Takeuchi M, Bayram H and Pinkerton KE: Animal models and mechanisms of tobacco smoke-induced chronic obstructive pulmonary disease (COPD). J Toxicol Environ Health B Crit Rev. 26:275–305. 2023. View Article : Google Scholar : PubMed/NCBI
26	Henrot P, Blervaque L, Dupin I, Zysman M, Esteves P, Gouzi F, Hayot M, Pomiès P and Berger P: Cellular interplay in skeletal muscle regeneration and wasting: Insights from animal models. J Cachexia Sarcopenia Muscle. 14:745–757. 2023. View Article : Google Scholar : PubMed/NCBI
27	Mineur YS, Abizaid A, Rao Y, Salas R, DiLeone RJ, Gündisch D, Diano S, De Biasi M, Horvath TL, Gao XB and Picciotto MR: Nicotine decreases food intake through activation of POMC neurons. Science. 332:1330–1332. 2011. View Article : Google Scholar : PubMed/NCBI
28	Chen H, Hansen MJ, Jones JE, Vlahos R, Bozinovski S, Anderson GP and Morris MJ: Cigarette smoke exposure reprograms the hypothalamic neuropeptide Y axis to promote weight loss. Am J Respir Crit Care Med. 173:1248–1254. 2006. View Article : Google Scholar : PubMed/NCBI
29	Jomova K, Raptova R, Alomar SY, Alwasel SH, Nepovimova E, Kuca K and Valko M: Reactive oxygen species, toxicity, oxidative stress, and antioxidants: Chronic diseases and aging. Arch Toxicol. 97:2499–2574. 2023. View Article : Google Scholar : PubMed/NCBI
30	Boatright KM and Salvesen GS: Caspase activation. Biochem Soc Symp. 233–242. 2003.PubMed/NCBI
31	Ruaro B, Salton F, Braga L, Wade B, Confalonieri P, Volpe MC, Baratella E, Maiocchi S and Confalonieri M: The history and mystery of alveolar epithelial type II cells: Focus on their physiologic and pathologic role in lung. Int J Mol Sci. 22:25662021. View Article : Google Scholar : PubMed/NCBI
32	Han S, Lee M, Shin Y, Giovanni R, Chakrabarty RP, Herrerias MM, Dada LA, Flozak AS, Reyfman PA, Khuder B, et al: Mitochondrial integrated stress response controls lung epithelial cell fate. Nature. 620:890–897. 2023. View Article : Google Scholar : PubMed/NCBI
33	Baglole CJ, Bushinsky SM, Garcia TM, Kode A, Rahman I, Sime PJ and Phipps RP: Differential induction of apoptosis by cigarette smoke extract in primary human lung fibroblast strains: Implications for emphysema. Am J Physiol Lung Cell Mol Physiol. 291:L19–L29. 2006. View Article : Google Scholar : PubMed/NCBI
34	Ezeriņa D, Takano Y, Hanaoka K, Urano Y and Dick TP: N-Acetyl cysteine functions as a fast-acting antioxidant by triggering intracellular H(2)S and sulfane sulfur production. Cell Chem Biol. 25:447–459.e4. 2018. View Article : Google Scholar : PubMed/NCBI
35	Wu CY, Cilic A, Pak O, Dartsch RC, Wilhelm J, Wujak M, Lo K, Brosien M, Zhang R, Alkoudmani I, et al: CEACAM6 as a novel therapeutic target to boost HO-1-mediated antioxidant defense in COPD. Am J Respir Crit Care Med. 207:1576–1590. 2023. View Article : Google Scholar : PubMed/NCBI
36	Consoli V, Sorrenti V, Grosso S and Vanella L: Heme oxygenase-1 signaling and redox homeostasis in physiopathological conditions. Biomolecules. 11:5892021. View Article : Google Scholar : PubMed/NCBI
37	Lee DW, Gelein RM and Opanashuk LA: Heme-oxygenase-1 promotes polychlorinated biphenyl mixture aroclor 1254-induced oxidative stress and dopaminergic cell injury. Toxicol Sci. 90:159–167. 2006. View Article : Google Scholar : PubMed/NCBI
38	Lin CC, Yang CC, Hsiao LD, Chen SY and Yang CM: Heme oxygenase-1 induction by carbon monoxide releasing molecule-3 suppresses interleukin-1β-mediated neuroinflammation. Front Mol Neurosci. 10:3872017. View Article : Google Scholar : PubMed/NCBI
39	Baglole CJ, Sime PJ and Phipps RP: Cigarette smoke-induced expression of heme oxygenase-1 in human lung fibroblasts is regulated by intracellular glutathione. Am J Physiol Lung Cell Mol Physiol. 295:L624–L636. 2008. View Article : Google Scholar : PubMed/NCBI
40	Jiang X, Stockwell BR and Conrad M: Ferroptosis: Mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 22:266–282. 2021. View Article : Google Scholar : PubMed/NCBI