Oxidative Stress Triggers Itch-mediated TXNIP Degradation and NF-κB Activation Promoting Chronic Obstructive Pulmonary Disease

Oxidative Stress Triggers Itch-mediated TXNIP Degradation and NF-κB Activation Promoting Chronic Obstructive Pulmonary Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oxidative Stress Triggers Itch-mediated TXNIP Degradation and NF-κB Activation Promoting Chronic Obstructive Pulmonary Disease Pei-Yun Lin, Kang-Yun Lee, Shu-Chuan Ho, Hsiao-Chi Chuang, Bing-Hua Su, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6476972/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Oct, 2025 Read the published version in Respiratory Research → Version 1 posted 9 You are reading this latest preprint version Abstract Background Chronic inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), are characterized by pulmonary structural changes, narrowing of the small airways, and destruction of the lung parenchyma caused by prolonged inflammation. Sustained inflammation mediated by macrophages is considered to play a critical role in COPD pathogenesis, while the inductive mechanisms of persistent inflammation remain unclear. Methods In vitro, RAW264.7 cells were treated with cigarette smoke extract (CSE), hydrogen peroxide, and 12-O-tetradecanoylphorbol-13-acetate. Loss-of-function assays were performed using MAPK inhibitors and Itch-specific knockdown. In vivo, lung tissues from mice exposed to whole-body cigarette smoke for 12 weeks, as well as clinical samples from healthy non-smokers, a healthy smoker, and COPD patients, were analyzed. Results We revealed that thioredoxin-interacting protein (TXNIP) participates in cigarette smoke-incited NF-κB activation that potentially conducted pulmonary inflammation. CSE markedly inhibits TXNIP expression in RAW264.7 cells through MAPKs-dependent regulation, accompanied by the induction of iNOS/NO and COX-2. The decrease in TXNIP was also detected in lung tissues and macrophages obtained from smoking mice, while higher NF-κB activation and lung inflammation occurred simultaneously. Additionally, cigarette smoke-associated oxidative stress initiated the proteasomal degradation of TXNIP followed by the MAPKs-regulated NF-κB activation concurrently. The expression of E3 ligase Itch was elevated in smoking mouse lungs and in hydrogen peroxide-stimulated cells, whereas specific silencing Itch significantly attenuated TXNIP degradation as well as NF-κB activation. Moreover, TXNIP was distinctly suppressed in lung tissues, bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells obtained from patients with COPD. Conclusion Accordingly, cigarette smoke-induced oxidative stress causes Itch-mediated TXNIP degradation, leading to NF-κB inflammation and potentially enabling COPD pathogenesis. ROS TXNIP NF-κB Itch COPD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease characterized by pulmonary structural changes, narrowing of the small airways, and destruction of alveolar tissue. The most commonly encountered risk factor for COPD is tobacco usage and secondhand smoke exposure [ 1 – 3 ]. Cigarette smoke is a complex, dynamic and reactive mixture containing an estimated 7,000 chemicals, including reactive oxygen species (ROS) and nicotine [ 4 ]. Cigarette smoke-induced oxidative stress plays a vital role in causing inflammation, cell senescence, and DNA damage, which contribute to COPD pathogenesis, particularly during acute exacerbations. Increased oxidative stress activates various intracellular signaling pathways, such as the mitogen-activated protein kinases (MAPKs) and transcription factor nuclear factor (NF)-κB, which frequently interact with redox-sensitive molecular targets, thereby driving the induction of inflammatory mediators [ 5 , 6 ]. So far, the effective molecular targeting of chronic and prolonged inflammation in the treatment of COPD has been widely considered. Therefore, the underlying mechanisms that control sustained inflammation need to be investigated more thoroughly. Thioredoxin-interacting protein (TXNIP) has been originally identified as a differentially expressed gene in 1α,25-dihydroxyvitamin D3-treated HL-60 cells, also called as vitamin D3 upregulated protein 1 [ 7 – 9 ]. Upregulation of TXNIP is associated with diseases such as diabetes mellitus, ischemic stroke, cardiovascular diseases, and neurodegenerative disorders, while reduced TXNIP expression often correlates with the onset of tumors [ 8 – 12 ]. Additionally, as a member of the α-arrestin protein family, TXNIP can function as a scaffold protein (independent of its binding to TRX) in specific subcellular compartments to enhance the formation of the NLR protein 3 (NLRP3) inflammasome, promoting interleukin (IL)-1β production under oxidative stress [ 13 ], and regulate the nuclear export and degradation of hypoxia-inducible factor 1-α (HIF1-α) during hypoxia [ 14 , 15 ]. Accordingly, the protein stability and gene expression of TXNIP can be regulated in response to multiple stimuli, subsequently contributing to signaling cascades that affect various biological processes. TXNIP expression has been reported to potentially affect the development of inflammation-related lung disorders. The induction of TXNIP-mediated NLRP3 inflammasome and pyroptosis contributes to acute lung inflammation and injury in septic mice [ 16 , 17 ]. Moreover, elevated TXNIP-regulated apoptosis facilitates titanium dioxide nanoparticle-exacerbated allergic airway inflammation in an ovalbumin-induced mouse model of asthma [ 18 ]. TXNIP overexpression in memory T helper (Th) 2 cells presents enhanced memory responses, including the increases of eosinophils infiltration, Th2 cytokines, and allergic airway inflammation in mice [ 19 ]. In addition, bleomycin-induced lung fibrosis develops alongside increased TXNIP/HIF1-α expression and oxidative stress in the lung tissues of rats [ 20 ]. The pathogenesis of inflammatory lung disorders is heterogeneous, and the role of TXNIP in regulating the induction of inflammation, oxidative stress, and lung disease progression, requires more investigation. Despite the established involvement of oxidative stress and the P2X7/inflammasome pathway in the development and exacerbation of cigarette smoke-induced COPD [ 5 , 21 ], the role of TXNIP in regulating cigarette smoke-associated lung inflammation and its molecular mechanisms remain unclear. In this study, we demonstrated that exposure to cigarette smoke resulted in a decrease in TXNIP levels in macrophages and murine lungs through a mechanism involving the generation of ROS and the activation of MAPKs. Oxidative stress induced the expression of the Itch E3 ligase, which regulated TXNIP for degradation and subsequently led to the activation of NF-κB. Further inhibition of Itch partially mitigated TXNIP degradation and NF-κB activation. Moreover, the Txnip gene exhibited relatively lower expression, while the Itch gene showed relatively higher expression in alveolar macrophages from smokers compared to non-smokers. TXNIP was significantly suppressed in the lung tissues, bronchoalveolar lavage fluid (BALF) cells, and peripheral blood mononuclear cells (PBMC) of COPD patients. This suggests that cigarette smoke-induced oxidative stress may trigger Itch-regulated TXNIP degradation, ultimately resulting in the activation of NF-κB-mediated inflammation that potentially contributes to the development of COPD. Materials and Methods Cell cultures and reagents Murine RAW264.7 macrophages (#ATCC TIB-71 TM ) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (#11965084, Gibco, Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS) (#04-001-1A, Biological Industries, Kibbutz Beit Haemek, Israel) at 37°C in a humidified atmosphere of 95% air and 5% CO2. MG132 (#1748), Lactacystin (#2267), PD98059 (#1213), SP600125 (#1496), SB203580 (#1202) are purchased from TOCRIS Bioscience (Bristol, UK). Hydrogen peroxide (#H1099), N-Acetyl-L-cysteine (NAC, #A7250), 12-O-Tetradecanoylphorbol 13-acetate (TPA, #P8139), and 4,6-diamidino-2-phenylindole (DAPI, #D9542) were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against phospho-ERK1/2 (Thr202/Tyr204) (#9101S), ERK1/2 (#9102S), phospho-JNK (Thr183/Tyr185) (#9251), JNK (#9252), phospho-p38 (Thr180/Tyr182) (# 9211), p38 (#9212), phospho-NF-kB (#3033), Itch (#12117), iNOS (#13120), COX-2 (#12282S) and the secondary antibodies HRP-conjugated goat anti-mouse (#7076S) and goat anti-rabbit (#7074S) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against TXNIP (#K0205-3) were purchased from MBL International Corporation (Woburn, MA, USA). Antibodies against hydroxynonenal (#ab46545) and nitrotyrosine (#ab125106) were purchased from Abcam (Cambridge, MA, USA), and GAPDH (#SC-32233) and β-actin (#SC-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Alexa Fluor 488- and 594-conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Smoking mouse and cigarette smoke extract Smoking mice were performed according to previous study [22]. In brief, six-week-old male BALB/c mice obtained from National Laboratory Animal Center (Taipei, Taiwan) were maintained on standard laboratory food and water ad libitum in the animal centers at the Taipei Medical University. All animal studies were performed in accordance with the rules of the Animal Protection Act of Taiwan, and the animal use protocols were approved by the Laboratory Animal Care and Use Committee of Taipei Medical University, Taiwan (LAC-2014-0168). Mice were whole-body exposed to cigarette smoke by a cigarette smoke chamber with the main-stream smoke from the combustion of 12 reference cigarettes (3R4F; Tobacco and Health Research Institute, KY, USA) for a period of approximately 50 minutes/day, 5 days/week, for 12 weeks. Control mice were exposed to cigarette smoke-free HEPA-filtered air. Cigarette smoke extract (CSE) was prepared according to previous study with the slight modification [23]. Two filtered cigarettes (Marlboro MX, tar: 10 mg, nicotine content: 0.8 mg; Philip Morris, Switzerland) was imported a vessel containing 6 ml medium by a vacuum pump. Freshly prepared CSE were next sterilized by passing through a 0.22-μm filter and recognized as 100% concentrations of CSE. Clinical samples Clinal samples including lung tissue, PBMC, and BALF were obtained from healthy non-smokers, a healthy smoker, and COPD patients. Patients with COPD were diagnosed and graded according to the guidelines of the Global Initiative for Obstructive Lung Disease [24], and the details of COPD patients were summarized in our previous study [22]. The clinical study protocol was approved by Taipei Medical University-Joint Institutional Review Board (TMU-JIRB No. 201310027) and performed in accordance with the relevant guidelines and regulations. The lung tissues were obtained from COPD patients with lung surgery for the peripheral lung tumor removal, whereas normal control tissues were derived from the noninvolved lung segments of the tumor lesion from non-COPD patients. Generation of Itch shRNA To stably express a lentivirus-based short hairpin RNA (shRNA) targeting Itch, TRCN0000026925 (5′-CCACCTGAAATACTTTCGTTT-3′), TRCN0000026908 (5′- CCCTACGAGTAAATTATGTTT-3′), and TRCN0000026914 (5′-GCGAAGGAATTAGAGGTTCTT-3′) were obtained from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan) followed by the preparation of lentiviral mouse Itch shRNAs from the RNAi Core of Research Center of Clinical Medicine (National Cheng Kung University Hospital, Taiwan). TRCN0000072247 (5′- GAATCGTCGTATGCAGTGAAA-3′) was used as the control luciferase shRNA (shLuc). RAW 264.7 cells were subsequently infected with an appropriate MOI for 24 h followed by puromycin (Calbiochem, San Diego, CA) selection. The protein expression was then measured by western blot analysis Western blot analysis The total proteins were extracted from RAW264.7 cells, BALF pellets, PBMCs, and mouse lung tissue homogenates using a Triton X-100 based lysis buffer with a protease inhibitor mix and a phosphatase-inhibitors cocktail I followed by the centrifugation at 13300 rpm for 10 min. Proteins were then resolved using SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). After blocking, the membranes were probed with the indicated primary antibodies (1:1000 dilution) followed by secondary antibodies (1:5000 dilution), and developed by an ECL Western blot detection kit (Pierce Chemical, Rockford, IL, USA) according to the manufacturer’s instructions. All immunoblotting studies were performed in at least two independent experiments, and the relative band intensity on the blots was quantified using Image J software (NIH, Bethesda, MD, USA). Histological analysis and immunostaining Lung tissues obtained from patients and smoking mice were fixed in 10% neutral-buffered formalin, embedded in paraffin wax, and sliced. For histopathology, sections (5 μm) were stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, lung tissues were deparaffinized and rehydrated with xylene and different concentrations of ethanol. After permeabilized with 0.1% Triton X-100 in PBS and incubated with 3% hydrogen peroxide, sections were blocked (1% BSA + 0.1% azide in PBS) and stained with specific antibodies against phospho-NF-kB (Ser536), TXNIP, CD11b (clone M1/70, BioLegend San Diego, CA, USA), hydroxynonenal, and nitrotyrosine followed by HRP- or Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody staining. RAW264.7 cells were fixed by 4% paraformaldehyde in PBS and permeabilized. Fixed cells were then stained with specific antibodies against phospho-NF-kB (Ser536) and TXNIP followed by secondary antibody staining. Hematoxylin and DAPI (5 μg/ml) were used for nuclear staining. Images were captured by using fluorescence microscopy (EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA) and LEICA TCS SP5 laser scanning confocal microscopy system (Leica, Heidelberg, Germany). NO and ROS detection Nitrite accumulation in the cell culture medium was used as an indicator of NO production, detected by the Griess reaction. Briefly, supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4) and incubated for 10 min at room temperature. The relative optical density (OD) of nitrite was measured at 540nm, and the concentration was evaluated by using sodium nitrite as a standard. For ROS detection, cells were treated with or without TPA and then co-incubated with 20 μM carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA, C6827, Thermo Scientific) fluoroprobe for 30min at 37°C in the dark. After washing, cells were collected and analyzed using flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA) with the excitation at 488 nm. The emission was detected with the FL-1 channel followed by CellQuest Pro 4.0.2 software (BD Biosciences) analysis, and quantification was performed using FlowJo software (Tree star, Inc., Ashland, Or, USA). The percentages of ROS-positive cells each group were shown. NF-κB reporter assay Expression vectors of pNF-κB-Luc plasmid (Stratagene, La Jolla, CA) and cytomegalovirus-Renilla luciferase construct (pRL-CMV) (Promega, Madison, WI) were transiently cotransfected into cells for 24 h using lipofectamine reagents (Invitrogen, Carlsbad, CA). The Renilla-derived luciferase reporter plasmid was used for transfection efficiency control. In the presence or absence of inhibitors treated for 1 h, cells were stimulated with H 2 O 2 for 4 h followed by the detection of firefly luciferase activity using Dual Luciferase® Reporter assay system (Promega, Madison, WI, USA) and the multimode reader (Varioskan Flash, Thermo Scientific) according to the manufacturer’s instructions. Statistical analysis Statistical analyses were performed using Student’s t-test (two groups) or one-way ANOVA (more than two groups) followed by a Tukey’s multiple comparison test with Prism 7.0 (GraphPad). The data are presented as the mean ± standard error of the mean (SEM) from three independent experiments. Statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001. Results Cigarette smoke extract initiates TXNIP reduction and iNOS expression TXNIP is involved in a wide variety of cellular processes. Overexpression of TXNIP enables cell apoptosis while TXNIP deficiency causes tumorigenesis and the exacerbation of endotoxic shock [25-27]. To investigate whether TXNIP participates in cigarette smoke-mediated inflammation, TXNIP expression was measured in cigarette smoke extract (CSE) stimulation. Freshly prepared CSE markedly attenuated TXNIP expression, accompanied by the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in murine RAW264.7 cells (Fig. 1A) . CSE significantly induced nitric oxide (NO) production in a dose-dependent manner (Fig. 1B) , while showing no significant induction of cytotoxicity. Similar to previous reports [28], CSE could effectively induce JNK, p38 MAPK, and ERK phosphorylation in RAW264.7 cells (Fig. 1C) . Moreover, the presence of JNK and p38 MAPK inhibitors, SP600125 and SB203580, markedly alleviated CSE-caused TXNIP reduction respectively (Fig. 1D) . TXNIP has been linked to regulate stress-activated apoptosis and inflammation through its cellular expression and distribution [9]. Interestingly, TXNIP was distinctly suppressed instead of being induced in coordination with the elevation of inflammatory mediators in CSE stimulation. This might suggest a novel role for TXNIP in cigarette smoke-regulated inflammation. Decreased TXNIP expression in the lungs of smoking mice Down-regulated TXNIP expression was observed in CSE-stimulated murine macrophage RAW264.7 cells. Subsequently, changes in pulmonary TXNIP expression in smoking mice were investigated. Following 12 weeks of cigarette smoke exposure, the mice exhibited significant immune cell infiltration in the lungs and abnormal enlargement of airspaces (Fig. 2A) . NF-κB activation was determined in the lungs of smoking mice, revealing elevated phosphorylation of p65 NF-κB at serine 536 (Fig. 2B) . This indicated that cigarette smoke exposure could effectively trigger NF-κB-regulated inflammation. Further assessments of TXNIP expression revealed significant decreases in TXNIP levels in the lung tissues of smoking mice compared to normal mice (Fig. 2C) . Additionally, TXNIP expression was intensely suppressed in lung macrophages expressing CD11b (Fig. 2D) . Consistent with previous results, the downregulation of TXNIP might potentially accompany NF-κB activation in macrophages, leading to pulmonary inflammation induced by cigarette smoke stimulation. Oxidative stress-regulated proteasomal degradation of TXNIP Cigarette smoke represents one of the most significant exogenous oxidants, containing abundant ROS, hydrogen peroxide (H 2 O 2 ), and NO that contribute to oxidative stress-mediated inflammation [29]. Given the marked downregulation of TXNIP in the lung tissue of smoking mice, we subsequently explored whether TXNIP expression is regulated by oxidative stress. The distinct elevations of a sensitive marker of oxidative damage and lipid peroxidation, 4-hydroxynonenal (4-HNE), and a versatile oxidative stress biomarker, nitrotyrosine (NitroTyo), were detected in smoking murine lung tissues by immunofluorescence staining (Fig. 3A) and western blot analysis (Fig. 3B) . These results suggested the potential coordination between TXNIP downregulation and oxidative stress. Exogeneous treatment of H 2 O 2 showed a dose-dependent TXNIP downregulation in RAW264.7 cells (Fig. 3C) . The presence of proteasome inhibitors, MG132 and lactacystin (LAC), distinctly reversed H 2 O 2 -caused TXNIP downregulation (Fig. 3D) , which suggested that oxidative stress might initiate TXNIP undergoing proteasomal degradation. Furthermore, the endogenous ROS induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) (Fig. 3E) similarly induced TXNIP degradation in RAW264.7 cells (Fig. 3F) , while the ROS inhibitor, N-acetylcysteine (NAC), markedly attenuated TPA-induced TXNIP degradation (Fig. 3G) . In addition, the various concentrates of freshly prepared CSE we used showed no significant cytotoxicity in RAW264.7 cells (Fig. 3H) . Notably, the presence of MG132, LAC, and NAC similarly attenuated the CSE-induced TXNIP degradation (Fig. 3I) . Therefore, cigarette smoking might induce oxidative stress-regulated TXNIP proteasomal degradation. ROS mediates MAPKs-regulated TXNIP degradation and NF- kB activation NF-kB is a critical transcriptional factor of the inflammatory process, while H 2 O 2 has been revealed as a fine-tuning regulator of NF-kB-dependent inflammation [30]. In H 2 O 2 -stimulated RAW264.7 cells, the nuclear translocation of NF-kB was significantly increased (Fig. 4A) . Furthermore, NF-κB nuclear translocation occurred concurrently with TXNIP degradation in the cells (Fig. 4B) . H 2 O 2 treatment effectively induced TXNIP degradation, which was in associated with the phosphorylation of JNK, p38 MAPK, and ERK (Fig 4C) . Pharmacological inhibition of MAPKs using specific inhibitors, SP600125, PD98059, and SB203580, which inhibited JNK, ERK, and p38 MAPK, respectively, suppressed H2O2-induced TXNIP degradation (Fig. 4D) , as well as subsequent NF-κB activation (Fig. 4E) . Accordingly, ROS-induced NF-κB activation may be regulated by MAPKs-mediated TXNIP degradation. Itch expression modulates TXNIP-mediated NF- kB activation TXNIP has been shown to interact with the ubiquitin E3 ligase Itch through a conserved PPXY motif in the C terminus of TXNIP and undergoes degradation [31]. Itch expression is initially slightly increased by ROS stimulation, but subsequently decreases following TXNIP suppression in cardiomyocytes [32]. Similarly, we observed a slight increase in the expression of Itch in lung tissue extracted from smoking mice (Fig. 5A) . H 2 O 2 and TPA could stimulate Itch induction in RAW264.7 cells (Fig. 5B) , while TPA-elevated Itch expression was partly inhibited by the presence of NAC (Fig. 5C) . ROS accumulation upon exposure to cigarette smoke might potentiate Itch induction accordingly. Since TXNIP degradation may occur in accordance with Itch expression, we further explored whether the specific knockdown of Itch might affect ROS-induced TXNIP degradation. By using RNA interference, Itch expression was specifically silenced in RAW264.7 cells (Fig. 5D) . H 2 O 2 -induced TXNIP degradation was markedly reversed in Itch knockdown RAW264.7 cells compared to the controls (Fig. 5E) . Moreover, H 2 O 2 -mediated NF-κB activation was significantly attenuated in Itch knockdown cells accompanying by the stabilization of TXNIP (Fig. 5F) . Therefore, ROS-mediated upregulation of Itch could potentially lead to TXNIP degradation, resulting in subsequent NF-κB activation in response to cigarette smoke exposure. Decreased TXNIP levels in COPD patients TXNIP protein expression exhibits distinct variations in different cells response to ROS stimulation [33,34], where we observed ROS-mediated TXNIP suppression in lung tissues of smoking mice as well as in murine macrophages. Exploration of gene profiles obtained from DataSet Record GDS3496 and GDS737 on Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) revealed that the significant relative lower expression of TXNIP and higher expression of Itch were shown in alveolar macrophages of cigarette smokers (n=13) compared to nonsmokers (n=11), while no significant changes of TXNIP and Itch were found in lung tissues from smokers with severe emphysema (n=18) compared to no or mild emphysema (n=12) (Fig. 6A) . To further confirm the expression status of TXNIP in oxidative stress-associated lung diseases, we collected lung tissues from non-COPD and COPD patients undergoing lung surgery for peripheral lung tumor removal. We found that TXNIP was markedly suppressed in lung tissues with COPD compared to non-COPD patients (Fig. 6B) . Similarly, decreased TXNIP expression was observed in BAL cells from COPD patients (Fig. 6C) . Furthermore, PBMCs obtained from healthy non-smokers, healthy smokers, and COPD patients revealed significantly greater inhibition of TXNIP in mild and severe COPD patients compared to healthy donors (Fig. 6D) . Accordingly, cigarette smoke exposure-mediated oxidative stress could induce Itch- and MAPKs-regulated TXNIP degradation, leading to NF-κB activation and subsequent inflammatory induction (Fig. 7) . Additionally, the decreased TXNIP levels observed in COPD patients suggested its potential role in regulating disease progression. Discussion The burden of cigarette smoke-induced oxidative stress, cellular damage, and inflammation are major contributors to COPD pathogenesis [ 35 ]. TXNIP possesses pro-oxidative and pro-inflammatory characteristics, leading to the initiation or exacerbation of inflammation and cellular damages in various disease progressions [ 36 , 37 ]. TXNIP induction has also been reported to play a role in the development of inflammation-related lung disorders, including acute lung injury, allergic exacerbation, and lung fibrosis [ 16 – 20 ]. In this study, we demonstrate that TXNIP reduction is associated with CSE-induced inflammation in RAW264.7 cells and lung inflammation in smoking mice. Cigarette smoke-mediated oxidative stress leads to proteasomal degradation of TXNIP, subsequently activating MAPKs-regulated NF-κB. Interestingly, upon cigarette smoke exposure and oxidative stimulation, the expression of Itch, the upstream E3 ubiquitin ligase of TXNIP, increases. Inhibition of Itch significantly attenuates TXNIP degradation and NF-κB activation. Furthermore, lung tissues, BALF cells, and PBMC obtained from COPD patients exhibit marked TXNIP suppression compared to healthy donors. Contrary to an increase, TXNIP is markedly suppressed through Itch regulation in response to cigarette smoke -mediated oxidative stress, subsequently activating MAPKs and NF-κB inflammation. The expression of TXNIP is often triggered by multiple stimuli, such as hyperglycemia, ischemia-reperfusion injury, hypoxia, ER stress, and ROS. Under stress conditions, increased TXNIP may either promote apoptotic signal-regulated kinase 1-mediated mitochondrial apoptosis through TRX-dependent signaling [ 25 , 37 ], or regulate TXNIP/NLRP3 inflammasome activation in a redox-independent fashion [ 13 , 36 ]. On the other hand, TXNIP degradation has been shown to contribute to TNF-α-stimulated NF-κB activation, while TXNIP deficiency exacerbates lipopolysaccharide (LPS)-induced endotoxic shock and mortality induced by E. coli infection through excessive NO production [ 27 , 38 ]. Moreover, toll-like receptor (TLR) 2- and ROS-mediated rapid TXNIP degradation in macrophages has shown to potentially expedite NF-κB activation in Streptococcus pyogenes infection [ 39 ]. Here, we found that CSE stimulation mediated dose-dependent degradation of TXNIP, accompanied by the induction of iNOS/NO and COX-2 in macrophages. The suppression of TXNIP could further promote MAPKs and NF-κB activation. Moreover, a significant increase in the development of emphysema has been found in USP13-deficient mice after mild CS exposure, where the reduction of TXNIP is also observed in lung epithelial cells with USP13 inhibition [ 40 ]. However, other reports have indicated that CSE upregulates TXNIP expression, and triggers TXNIP-NLRP3-gasdermin D axis to promote inflammation and pyroptosis of islet β-cells [ 41 , 42 ]. So far, the protein stability and functions of TXNIP in response to cigarette smoke stimuli remain controversial and appear to exhibit cell-type-specific regulation accordingly. Itch, a HECT-type ubiquitin E3 ligase, can interact with TXNIP and regulate its ubiquitin labeling, promoting proteasomal degradation [ 31 , 32 , 43 ]. Itch has long been respected as a critical suppressor of inflammation, which limits Th2 immunity by regulating T cells, B cells, and macrophages. Itch deficiency presents abnormal Th2-related lung and skin inflammation and spontaneous gastrointestinal tract inflammation in mice, along with immune abnormalities in humans, while Itch overexpression has been observed in several human cancers [ 44 , 45 ]. The promotion or repression of Itch activity is regulated by the phosphorylation status in response to various stimulations of growth factors, death receptors, DNA damage, and oxidative stress [ 46 – 48 ]. In addition to post-translational modification, ROS-mediated increased expression of Itch has also been reported to trigger the degradation of FLICE-like inhibitory protein [ 49 ]. Upon cigarette smoke and ROS stimulation, we found that increased Itch could be detected in lung tissues from smoking mice and in RAW264.7 cells, along with the TXNIP degradation. Additionally, GEO profiles similarly indicated that alveolar macrophages of smokers exhibit higher Itch gene expression than non-smokers, even though Itch gene expression seemed to show no significant changes in association with emphysema progression. Furthermore, ROS-induced Itch expression effectively contributed to downstream NF-κB activation, suggesting the potential proinflammatory role of Itch in cigarette smoke exposure. Chronic inflammation induced by abundant ROS stimulation and MAPKs activation is usually the major cause leading to consistent alveolar macrophage activation and alveolar epithelial cell damage in COPD pathogenesis [ 5 , 6 , 50 ]. Targeting oxidative stress with therapeutic agents has been recommended as an effective approach in treating COPD, and several antioxidants, including NAC, have been applied in clinical trials with some beneficial outcomes [ 50 ]. In addition to its well-known function of elevating intracellular glutathione levels [ 5 , 50 ], we demonstrated here that NAC has the capacity to stabilize the ROS-regulated Itch/TXNIP/NF-κB axis, thereby attenuating inflammation. Meanwhile, targeting MAPK signaling is also speculated as another efficient approach for treating inflammatory lung diseases, with clinical trials showing the therapeutic potential of p38 MAPK inhibitors in improving lung function and reducing exacerbations in COPD patients [ 51 , 52 ]. Here, we investigated that ROS-initiated Itch/TXNIP/NF-κB axis was partly regulated by the activation of JNK, p38 MAPK, and ERK, while the blockage of MAPKs could potentially attenuate NF-κB activation. Given the heterogeneity of COPD pathogenesis, both antioxidants and MAPK inhibitors exhibit only limited therapeutic potential. Therefore, combining these treatments with other anti-inflammatory medications appears to be a promising future therapeutic approach. TXNIP expression varies across different types of respiratory disorders. In lung cancer, TXNIP expression is significantly reduced. The downregulation of TXNIP promotes tumor proliferation and migration while inhibiting apoptosis in lung cancer cell lines [ 12 ]. In allergic airway inflammation and lung fibrosis, elevated TXNIP levels contribute to Th2 immune responses in the airway and oxidative stress in fibrotic lungs, respectively [ 19 , 20 ]. In this study, we first revealed the downregulation of TXNIP associated with cigarette smoke-induced oxidative stress and the development of inflammation in the lung tissue of smoking mice and COPD patients. Although P2X7/caspase-1 activation has been reported to be involved in cigarette smoke-induced lung inflammation in mice and smoking donors [ 21 ], whether TXNIP plays a role in cigarette smoke-mediated inflammasome activation remains unclear. Moreover, we have observed the ROS/Itch/TXNIP axis expressed upstream of NF-κB activation, potentially providing another pathway for the initiation of NF-κB, which is considered to be of paramount importance in COPD inflammation. However, due to the limited number of clinical samples, the expression and functions of TXNIP and Itch in COPD pathogenesis still require further investigation. Conclusion In summary, we have demonstrated that cigarette smoke-induced oxidative stress triggers Itch expression and MAPK activation, leading to TXNIP degradation in macrophages. Itch- and MAPK-dependent TXNIP proteasomal degradation facilitates NF-κB activation and inflammatory responses, potentially contributing to COPD pathogenesis. Stabilizing TXNIP using a ROS scavenger or Itch-specific knockdown can attenuate NF-κB-mediated inflammation, suggesting the potential therapeutic target for COPD treatment. Declarations Conflict of interests statement : The authors declare that they have no known conflict of interests. Acknowledgements We extend our gratitude to Taipei Medical University-Joint Institutional Review Board and the Laboratory Animal Center, Taipei Medical University, for their technical assistance. Additionally, we are thankful for the human samples and animals that contributed to this study. Authors’ contributions P.-Y. Lin and C.-L. Chen developed the concepts, conducted the experiment, analyzed the data, and drafted the manuscript. C.-L. Chen also designed the experiment, interpreted the data, and revised the manuscript. K.-Y. Lee, S.-C. Ho, H.-C. Chuan, B.-H. Su, and R. Satria assisted with material preparation, provided resources, and reviewed the manuscript. P.-C. Tseng, Y.-J. Wu, T.-T. Tsai, and F.-C. Shih assisted in conducting the experiment and analyzing the data. All authors have read and approved the final manuscript. Funding information This work was funded by the Ministry of Science and Technology of Taiwan (MOST 111-2314-B-038-120-MY3, NSTC 113-2320-B-038-003, and Taipei Medical University (DP2-TMU-112-T-05). Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All experiment procedures and animals were in compliance with the animal and ethics review committee of the Laboratory Animal Center at Taipei Medical University, Taipei, Taiwan. Consent for publication Not available. Competing Interest The authors declare that they have no known conflict of interests. References Christenson SA, Smith BM, Bafadhel M, et al. Chronic obstructive pulmonary disease. Lancet 2022; 399: 2227-2242. Nguyen JMK, Robinson DN, Sidhaye VK. Why new biology must be uncovered to advance therapeutic strategies for chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol 2021; 320: L1-l11. Brandsma CA, Van den Berge M, Hackett TL, et al. Recent advances in chronic obstructive pulmonary disease pathogenesis: from disease mechanisms to precision medicine. J Pathol 2020; 250: 624-635. Borgerding M, Klus H. Analysis of complex mixtures--cigarette smoke. Exp Toxicol Pathol 2005; 57 Suppl 1: 43-73. 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SUV39H1 Reduction Is Implicated in Abnormal Inflammation in COPD. Sci Rep 2017; 7: 46667. Su BH, Tseng YL, Shieh GS, et al. Prothymosin α overexpression contributes to the development of pulmonary emphysema. Nat Commun 2013; 4: 1906. Halpin DMG, Criner GJ, Papi A, et al. Global Initiative for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease. The 2020 GOLD Science Committee Report on COVID-19 and Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 2021; 203: 24-36. Chen CL, Lin CF, Chang WT, et al. Ceramide induces p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated pathway. Blood 2008; 111: 4365-4374. Sheth SS, Bodnar JS, Ghazalpour A, et al. Hepatocellular carcinoma in Txnip-deficient mice. Oncogene 2006; 25: 3528-3536. Park YJ, Yoon SJ, Suh HW, et al. TXNIP deficiency exacerbates endotoxic shock via the induction of excessive nitric oxide synthesis. PLoS Pathog 2013; 9: e1003646. Lin CC, Lee IT, Yang YL, et al. Induction of COX-2/PGE(2)/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation. Free Radic Biol Med 2010; 48: 240-254. Taniguchi A, Tsuge M, Miyahara N, et al. Reactive Oxygen Species and Antioxidative Defense in Chronic Obstructive Pulmonary Disease. Antioxidants (Basel) 2021; 10. Oliveira-Marques V, Marinho HS, Cyrne L, et al. Role of hydrogen peroxide in NF-kappaB activation: from inducer to modulator. Antioxid Redox Signal 2009; 11: 2223-2243. Zhang P, Wang C, Gao K, et al. The ubiquitin ligase itch regulates apoptosis by targeting thioredoxin-interacting protein for ubiquitin-dependent degradation. J Biol Chem 2010; 285: 8869-8879. Otaki Y, Takahashi H, Watanabe T, et al. HECT-Type Ubiquitin E3 Ligase ITCH Interacts With Thioredoxin-Interacting Protein and Ameliorates Reactive Oxygen Species-Induced Cardiotoxicity. J Am Heart Assoc 2016; 5. Zaragoza-Campillo MA, Morán J. Reactive Oxygen Species Evoked by Potassium Deprivation and Staurosporine Inactivate Akt and Induce the Expression of TXNIP in Cerebellar Granule Neurons. Oxid Med Cell Longev 2017; 2017: 8930406. Ogata FT, Batista WL, Sartori A, et al. Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization. PLoS One 2013; 8: e84588. Hikichi M, Mizumura K, Maruoka S, et al. Pathogenesis of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke. J Thorac Dis 2019; 11: S2129-s2140. Mohamed IN, Li L, Ismael S, et al. Thioredoxin interacting protein, a key molecular switch between oxidative stress and sterile inflammation in cellular response. World J Diabetes 2021; 12: 1979-1999. Choi EH, Park SJ. TXNIP: A key protein in the cellular stress response pathway and a potential therapeutic target. Exp Mol Med 2023; 55: 1348-1356. Kelleher ZT, Sha Y, Foster MW, et al. Thioredoxin-mediated denitrosylation regulates cytokine-induced nuclear factor κB (NF-κB) activation. J Biol Chem 2014; 289: 3066-3072. Tseng PC, Kuo CF, Cheng MH, et al. HECT E3 Ubiquitin Ligase-Regulated Txnip Degradation Facilitates TLR2-Mediated Inflammation During Group A Streptococcal Infection. Front Immunol 2019; 10: 2147. Gregory AD, Tran KC, Tamaskar AS, et al. USP13 Deficiency Aggravates Cigarette-smoke-induced Alveolar Space Enlargement. Cell Biochem Biophys 2021; 79: 485-491. Sun Q, Xu H, Xue J, et al. MALAT1 via microRNA-17 regulation of insulin transcription is involved in the dysfunction of pancreatic β-cells induced by cigarette smoke extract. J Cell Physiol 2018; 233: 8862-8873. Xu W, Wang H, Sun Q, et al. TXNIP-NLRP3-GSDMD axis-mediated inflammation and pyroptosis of islet β-cells is involved in cigarette smoke-induced hyperglycemia, which is alleviated by andrographolide. Environ Toxicol 2024; 39: 1415-1428. Liu Y, Lau J, Li W, et al. Structural basis for the regulatory role of the PPxY motifs in the thioredoxin-interacting protein TXNIP. Biochem J 2016; 473: 179-187. Field NS, Moser EK, Oliver PM. Itch regulation of innate and adaptive immune responses in mice and humans. J Leukoc Biol 2020; 108: 353-362. Yin Q, Wyatt CJ, Han T, et al. ITCH as a potential therapeutic target in human cancers. Semin Cancer Biol 2020; 67: 117-130. Melino G, Gallagher E, Aqeilan RI, et al. Itch: a HECT-type E3 ligase regulating immunity, skin and cancer. Cell Death Differ 2008; 15: 1103-1112. Stagni V, Santini S, Barilà D. ITCH E3 ligase in ATM network. Oncoscience 2014; 1: 394-395. Perez JM, Chirieleison SM, Abbott DW. An IκB Kinase-Regulated Feedforward Circuit Prolongs Inflammation. Cell Rep 2015; 12: 537-544. Paul T, Roy R, Sarkar RD, et al. H(2)O(2) mediated FLIP and XIAP down-regulation involves increased ITCH expression and ERK-Akt crosstalk in imatinib resistant Chronic Myeloid Leukemia cell line K562. Free Radic Biol Med 2021; 166: 265-276. Dailah HG. Therapeutic Potential of Small Molecules Targeting Oxidative Stress in the Treatment of Chronic Obstructive Pulmonary Disease (COPD): A Comprehensive Review. Molecules 2022; 27. Ahmadi A, Ahrari S, Salimian J, et al. p38 MAPK signaling in chronic obstructive pulmonary disease pathogenesis and inhibitor therapeutics. Cell Commun Signal 2023; 21: 314. Saleem S. Targeting MAPK signaling: A promising approach for treating inflammatory lung disease. Pathol Res Pract 2024; 254: 155122. Additional Declarations No competing interests reported. Supplementary Files 2025LinetalCSSupplementaryFile.pdf floatimage1.jpeg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 17 Oct, 2025 Read the published version in Respiratory Research → Version 1 posted Editorial decision: Revision requested 11 Jul, 2025 Reviews received at journal 11 Jul, 2025 Reviewers agreed at journal 14 Jun, 2025 Reviews received at journal 26 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviewers invited by journal 28 Apr, 2025 Editor assigned by journal 25 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 18 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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07:38:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6476972/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6476972/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12931-025-03369-5","type":"published","date":"2025-10-17T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82144929,"identity":"dc11189d-9223-4087-9aeb-b19685cf3acc","added_by":"auto","created_at":"2025-05-07 06:49:25","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCSE initiates TXNIP downregulation. (A)\u003c/strong\u003e RAW264.7 cells were treated with indicated dosages of cigarette smoke extract (CSE) for 24 h, the expressions of TXNIP, iNOS, and COX-2 were detected. GAPDH was used as an internal control. \u003cstrong\u003e(B)\u003c/strong\u003e NO release was detected after 24 h CSE exposure. \u003cem\u003e***\u003c/em\u003ep \u0026lt; 0.001 vs the untreated group. \u003cstrong\u003e(C)\u003c/strong\u003e The expression of phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, and p38 after 2 h CSE exposure were detected by western blot analysis. GAPDH was used as an internal control. \u003cstrong\u003e(D)\u003c/strong\u003e Cells were pretreated with or without SP600125 (SP), SB203580 (SB), and PD98059 (PD) for 1 h followed by CSE exposure for 24 h. The expression of TXNIP and GAPDH was detected. Protein molecular weights are indicated as kilodaltons (kDa).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/1e2c830b5931f27212de63b9.jpeg"},{"id":82143418,"identity":"c7d371ce-fdcc-4e30-8c5f-055c921d277b","added_by":"auto","created_at":"2025-05-07 06:41:25","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":564819,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSmoking mice present lung inflammation, NF-kB activation, and TXNIP downregulation. (A)\u003c/strong\u003e The hematoxylin and eosin (H\u0026amp;E) staining of lung sections from mice with or without a 12-week time course of smoking exposure were shown. Microphotographs are shown at 100× magnification. \u003cstrong\u003e(B)\u003c/strong\u003e Immunofluorescence staining was performed to detect the expression of phospho-NF-kB p65 (Ser536) in lung tissues of normal and smoking mice using specific antibodies followed by Alexa594-conjugated secondary antibodies staining (\u003cem\u003eRed\u003c/em\u003e). DAPI staining was used to determine the nuclei. Scale bar is 200 μm. \u003cstrong\u003e(C)\u003c/strong\u003e TXNIP expression in total protein isolated from normal (n=3) and smoking mouse lung tissues (n=3) were measured by immunoblotting. b-actin was used as an internal control, and the quantitativeratios of TXNIP/b-actin were shown as the means ± SEM from three mice. *p \u0026lt; 0.05. \u003cstrong\u003e(D)\u003c/strong\u003e Lung tissues obtained from normal and smoking mice were stained with TXNIP antibodies followed by Alexa488-conjugated secondary and CD11b-Alexa594-conjugated antibodies. DAPI was used for nuclear staining. The immunofluorescence and differential interference contrast (DIC) images were determined by confocal microscope and shown. Scale bar is 25 μm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/536769aea26c55ad91676b34.jpeg"},{"id":82146805,"identity":"c8eac874-c61a-4f3e-8d05-db299a6104bd","added_by":"auto","created_at":"2025-05-07 06:57:25","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":619597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress mediates TXNIP proteasomal degradation. (A) \u003c/strong\u003eImmunofluorescence staining was performed to detect the expression of 4-hydroxynonenal (4-HNE) and nitrotyrosine (NitroTyr) in lung tissues of normal and smoking mice respectively using specific antibodies followed by Alexa488-conjugated secondary antibodies staining (\u003cem\u003eGreen\u003c/em\u003e). DAPI staining was used to determine the nuclei. Scale bar is 200 μm. \u003cstrong\u003e(B)\u003c/strong\u003e The expression of 4-HNE and NitroTyr in total protein isolated from normal (n=3) and smoking mouse lung tissues (n=3) were measured, and b-actin was used as an internal control. \u003cstrong\u003e(C)\u003c/strong\u003e RAW264.7 cells were treated with various dosages of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for indicated time points, and the protein expression of TXNIP and GAPDH were detected. \u003cstrong\u003e(D)\u003c/strong\u003e In the presence of MG132 (10 mM) and lactacystin (10 mM), cells were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1 mM) for 2 h followed by the determination of TXNIP and GAPDH expression. \u003cstrong\u003e(E)\u003c/strong\u003e RAW264.7 cells were treated with TPA (50 ng/ml) for indicated time points followed by the ROS detection. The relative percentages of ROS production were measured, and shown as the means ± SEM from triplicate cultures. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u003cem\u003e \u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003e(F)\u003c/strong\u003e TXNIP expression in TPA (50 ng/ml)-treated RAW264.7 cells was detected at indicated time points. \u003cstrong\u003e(G)\u003c/strong\u003e Cells were pretreated with or without NAC (5 mM) for 1 h followed by TPA (50 ng/ml) stimulation for 2 h. TXNIP expression was detected, and GAPDH was used as an internal control. \u003cstrong\u003e(H)\u003c/strong\u003e RAW264.7 cells were exposed to indicated dosages of CSE for 24 h followed by the measurements of LDH release. \u003cstrong\u003e(I)\u003c/strong\u003e In the presence or absence of MG132 (10 mM), lactacystin (10 mM), and NAC (5 mM), cells were exposed to indicated dosages of CSE for 24 h followed by detecting TXNIP expression. GAPDH was used as an internal control. Protein molecular weights are indicated as kilodaltons (kDa).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/0cf403dfe650fe36b5e73f6a.jpeg"},{"id":82143428,"identity":"48c3e97b-5965-4aa0-adae-27e9ed8ee5c3","added_by":"auto","created_at":"2025-05-07 06:41:25","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":580295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAP-Kinases regulate oxidative stress-mediated TXNIP degradation and NF-kB activation. (A)\u003c/strong\u003e The nuclear translocation of NF-kB in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated RAW264.7 cells were assayed at indicated time points using a specific antibody against NF-kB (\u003cem\u003eGreen\u003c/em\u003e) followed by fluorescence microscopic observation. DAPI was used as a nuclear staining, and scale bar was shown. Quantitative measurements of NF-κB nuclear translocation (percentages based on more than 150 cells in total) were performed and shown as the means ± SEM of triplicate cultures. **p \u0026lt; 0.01. \u003cstrong\u003e(B)\u003c/strong\u003e RAW264.7 cells were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1 mM) for 4h and subsequently stained with TXNIP (\u003cem\u003eRed\u003c/em\u003e) and NF-kB (\u003cem\u003eGreen\u003c/em\u003e) respectively. DAPI was used as a nuclear staining, and scale bar was shown. \u003cstrong\u003e(C)\u003c/strong\u003e RAW264.7 cells were treated with different dosages of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for indicated time points, the expressions of TXNIP, phospho-JNK (Thr183/Tyr185), JNK, phospho-ERK (Thr202/Tyr204), ERK, phospho-p38 (Thr180/Tyr182), and p38 were determined. GAPDH was used as an internal control. Protein molecular weights are indicated as kilodaltons (kDa). \u003cstrong\u003e(D)\u003c/strong\u003e RAW264.7 cells were preincubated with 25 mM of SP600125 (SP), PD98059 (PD), and SB 253580 (SB) for 1 h followed by 1 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 2 h. The expression of TXNIP and GAPDH were measured. \u003cstrong\u003e(E)\u003c/strong\u003e The luciferase reporter assay of NF-κB was measured after 1 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 4 h in the presence or absence of inhibitors. Triplicate cultures were performed and shown as the means ± SEM. **p \u0026lt; 0.01 as compared to control;\u003csup\u003e ##\u003c/sup\u003e p \u0026lt; 0.01 and \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 as compared to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e groups.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/75c7fdea2ac89862406df722.jpeg"},{"id":82144932,"identity":"279a01e7-65f5-4e16-bcbc-86c84e1bcc48","added_by":"auto","created_at":"2025-05-07 06:49:25","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":543916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eItch expression modulates TXNIP-mediated NF-kB activation. (A)\u003c/strong\u003e The protein expression of Itch in normal and smoking mouse lung tissues was measured, and the quantified results of Itch relative to β-actin were shown (n=3 each group). *p\u003cem\u003e \u003c/em\u003e\u0026lt; 0.05. β-actin was used as an internal control. \u003cstrong\u003e(B)\u003c/strong\u003e The expression of Itch and GAPDH were measured in RAW 264.7 cells treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1 mM) and TPA (50 ng/ml) for 2 h. \u003cstrong\u003e(C)\u003c/strong\u003e In the presence of 5 mM NAC, the expression of Itch and GAPDH were detected in cells treated with TPA for 2 h. \u003cstrong\u003e(D)\u003c/strong\u003e RAW264.7 cells expressed with shRNA targeting luciferase (shLuc) and shRNA targeting Itch (shItch#1, shItch#2, and shItch#3) were generated, and the expression of Itch and GAPDH were confirmed by immunoblotting. \u003cstrong\u003e(E)\u003c/strong\u003e Cells expressed shLuc and shItch#1 were treated with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 2 h. The expression of Itch, TXNIP, and GAPDH were detected. Protein molecular weights are indicated as kilodaltons (kDa). \u003cstrong\u003e(F)\u003c/strong\u003e The luciferase reporter assay of NF-κB was measured in shLuc- and shItch-cells stimulated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (1 mM) for 4 h. The quantitative data are presented as the means ± SEM from triplicate cultures. ***p \u0026lt; 0.001 as compared to untreated.\u003csup\u003e ###\u003c/sup\u003ep \u0026lt; 0.001 as compared to the shLuc-group.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/315deb0b7f89cb9baf8efa35.jpeg"},{"id":82143424,"identity":"91927980-6c72-402c-a231-570f65c3fe7a","added_by":"auto","created_at":"2025-05-07 06:41:25","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":668246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased expression of TXNIP in COPD patients.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Gene expression of TXNIP (GDS3496/201010_s_at) and Itch (GDS3496/217094_s_at) in alveolar macrophages of cigarette smokers obtained from the public GEO profiles was assayed. Gene expression of TXNIP (GDS737/201010_s_at) and Itch (GDS737/209743_s_at) in smoker lung tissues with no/mild and severe emphysema obtained from the public GEO profiles was assayed. The relative fold changes were shown as means ± SEM. *p\u003cem\u003e \u003c/em\u003e\u0026lt; 0.05, **p \u0026lt; 0.01. \u003cstrong\u003e(B)\u003c/strong\u003eImmunohistochemical staining was performed using specific antibodies to detect TXNIP expression in the fixed lung tissue sections obtained from COPD patients. Normal controls were obtained from the noninvolved lung sections of the tumor lesion. Microphotographs are shown at 200× and enlarged magnification. \u003cstrong\u003e(C)\u003c/strong\u003e Bronchoalveolar lavage fluid (BALF) cells were collected from donors with or without COPD, and subsequently performed the immunoblotting to measure TXNIP expression. GAPDH was used as an internal control. \u003cstrong\u003e(D)\u003c/strong\u003e Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors (n=2), smokers (n=1), mild COPD patients (n=1), and severe COPD patients (n=2). TXNIP expression was detected and b-actin was used as an internal control. Protein molecular weights are indicated as kilodaltons (kDa).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/ac6f418c50b2ba6bc09eace8.jpeg"},{"id":82143420,"identity":"74343f35-58b2-4699-89db-6c878481258e","added_by":"auto","created_at":"2025-05-07 06:41:25","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":292809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased expression of TXNIP in COPD patients.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Gene expression of TXNIP (GDS3496/201010_s_at) and Itch (GDS3496/217094_s_at) in alveolar macrophages of cigarette smokers obtained from the public GEO profiles was assayed. Gene expression of TXNIP (GDS737/201010_s_at) and Itch (GDS737/209743_s_at) in smoker lung tissues with no/mild and severe emphysema obtained from the public GEO profiles was assayed. The relative fold changes were shown as means ± SEM. *p\u003cem\u003e \u003c/em\u003e\u0026lt; 0.05, **p \u0026lt; 0.01. \u003cstrong\u003e(B)\u003c/strong\u003eImmunohistochemical staining was performed using specific antibodies to detect TXNIP expression in the fixed lung tissue sections obtained from COPD patients. Normal controls were obtained from the noninvolved lung sections of the tumor lesion. Microphotographs are shown at 200× and enlarged magnification. \u003cstrong\u003e(C)\u003c/strong\u003e Bronchoalveolar lavage fluid (BALF) cells were collected from donors with or without COPD, and subsequently performed the immunoblotting to measure TXNIP expression. GAPDH was used as an internal control. \u003cstrong\u003e(D)\u003c/strong\u003e Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors (n=2), smokers (n=1), mild COPD patients (n=1), and severe COPD patients (n=2). TXNIP expression was detected and b-actin was used as an internal control. Protein molecular weights are indicated as kilodaltons (kDa).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/a887a4128d83fa2e26d3ee85.jpeg"},{"id":93956673,"identity":"34b432ff-1b64-45fe-af22-0fea440ab76e","added_by":"auto","created_at":"2025-10-20 16:11:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4793215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/3cb2f3f2-4078-46f1-b596-44ce74311c8f.pdf"},{"id":82144935,"identity":"c24da84b-11b3-40ab-bcec-9866b95d5767","added_by":"auto","created_at":"2025-05-07 06:49:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3927405,"visible":true,"origin":"","legend":"","description":"","filename":"2025LinetalCSSupplementaryFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/4d17cd7310d1b0328cc4d9d9.pdf"},{"id":82143416,"identity":"1933c0ab-9d3d-47ed-9967-fb10c5cb91a3","added_by":"auto","created_at":"2025-05-07 06:41:25","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":555940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6476972/v1/f2ec382d44ef236260e0ea96.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oxidative Stress Triggers Itch-mediated TXNIP Degradation and NF-κB Activation Promoting Chronic Obstructive Pulmonary Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease characterized by pulmonary structural changes, narrowing of the small airways, and destruction of alveolar tissue. The most commonly encountered risk factor for COPD is tobacco usage and secondhand smoke exposure [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Cigarette smoke is a complex, dynamic and reactive mixture containing an estimated 7,000 chemicals, including reactive oxygen species (ROS) and nicotine [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cigarette smoke-induced oxidative stress plays a vital role in causing inflammation, cell senescence, and DNA damage, which contribute to COPD pathogenesis, particularly during acute exacerbations. Increased oxidative stress activates various intracellular signaling pathways, such as the mitogen-activated protein kinases (MAPKs) and transcription factor nuclear factor (NF)-κB, which frequently interact with redox-sensitive molecular targets, thereby driving the induction of inflammatory mediators [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. So far, the effective molecular targeting of chronic and prolonged inflammation in the treatment of COPD has been widely considered. Therefore, the underlying mechanisms that control sustained inflammation need to be investigated more thoroughly.\u003c/p\u003e \u003cp\u003eThioredoxin-interacting protein (TXNIP) has been originally identified as a differentially expressed gene in 1α,25-dihydroxyvitamin D3-treated HL-60 cells, also called as vitamin D3 upregulated protein 1 [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Upregulation of TXNIP is associated with diseases such as diabetes mellitus, ischemic stroke, cardiovascular diseases, and neurodegenerative disorders, while reduced TXNIP expression often correlates with the onset of tumors [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, as a member of the α-arrestin protein family, TXNIP can function as a scaffold protein (independent of its binding to TRX) in specific subcellular compartments to enhance the formation of the NLR protein 3 (NLRP3) inflammasome, promoting interleukin (IL)-1β production under oxidative stress [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and regulate the nuclear export and degradation of hypoxia-inducible factor 1-α (HIF1-α) during hypoxia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Accordingly, the protein stability and gene expression of TXNIP can be regulated in response to multiple stimuli, subsequently contributing to signaling cascades that affect various biological processes.\u003c/p\u003e \u003cp\u003eTXNIP expression has been reported to potentially affect the development of inflammation-related lung disorders. The induction of TXNIP-mediated NLRP3 inflammasome and pyroptosis contributes to acute lung inflammation and injury in septic mice [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, elevated TXNIP-regulated apoptosis facilitates titanium dioxide nanoparticle-exacerbated allergic airway inflammation in an ovalbumin-induced mouse model of asthma [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. TXNIP overexpression in memory T helper (Th) 2 cells presents enhanced memory responses, including the increases of eosinophils infiltration, Th2 cytokines, and allergic airway inflammation in mice [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, bleomycin-induced lung fibrosis develops alongside increased TXNIP/HIF1-α expression and oxidative stress in the lung tissues of rats [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The pathogenesis of inflammatory lung disorders is heterogeneous, and the role of TXNIP in regulating the induction of inflammation, oxidative stress, and lung disease progression, requires more investigation.\u003c/p\u003e \u003cp\u003eDespite the established involvement of oxidative stress and the P2X7/inflammasome pathway in the development and exacerbation of cigarette smoke-induced COPD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the role of TXNIP in regulating cigarette smoke-associated lung inflammation and its molecular mechanisms remain unclear. In this study, we demonstrated that exposure to cigarette smoke resulted in a decrease in TXNIP levels in macrophages and murine lungs through a mechanism involving the generation of ROS and the activation of MAPKs. Oxidative stress induced the expression of the Itch E3 ligase, which regulated TXNIP for degradation and subsequently led to the activation of NF-κB. Further inhibition of Itch partially mitigated TXNIP degradation and NF-κB activation. Moreover, the \u003cem\u003eTxnip\u003c/em\u003e gene exhibited relatively lower expression, while the \u003cem\u003eItch\u003c/em\u003e gene showed relatively higher expression in alveolar macrophages from smokers compared to non-smokers. TXNIP was significantly suppressed in the lung tissues, bronchoalveolar lavage fluid (BALF) cells, and peripheral blood mononuclear cells (PBMC) of COPD patients. This suggests that cigarette smoke-induced oxidative stress may trigger Itch-regulated TXNIP degradation, ultimately resulting in the activation of NF-κB-mediated inflammation that potentially contributes to the development of COPD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell cultures and reagents\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMurine RAW264.7 macrophages (#ATCC TIB-71\u003csup\u003eTM\u003c/sup\u003e) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (#11965084, Gibco, Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS) (#04-001-1A, Biological Industries, Kibbutz Beit Haemek, Israel) at 37\u0026deg;C in a humidified atmosphere of 95% air and 5% CO2. MG132 (#1748), Lactacystin (#2267), PD98059 (#1213), SP600125 (#1496), SB203580 (#1202) are purchased from TOCRIS Bioscience (Bristol, UK). Hydrogen peroxide (#H1099), N-Acetyl-L-cysteine (NAC, #A7250), 12-O-Tetradecanoylphorbol 13-acetate (TPA, #P8139), and 4,6-diamidino-2-phenylindole (DAPI, #D9542) were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against phospho-ERK1/2 (Thr202/Tyr204) (#9101S), ERK1/2 (#9102S), phospho-JNK (Thr183/Tyr185) (#9251), JNK (#9252), phospho-p38 (Thr180/Tyr182) (# 9211), p38 (#9212), phospho-NF-kB (#3033), Itch (#12117), iNOS (#13120), COX-2 (#12282S) and the secondary antibodies HRP-conjugated goat anti-mouse (#7076S) and goat anti-rabbit (#7074S) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against TXNIP (#K0205-3) were purchased from MBL International Corporation (Woburn, MA, USA). Antibodies against hydroxynonenal (#ab46545) and nitrotyrosine (#ab125106) were purchased from Abcam (Cambridge, MA, USA), and GAPDH (#SC-32233) and \u0026beta;-actin (#SC-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Alexa Fluor 488- and 594-conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmoking mouse and cigarette smoke extract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSmoking mice were performed according to previous study [22]. In brief, six-week-old male BALB/c mice obtained from National Laboratory Animal Center (Taipei, Taiwan) were maintained on standard laboratory food and water ad libitum in the animal centers at the Taipei Medical University. All animal studies were performed in accordance with the rules of the Animal Protection Act of Taiwan, and the animal use protocols were approved by the Laboratory Animal Care and Use Committee of Taipei Medical University, Taiwan (LAC-2014-0168). Mice were whole-body exposed to cigarette smoke by a cigarette smoke chamber with the main-stream smoke from the combustion of 12 reference cigarettes (3R4F; Tobacco and Health Research Institute, KY, USA) for a period of approximately 50 minutes/day, 5 days/week, for 12 weeks. Control mice were exposed to cigarette smoke-free HEPA-filtered air. Cigarette smoke extract (CSE) was prepared according to previous study with the slight modification [23]. Two filtered cigarettes (Marlboro MX, tar: 10 mg, nicotine content: 0.8 mg; Philip Morris, Switzerland) was imported a vessel containing 6 ml medium by a vacuum pump. Freshly prepared CSE were next sterilized by passing through a 0.22-\u0026mu;m filter and recognized as 100% concentrations of CSE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinal samples including lung tissue, PBMC, and BALF were obtained from healthy non-smokers, a healthy smoker, and COPD patients. Patients with COPD were diagnosed and graded according to the guidelines of the Global Initiative for Obstructive Lung Disease [24], and the details of COPD patients were summarized in our previous study [22]. The clinical study protocol was approved by Taipei Medical University-Joint Institutional Review Board (TMU-JIRB No. 201310027) and performed in accordance with the relevant guidelines and regulations. The lung tissues were obtained from COPD patients with lung surgery for the peripheral lung tumor removal, whereas normal control tissues were derived from the noninvolved lung segments of the tumor lesion from non-COPD patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of Itch shRNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo stably express a lentivirus-based short hairpin RNA (shRNA) targeting Itch, TRCN0000026925 (5\u0026prime;-CCACCTGAAATACTTTCGTTT-3\u0026prime;), TRCN0000026908 (5\u0026prime;- CCCTACGAGTAAATTATGTTT-3\u0026prime;), and TRCN0000026914 (5\u0026prime;-GCGAAGGAATTAGAGGTTCTT-3\u0026prime;) were obtained from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan) followed by the preparation of lentiviral mouse Itch shRNAs from the RNAi Core of Research Center of Clinical Medicine (National Cheng Kung University Hospital, Taiwan). TRCN0000072247 (5\u0026prime;- GAATCGTCGTATGCAGTGAAA-3\u0026prime;) was used as the control luciferase shRNA (shLuc). RAW 264.7 cells were subsequently infected with an appropriate MOI for 24 h followed by puromycin (Calbiochem, San Diego, CA) selection. The protein expression was then measured by western blot analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total proteins were extracted from RAW264.7 cells, BALF pellets, PBMCs, and mouse lung tissue homogenates using a Triton X-100 based lysis buffer with a protease inhibitor mix and a phosphatase-inhibitors cocktail I followed by the centrifugation at 13300 rpm for 10 min. Proteins were then resolved using SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). After blocking, the membranes were probed with the indicated primary antibodies (1:1000 dilution) followed by secondary antibodies (1:5000 dilution), and developed by an ECL Western blot detection kit (Pierce Chemical, Rockford, IL, USA) according to the manufacturer\u0026rsquo;s instructions. All immunoblotting studies were performed in at least two independent experiments, and the relative band intensity on the blots was quantified using Image J software (NIH, Bethesda, MD, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis and immunostaining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLung tissues obtained from patients and smoking mice were fixed in 10% neutral-buffered formalin, embedded in paraffin wax, and sliced. For histopathology, sections (5 \u0026mu;m) were stained with hematoxylin and eosin (H\u0026amp;E). For immunohistochemical analysis, lung tissues were deparaffinized and rehydrated with xylene and different concentrations of ethanol. After permeabilized with 0.1% Triton X-100 in PBS and incubated with 3% hydrogen peroxide, sections were blocked (1% BSA + 0.1% azide in PBS) and stained with specific antibodies against phospho-NF-kB (Ser536), TXNIP, CD11b (clone M1/70, BioLegend San Diego, CA, USA), hydroxynonenal, and nitrotyrosine followed by HRP- or Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody staining. RAW264.7 cells were fixed by 4% paraformaldehyde in PBS and permeabilized. Fixed cells were then stained with specific antibodies against phospho-NF-kB (Ser536) and TXNIP followed by secondary antibody staining. Hematoxylin and DAPI (5 \u0026mu;g/ml) were used for nuclear staining. Images were captured by using fluorescence microscopy (EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA) and LEICA TCS SP5 laser scanning confocal microscopy system (Leica, Heidelberg, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNO and ROS detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNitrite accumulation in the cell culture medium was used as an indicator of NO production, detected by the Griess reaction. Briefly, supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% H3PO4) and incubated for 10 min at room temperature. The relative optical density (OD) of nitrite was measured at 540nm, and the concentration was evaluated by using sodium nitrite as a standard. For ROS detection, cells were treated with or without TPA and then co-incubated with 20 \u0026mu;M carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA, C6827, Thermo Scientific) fluoroprobe for 30min at 37\u0026deg;C in the dark. After washing, cells were collected and analyzed using flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA) with the excitation at 488 nm. The emission was detected with the FL-1 channel followed by CellQuest Pro 4.0.2 software (BD Biosciences) analysis, and quantification was performed using FlowJo software (Tree star, Inc., Ashland, Or, USA). The percentages of ROS-positive cells each group were shown. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNF-\u0026kappa;B reporter assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression vectors of pNF-\u0026kappa;B-Luc plasmid (Stratagene, La Jolla, CA) and cytomegalovirus-Renilla luciferase construct (pRL-CMV) (Promega, Madison, WI) were transiently cotransfected into cells for 24 h using lipofectamine reagents (Invitrogen, Carlsbad, CA). The Renilla-derived luciferase reporter plasmid was used for transfection efficiency control. In the presence or absence of inhibitors treated for 1 h, cells were stimulated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h followed by the detection of firefly luciferase activity using Dual Luciferase\u0026reg; Reporter assay system (Promega, Madison, WI, USA) and the multimode reader (Varioskan Flash, Thermo Scientific) according to the manufacturer\u0026rsquo;s instructions. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using Student\u0026rsquo;s t-test (two groups) or one-way ANOVA (more than two groups) followed by a Tukey\u0026rsquo;s multiple comparison test with Prism 7.0 (GraphPad). The data are presented as the mean \u0026plusmn; standard error of the mean (SEM) from three independent experiments. Statistical significance was set at *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCigarette smoke extract initiates TXNIP reduction and iNOS expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTXNIP is involved in a wide variety of cellular processes. Overexpression of TXNIP enables cell apoptosis while TXNIP deficiency causes tumorigenesis and the exacerbation of endotoxic shock [25-27]. To investigate whether TXNIP participates in cigarette smoke-mediated inflammation, TXNIP expression was measured in cigarette smoke extract (CSE) stimulation. Freshly prepared CSE markedly attenuated TXNIP expression, accompanied by the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in murine RAW264.7 cells \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. CSE significantly induced nitric oxide (NO) production in a dose-dependent manner \u003cstrong\u003e(Fig. 1B)\u003c/strong\u003e, while showing no significant induction of cytotoxicity. Similar to previous reports [28], CSE could effectively induce JNK, p38 MAPK, and ERK phosphorylation in RAW264.7 cells \u003cstrong\u003e(Fig. 1C)\u003c/strong\u003e. Moreover, the presence of JNK and p38 MAPK inhibitors, SP600125 and SB203580, markedly alleviated CSE-caused TXNIP reduction respectively \u003cstrong\u003e(Fig. 1D)\u003c/strong\u003e. TXNIP has been linked to regulate stress-activated apoptosis and inflammation through its cellular expression and distribution [9]. Interestingly, TXNIP was distinctly suppressed instead of being induced in coordination with the elevation of inflammatory mediators in CSE stimulation. This might suggest a novel role for TXNIP in cigarette smoke-regulated inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecreased TXNIP expression in the lungs of smoking mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDown-regulated TXNIP expression was observed in CSE-stimulated murine macrophage RAW264.7 cells. Subsequently, changes in pulmonary TXNIP expression in smoking mice were investigated. Following 12 weeks of cigarette smoke exposure, the mice exhibited significant immune cell infiltration in the lungs and abnormal enlargement of airspaces \u003cstrong\u003e(Fig. 2A)\u003c/strong\u003e. NF-\u0026kappa;B activation was determined in the lungs of smoking mice, revealing elevated phosphorylation of p65 NF-\u0026kappa;B at serine 536 \u003cstrong\u003e(Fig. 2B)\u003c/strong\u003e. This indicated that cigarette smoke exposure could effectively trigger NF-\u0026kappa;B-regulated inflammation. Further assessments of TXNIP expression revealed significant decreases in TXNIP levels in the lung tissues of smoking mice compared to normal mice \u003cstrong\u003e(Fig. 2C)\u003c/strong\u003e. Additionally, TXNIP expression was intensely suppressed in lung macrophages expressing CD11b \u003cstrong\u003e(Fig. 2D)\u003c/strong\u003e. Consistent with previous results, the downregulation of TXNIP might potentially accompany NF-\u0026kappa;B activation in macrophages, leading to pulmonary inflammation induced by cigarette smoke stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress-regulated proteasomal degradation of TXNIP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCigarette smoke represents one of the most significant exogenous oxidants, containing abundant ROS, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and NO that contribute to oxidative stress-mediated inflammation [29]. Given the marked downregulation of TXNIP in the lung tissue of smoking mice, we subsequently explored whether TXNIP expression is regulated by oxidative stress. The distinct elevations of a sensitive marker of oxidative damage and lipid peroxidation, 4-hydroxynonenal (4-HNE), and a versatile oxidative stress biomarker, nitrotyrosine (NitroTyo), were detected in smoking murine lung tissues by immunofluorescence staining \u003cstrong\u003e(Fig. 3A)\u003c/strong\u003e and western blot analysis \u003cstrong\u003e(Fig. 3B)\u003c/strong\u003e. These results suggested the potential coordination between TXNIP downregulation and oxidative stress. Exogeneous treatment of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e showed a dose-dependent TXNIP downregulation in RAW264.7 cells \u003cstrong\u003e(Fig. 3C)\u003c/strong\u003e. The presence of proteasome inhibitors, MG132 and lactacystin (LAC), distinctly reversed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-caused TXNIP downregulation \u003cstrong\u003e(Fig. 3D)\u003c/strong\u003e, which suggested that oxidative stress might initiate TXNIP undergoing proteasomal degradation. Furthermore, the endogenous ROS induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) \u003cstrong\u003e(Fig. 3E)\u003c/strong\u003e similarly induced TXNIP degradation in RAW264.7 cells \u003cstrong\u003e(Fig. 3F)\u003c/strong\u003e, while the ROS inhibitor, N-acetylcysteine (NAC), markedly attenuated TPA-induced TXNIP degradation \u003cstrong\u003e(Fig. 3G)\u003c/strong\u003e. In addition, the various concentrates of freshly prepared CSE we used showed no significant cytotoxicity in RAW264.7 cells \u003cstrong\u003e(Fig. 3H)\u003c/strong\u003e. Notably, the presence of MG132, LAC, and NAC similarly attenuated the CSE-induced TXNIP degradation \u003cstrong\u003e(Fig. 3I)\u003c/strong\u003e. Therefore, cigarette smoking might induce oxidative stress-regulated TXNIP proteasomal degradation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS mediates MAPKs-regulated TXNIP degradation and NF-\u003c/strong\u003e\u003cstrong\u003ekB activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNF-kB is a critical transcriptional factor of the inflammatory process, while H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e has been revealed as a fine-tuning regulator of NF-kB-dependent inflammation [30]. In H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW264.7 cells, the nuclear translocation of NF-kB was significantly increased \u003cstrong\u003e(Fig. 4A)\u003c/strong\u003e. Furthermore, NF-\u0026kappa;B nuclear translocation occurred concurrently with TXNIP degradation in the cells \u003cstrong\u003e(Fig. 4B)\u003c/strong\u003e.\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment effectively induced TXNIP degradation, which was in associated with the phosphorylation of JNK, p38 MAPK, and ERK \u003cstrong\u003e(Fig 4C)\u003c/strong\u003e. Pharmacological inhibition of MAPKs using specific inhibitors, SP600125, PD98059, and SB203580, which inhibited JNK, ERK, and p38 MAPK, respectively, suppressed H2O2-induced TXNIP degradation \u003cstrong\u003e(Fig. 4D)\u003c/strong\u003e, as well as subsequent NF-\u0026kappa;B activation \u003cstrong\u003e(Fig. 4E)\u003c/strong\u003e. Accordingly, ROS-induced NF-\u0026kappa;B activation may be regulated by MAPKs-mediated TXNIP degradation. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eItch expression modulates TXNIP-mediated NF-\u003c/strong\u003e\u003cstrong\u003ekB activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTXNIP has been shown to interact with the ubiquitin E3 ligase Itch through a conserved PPXY motif in the C terminus of TXNIP and undergoes degradation [31]. Itch expression is initially slightly increased by ROS stimulation, but subsequently decreases following TXNIP suppression in cardiomyocytes [32]. Similarly, we observed a slight increase in the expression of Itch in lung tissue extracted from smoking mice \u003cstrong\u003e(Fig. 5A)\u003c/strong\u003e. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and TPA could stimulate Itch induction in RAW264.7 cells \u003cstrong\u003e(Fig. 5B)\u003c/strong\u003e, while TPA-elevated Itch expression was partly inhibited by the presence of NAC \u003cstrong\u003e(Fig. 5C)\u003c/strong\u003e. ROS accumulation upon exposure to cigarette smoke might potentiate Itch induction accordingly. Since TXNIP degradation may occur in accordance with Itch expression, we further explored whether the specific knockdown of Itch might affect ROS-induced TXNIP degradation. By using RNA interference, Itch expression was specifically silenced in RAW264.7 cells \u003cstrong\u003e(Fig. 5D)\u003c/strong\u003e. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced TXNIP degradation was markedly reversed in Itch knockdown RAW264.7 cells compared to the controls \u003cstrong\u003e(Fig. 5E)\u003c/strong\u003e. Moreover, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated NF-\u0026kappa;B activation was significantly attenuated in Itch knockdown cells accompanying by the stabilization of TXNIP \u003cstrong\u003e(Fig. 5F)\u003c/strong\u003e. Therefore, ROS-mediated upregulation of Itch could potentially lead to TXNIP degradation, resulting in subsequent NF-\u0026kappa;B activation in response to cigarette smoke exposure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecreased TXNIP levels in COPD patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTXNIP protein expression exhibits distinct variations in different cells response to ROS stimulation [33,34], where we observed ROS-mediated TXNIP suppression in lung tissues of smoking mice as well as in murine macrophages. Exploration of gene profiles obtained from DataSet Record GDS3496 and GDS737 on Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) revealed that the significant relative lower expression of TXNIP and higher expression of Itch were shown in alveolar macrophages of cigarette smokers (n=13) compared to nonsmokers (n=11), while no significant changes of TXNIP and Itch were found in lung tissues from smokers with severe emphysema (n=18) compared to no or mild emphysema (n=12) \u003cstrong\u003e(Fig. 6A)\u003c/strong\u003e. To further confirm the expression status of TXNIP in oxidative stress-associated lung diseases, we collected lung tissues from non-COPD and COPD patients undergoing lung surgery for peripheral lung tumor removal. We found that TXNIP was markedly suppressed in lung tissues with COPD compared to non-COPD patients \u003cstrong\u003e(Fig. 6B)\u003c/strong\u003e. Similarly, decreased TXNIP expression was observed in BAL cells from COPD patients \u003cstrong\u003e(Fig. 6C)\u003c/strong\u003e. Furthermore, PBMCs obtained from healthy non-smokers, healthy smokers, and COPD patients revealed significantly greater inhibition of TXNIP in mild and severe COPD patients compared to healthy donors \u003cstrong\u003e(Fig. 6D)\u003c/strong\u003e. Accordingly, cigarette smoke exposure-mediated oxidative stress could induce Itch- and MAPKs-regulated TXNIP degradation, leading to NF-\u0026kappa;B activation and subsequent inflammatory induction \u003cstrong\u003e(Fig. 7)\u003c/strong\u003e. Additionally, the decreased TXNIP levels observed in COPD patients suggested its potential role in regulating disease progression.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe burden of cigarette smoke-induced oxidative stress, cellular damage, and inflammation are major contributors to COPD pathogenesis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. TXNIP possesses pro-oxidative and pro-inflammatory characteristics, leading to the initiation or exacerbation of inflammation and cellular damages in various disease progressions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. TXNIP induction has also been reported to play a role in the development of inflammation-related lung disorders, including acute lung injury, allergic exacerbation, and lung fibrosis [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we demonstrate that TXNIP reduction is associated with CSE-induced inflammation in RAW264.7 cells and lung inflammation in smoking mice. Cigarette smoke-mediated oxidative stress leads to proteasomal degradation of TXNIP, subsequently activating MAPKs-regulated NF-κB. Interestingly, upon cigarette smoke exposure and oxidative stimulation, the expression of Itch, the upstream E3 ubiquitin ligase of TXNIP, increases. Inhibition of Itch significantly attenuates TXNIP degradation and NF-κB activation. Furthermore, lung tissues, BALF cells, and PBMC obtained from COPD patients exhibit marked TXNIP suppression compared to healthy donors. Contrary to an increase, TXNIP is markedly suppressed through Itch regulation in response to cigarette smoke -mediated oxidative stress, subsequently activating MAPKs and NF-κB inflammation.\u003c/p\u003e \u003cp\u003eThe expression of TXNIP is often triggered by multiple stimuli, such as hyperglycemia, ischemia-reperfusion injury, hypoxia, ER stress, and ROS. Under stress conditions, increased TXNIP may either promote apoptotic signal-regulated kinase 1-mediated mitochondrial apoptosis through TRX-dependent signaling [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], or regulate TXNIP/NLRP3 inflammasome activation in a redox-independent fashion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. On the other hand, TXNIP degradation has been shown to contribute to TNF-α-stimulated NF-κB activation, while TXNIP deficiency exacerbates lipopolysaccharide (LPS)-induced endotoxic shock and mortality induced by E. coli infection through excessive NO production [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, toll-like receptor (TLR) 2- and ROS-mediated rapid TXNIP degradation in macrophages has shown to potentially expedite NF-κB activation in \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e infection [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Here, we found that CSE stimulation mediated dose-dependent degradation of TXNIP, accompanied by the induction of iNOS/NO and COX-2 in macrophages. The suppression of TXNIP could further promote MAPKs and NF-κB activation. Moreover, a significant increase in the development of emphysema has been found in USP13-deficient mice after mild CS exposure, where the reduction of TXNIP is also observed in lung epithelial cells with USP13 inhibition [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, other reports have indicated that CSE upregulates TXNIP expression, and triggers TXNIP-NLRP3-gasdermin D axis to promote inflammation and pyroptosis of islet β-cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. So far, the protein stability and functions of TXNIP in response to cigarette smoke stimuli remain controversial and appear to exhibit cell-type-specific regulation accordingly.\u003c/p\u003e \u003cp\u003eItch, a HECT-type ubiquitin E3 ligase, can interact with TXNIP and regulate its ubiquitin labeling, promoting proteasomal degradation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Itch has long been respected as a critical suppressor of inflammation, which limits Th2 immunity by regulating T cells, B cells, and macrophages. Itch deficiency presents abnormal Th2-related lung and skin inflammation and spontaneous gastrointestinal tract inflammation in mice, along with immune abnormalities in humans, while Itch overexpression has been observed in several human cancers [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The promotion or repression of Itch activity is regulated by the phosphorylation status in response to various stimulations of growth factors, death receptors, DNA damage, and oxidative stress [\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In addition to post-translational modification, ROS-mediated increased expression of Itch has also been reported to trigger the degradation of FLICE-like inhibitory protein [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Upon cigarette smoke and ROS stimulation, we found that increased Itch could be detected in lung tissues from smoking mice and in RAW264.7 cells, along with the TXNIP degradation. Additionally, GEO profiles similarly indicated that alveolar macrophages of smokers exhibit higher Itch gene expression than non-smokers, even though Itch gene expression seemed to show no significant changes in association with emphysema progression. Furthermore, ROS-induced Itch expression effectively contributed to downstream NF-κB activation, suggesting the potential proinflammatory role of Itch in cigarette smoke exposure.\u003c/p\u003e \u003cp\u003eChronic inflammation induced by abundant ROS stimulation and MAPKs activation is usually the major cause leading to consistent alveolar macrophage activation and alveolar epithelial cell damage in COPD pathogenesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Targeting oxidative stress with therapeutic agents has been recommended as an effective approach in treating COPD, and several antioxidants, including NAC, have been applied in clinical trials with some beneficial outcomes [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In addition to its well-known function of elevating intracellular glutathione levels [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], we demonstrated here that NAC has the capacity to stabilize the ROS-regulated Itch/TXNIP/NF-κB axis, thereby attenuating inflammation. Meanwhile, targeting MAPK signaling is also speculated as another efficient approach for treating inflammatory lung diseases, with clinical trials showing the therapeutic potential of p38 MAPK inhibitors in improving lung function and reducing exacerbations in COPD patients [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Here, we investigated that ROS-initiated Itch/TXNIP/NF-κB axis was partly regulated by the activation of JNK, p38 MAPK, and ERK, while the blockage of MAPKs could potentially attenuate NF-κB activation. Given the heterogeneity of COPD pathogenesis, both antioxidants and MAPK inhibitors exhibit only limited therapeutic potential. Therefore, combining these treatments with other anti-inflammatory medications appears to be a promising future therapeutic approach.\u003c/p\u003e \u003cp\u003eTXNIP expression varies across different types of respiratory disorders. In lung cancer, TXNIP expression is significantly reduced. The downregulation of TXNIP promotes tumor proliferation and migration while inhibiting apoptosis in lung cancer cell lines [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In allergic airway inflammation and lung fibrosis, elevated TXNIP levels contribute to Th2 immune responses in the airway and oxidative stress in fibrotic lungs, respectively [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we first revealed the downregulation of TXNIP associated with cigarette smoke-induced oxidative stress and the development of inflammation in the lung tissue of smoking mice and COPD patients. Although P2X7/caspase-1 activation has been reported to be involved in cigarette smoke-induced lung inflammation in mice and smoking donors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], whether TXNIP plays a role in cigarette smoke-mediated inflammasome activation remains unclear. Moreover, we have observed the ROS/Itch/TXNIP axis expressed upstream of NF-κB activation, potentially providing another pathway for the initiation of NF-κB, which is considered to be of paramount importance in COPD inflammation. However, due to the limited number of clinical samples, the expression and functions of TXNIP and Itch in COPD pathogenesis still require further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we have demonstrated that cigarette smoke-induced oxidative stress triggers Itch expression and MAPK activation, leading to TXNIP degradation in macrophages. Itch- and MAPK-dependent TXNIP proteasomal degradation facilitates NF-κB activation and inflammatory responses, potentially contributing to COPD pathogenesis. Stabilizing TXNIP using a ROS scavenger or Itch-specific knockdown can attenuate NF-κB-mediated inflammation, suggesting the potential therapeutic target for COPD treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interests statement\u003c/strong\u003e: The authors declare that they have no known conflict of interests.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to Taipei Medical University-Joint Institutional Review Board and the Laboratory Animal Center, Taipei Medical University, for their technical assistance. Additionally, we are thankful for the human samples and animals that contributed to this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.-Y. Lin and C.-L. Chen developed the concepts, conducted the experiment, analyzed the data, and drafted the manuscript. C.-L. Chen also designed the experiment, interpreted the data, and revised the manuscript. K.-Y. Lee, S.-C. Ho, H.-C. Chuan, B.-H. Su, and R. Satria assisted with material preparation, provided resources, and reviewed the manuscript. P.-C. Tseng, Y.-J. Wu, T.-T. Tsai, and F.-C. Shih assisted in conducting the experiment and analyzing the data. All authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Ministry of Science and Technology of Taiwan (MOST 111-2314-B-038-120-MY3, NSTC 113-2320-B-038-003, and Taipei Medical University (DP2-TMU-112-T-05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiment procedures and animals were in compliance with the animal and ethics review committee of the Laboratory Animal Center at Taipei Medical University, Taipei, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known conflict of interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChristenson SA, Smith BM, Bafadhel M, et al. 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Targeting MAPK signaling: A promising approach for treating inflammatory lung disease. Pathol Res Pract 2024; 254: 155122.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ROS, TXNIP, NF-κB, Itch, COPD","lastPublishedDoi":"10.21203/rs.3.rs-6476972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6476972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eChronic inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), are characterized by pulmonary structural changes, narrowing of the small airways, and destruction of the lung parenchyma caused by prolonged inflammation. Sustained inflammation mediated by macrophages is considered to play a critical role in COPD pathogenesis, while the inductive mechanisms of persistent inflammation remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eIn vitro, RAW264.7 cells were treated with cigarette smoke extract (CSE), hydrogen peroxide, and 12-O-tetradecanoylphorbol-13-acetate. Loss-of-function assays were performed using MAPK inhibitors and Itch-specific knockdown. In vivo, lung tissues from mice exposed to whole-body cigarette smoke for 12 weeks, as well as clinical samples from healthy non-smokers, a healthy smoker, and COPD patients, were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e We revealed that thioredoxin-interacting protein (TXNIP) participates in cigarette smoke-incited NF-κB activation that potentially conducted pulmonary inflammation. CSE markedly inhibits TXNIP expression in RAW264.7 cells through MAPKs-dependent regulation, accompanied by the induction of iNOS/NO and COX-2. The decrease in TXNIP was also detected in lung tissues and macrophages obtained from smoking mice, while higher NF-κB activation and lung inflammation occurred simultaneously. Additionally, cigarette smoke-associated oxidative stress initiated the proteasomal degradation of TXNIP followed by the MAPKs-regulated NF-κB activation concurrently. The expression of E3 ligase Itch was elevated in smoking mouse lungs and in hydrogen peroxide-stimulated cells, whereas specific silencing Itch significantly attenuated TXNIP degradation as well as NF-κB activation. Moreover, TXNIP was distinctly suppressed in lung tissues, bronchoalveolar lavage fluid cells and peripheral blood mononuclear cells obtained from patients with COPD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Accordingly, cigarette smoke-induced oxidative stress causes Itch-mediated TXNIP degradation, leading to NF-κB inflammation and potentially enabling COPD pathogenesis.\u003c/p\u003e","manuscriptTitle":"Oxidative Stress Triggers Itch-mediated TXNIP Degradation and NF-κB Activation Promoting Chronic Obstructive Pulmonary Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:41:20","doi":"10.21203/rs.3.rs-6476972/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-11T22:04:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T18:54:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154959660780150271525773268086292788315","date":"2025-06-14T07:39:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-26T07:18:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202227978198804159806950002569436811659","date":"2025-04-30T04:59:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T23:48:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-25T11:41:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T12:25:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Respiratory Research","date":"2025-04-18T07:22:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc1b74a6-75e0-4a13-8cef-da11d12595ee","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:08:49+00:00","versionOfRecord":{"articleIdentity":"rs-6476972","link":"https://doi.org/10.1186/s12931-025-03369-5","journal":{"identity":"respiratory-research","isVorOnly":false,"title":"Respiratory Research"},"publishedOn":"2025-10-17 15:57:32","publishedOnDateReadable":"October 17th, 2025"},"versionCreatedAt":"2025-05-07 06:41:20","video":"","vorDoi":"10.1186/s12931-025-03369-5","vorDoiUrl":"https://doi.org/10.1186/s12931-025-03369-5","workflowStages":[]},"version":"v1","identity":"rs-6476972","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6476972","identity":"rs-6476972","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}