The NDUFS1/BRK1 Axis in COPD Pathogenesis: A Multi-Omics Approach Linking Mitochondrial Dysfunction to Actin Cytoskeleton Disruption

The NDUFS1/BRK1 Axis in COPD Pathogenesis: A Multi-Omics Approach Linking Mitochondrial Dysfunction to Actin Cytoskeleton Disruption | 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 The NDUFS1/BRK1 Axis in COPD Pathogenesis: A Multi-Omics Approach Linking Mitochondrial Dysfunction to Actin Cytoskeleton Disruption Conghui Liu, Aishuang Fu, Jin Ye, Yang Wen, Xiaojie Zhang, Tingyou Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8990913/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Chronic obstructive pulmonary disease (COPD) is a major global health burden, affecting over 300 million people and ranking as the third leading cause of death worldwide. Current therapies do not modify disease progression, and molecular mechanisms linking mitochondrial dysfunction and cytoskeletal disruption remain unclear. Emerging evidence implicates NDUFS1, a subunit of mitochondrial complex I, and BRK1, a key regulator of actin cytoskeleton dynamics, in COPD pathogenesis, potentially through related pathways. Methods: We conducted an integrative multi-omics study combining transcriptomic profiling of lung tissues from COPD patients and healthy controls, two-sample Mendelian randomization(MR) using genome-wide association study summary statistics, and network pharmacology coupled with molecular docking. Transcriptomic analyses assessed differential expression of NDUFS1 and BRK1, their correlation with clinical parameters such as forced vital capacity and GOLD stages, and pathway-level changes via gene set enrichment analysis. MR used inverse variance weighted method as primary analysis, supported by MR-Egger, weighted median, and mode-based approaches, with sensitivity tests for pleiotropy. Network pharmacology identified compounds targeting NDUFS1, followed by docking to evaluate binding affinities. Results: Transcriptomic results showed NDUFS1 upregulation and BRK1 downregulation in COPD. BRK1 decreased across GOLD stages and correlated positively with forced vital capacity. Gene set enrichment analysis revealed suppression of the actin cytoskeleton pathway, strongly negatively correlated with NDUFS1 and positively with BRK1. MR indicated a protective causal effect of BRK1 on COPD risk, with consistent directional support across methods. Higher NDUFS1 expression causally reduced BRK1 levels, with no significant pleiotropy. β-sitosterol showed the strongest predicted binding affinity to NDUFS1 among screened phytochemicals. Conclusion: Dysregulation of NDUFS1 and BRK1 may contribute to COPD via mitochondrial-cytoskeletal crosstalk. Causal evidence supports further investigation, with β-sitosterol emerging as a candidate for therapeutic development. Chronic obstructive pulmonary disease NDUFS1 BRK1 Mitochondrial dysfunction Actin cytoskeleton Multi-omics Network pharmacology β-sitosterol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Chronic obstructive pulmonary disease (COPD) is a significant global health concern characterized by progressive airflow limitation and chronic inflammation, leading to substantial morbidity and mortality( 1 , 2 ). With over 300 million affected individuals and ranking as the third leading cause of death worldwide, the burden of this disease is profound, particularly as healthcare costs in the United States surpass $ 50 billion annually( 1 , 3 ). Current therapeutic strategies, including bronchodilators and corticosteroids, predominantly focus on symptom management and do not address the underlying pathophysiological processes that drive disease progression or reverse lung damage, highlighting an urgent need for novel therapeutic targets and mechanisms to combat this debilitating condition( 4 , 5 ). Traditional Chinese medicine (TCM) has recently demonstrated unique value and potential in the treatment of COPD, with its holistic perspective and multi-target regulatory approach aligning well with the complex pathological network of COPD( 6 – 9 ). However, the complex composition of herbal formulas and the unclear material basis and molecular mechanisms underlying their effects have long limited the modernization and internationalization of TCM( 10 , 11 ). Network pharmacology, as an emerging research paradigm, systematically elucidates the synergistic mechanisms of multi-component, multi-target, and multi-pathway actions by constructing multidimensional “drug–component–target–disease” networks, providing a powerful tool to translate traditional empirical knowledge into modern scientific language( 9 , 12 ). This study takes seabuckthorn, a traditional Chinese herb known for its cough-relieving, expectorant, blood-activating, and stasis-resolving properties, as an example, applying network pharmacology combined with molecular docking techniques to elucidate the potential bioactive compounds and molecular mechanisms underlying its therapeutic effects on COPD at the molecular level, thereby offering new insights for the modernization and clinical translation of TCM( 13 ). Recent research efforts have sought to elucidate the molecular mechanisms underlying COPD pathogenesis, revealing critical insights into various regulatory pathways( 14 ). Among these, the NDUFS1/BRK1 axis emerges as a notable area of interest. NDUFS1, a mitochondrial complex I subunit, and BRK1, an actin cytoskeleton regulator, have been linked to oxidative stress and cellular integrity in other disease( 15 ). Preliminary findings suggest that the reciprocal expression patterns of these two proteins may contribute to tissue destruction associated with COPD, thereby positioning this regulatory axis as a promising target for therapeutic intervention.To investigate the NDUFS1/BRK1 axis, this study employs an integrative approach that combines transcriptomics with Mendelian randomization(MR) and network pharmacology. The use of MR allows researchers to leverage genetic variants as instrumental variables to infer causal relationships, effectively mitigating confounding biases typically present in observational studies( 16 , 17 ). Additionally, molecular docking techniques are utilized to validate potential therapeutic candidates, thereby bridging the gap between molecular discovery and practical application in clinical settings. The primary objectives of this research are threefold: first, to establish the NDUFS1/BRK1 axis as a causal driver of COPD; second, to elucidate the mechanistic link between this axis and actin cytoskeleton dysfunction; and third, to identify small-molecule modulators that could serve as potential therapeutic agents. By elucidating the complex interplay between mitochondrial dysfunction and cytoskeletal integrity, this study aims to contribute significantly to the understanding of COPD pathogenesis and foster the development of targeted therapeutic strategies that could significantly impact patient outcomes. Ultimately, the findings from this study could redefine the molecular landscape of COPD, offering new insights and potential pathways for intervention that address the chronic and progressive nature of this disease. By targeting the NDUFS1/BRK1 axis, we may pave the way for innovative strategies that not only manage symptoms but also potentially halt disease progression and restore lung function in affected individuals. 2. Methods 2.1 Transcriptomic Profiling and Differential Expression Analysis Differential expression analysis of NDUFS1 and BRK1 was performed using DESeq2 with false discovery rate (FDR) adjustment, applying a significance threshold of FDR 1. 2.2 Clinical Correlation and Pathway Analysis Correlation analyses between gene expression levels and clinical parameters including forced vital capacity (FVC) and GOLD stages were conducted using Pearson's correlation coefficient for normally distributed data and Spearman's rank correlation for non-parametric data. Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler R package to evaluate enrichment of the actin cytoskeleton pathway (GO:0015629). Normalized Enrichment Scores (NES) were calculated with 1000 permutations, and significance was determined at FDR < 0.001. 2.3 MR Study Design We conducted a two-sample MR study to investigate the causal effects of gene expression levels of NDUFS1 and BRK1 on the risk of Chronic Obstructive Pulmonary Disease (COPD), as well as the causal effect of NDUFS1 on BRK1. This study was reported in accordance with the STROBE-MR guidelines (Strengthening the Reporting of Observational Studies in Epidemiology using MR). A completed STROBE-MR checklist is provided in Supplementary File 1. 2.4 Data Sources Summary-level data for all exposures and outcomes were obtained from publicly available genome-wide association study (GWAS) repositories. No specific ethical approval or individual participant consent was required for this study as only aggregated summary statistics were used.The characteristics of the datasets are summarized in Table 1 : Table 1 Characteristics of the GWAS datasets used in the MR analysis. Trait Gene/ID Role OpenGWAS ID Source Sample Size (Total) Cases Controls Ancestry Year PMID NDUF-S1 ENSG00000023228 Exposure eqtl-a-ENSG00000023228 Univ. of Washington 26,609 - - European 2018 - BRK1 ENSG00000254999 Exposure eqtl-a-ENSG00000254999 Univ. of Washington 25,690 - - European 2018 - COPD - Outcome ebi-a-GCST90018807 Sakaue S et al. 468,475 13,530 454,945 European 2021 34594039 Exposure 1 (NDUFS1): Summary statistics for NDUFS1 (ENSG00000023228) gene expression were sourced from the University of Washington (OpenGWAS ID: eqtl-a-ENSG00000023228). The dataset included 26,609 individuals of European ancestry (Year: 2018). Exposure 2 (BRK1): Summary statistics for BRK1 (ENSG00000254999) gene expression were obtained from the University of Washington (OpenGWAS ID: eqtl-a-ENSG00000254999), comprising 25,690 individuals of European ancestry (Year: 2018). Outcome (COPD): Summary statistics for COPD were derived from the Sakaue S et al. study (OpenGWAS ID: ebi-a-GCST90018807). This study included a total of 468,475 participants, consisting of 13,530 cases and 454,945 controls of European ancestry. Citation: Sakaue S, et al. Nature Genetics. 2021; PMID: 34594039( 18 ). Given that the eQTL data (N≈26k) and the COPD GWAS (N≈468k) were derived from distinct consortia and study populations, significant sample overlap was unlikely, minimizing the risk of weak instrument bias due to overlap. Potential sample overlap is considered negligible as the studies were conducted by independent consortia with distinct recruitment criteria. 2.5 MR Statistical Analysis and Sensitivity Testing Instrumental Variable Selection Instrumental variable selection was based on the three core MR assumptions: ( 1 ) Relevance (SNPs strongly associated with exposure), ( 2 ) Independence (SNPs not associated with confounders), and ( 3 ) Exclusion Restriction (SNPs affect outcome only through exposure). SNPs were selected based on genome-wide significance. Instrumental variables (IVs) were selected using the following criteria: 1) genome-wide significance ( P < 5×10⁻⁸); 2) independence (linkage disequilibrium r² 10 ). A total of 9 independent SNPs were identified as valid IVs for the analysis. The F-statistic was calculated using the formula: F = ( β ² /SE ²), where β represents the effect size and SE the standard error. The inverse variance weighted (IVW) method served as the primary analysis approach. Complementary methods including weighted median, MR-Egger, weighted mode, and simple mode were employed for sensitivity analysis. Heterogeneity was assessed using Cochran's Q statistic, with P < 0.05 indicating significant heterogeneity. Horizontal pleiotropy was evaluated through MR-Egger intercept testing, and leave-one-out analysis was performed to examine the influence of individual SNPs on the overall causal estimate. 2.6 Network Pharmacology and Compound Screening Bioactive compounds targeting NDUFS1 were screened from multiple databases including TCMSP, TCM Database@Taiwan, and Coremine Medical. Selection criteria included oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. Compound-target networks were constructed using Cytoscape 3.9.1, and topological analysis was performed using CytoNCA plugin to calculate degree centrality. The top five compounds based on degree values were selected for further analysis. 2.7 Molecular Docking and Binding Affinity Assessment The three-dimensional structure of NDUFS1 (UniProt ID: P28331) was retrieved from the Protein Data Bank (PDB ID: 5LDX). Ligand structures were obtained from PubChem database and optimized using ChemBio3D Ultra 14.0. Molecular docking simulations were performed using AutoDock Vina with the following parameters: grid box size 60×60×60 A, exhaustiveness setting of 20. Each ligand-receptor pair underwent 20 independent docking runs, and the conformation with the lowest binding energy was selected for interaction analysis. Hydrogen bonding, hydrophobic interactions, and binding energies were analyzed using Discovery Studio 2016. 2.8 Statistical Software and Reproducibility All statistical analyses were conducted using R version 4.2.1. Specific packages included DESeq2 (v1.36.0) for differential expression, clusterProfiler (v4.4.4) for enrichment analysis, and Two Sample MR (v0.5.6) for MR. Visualization was performed using gplot2 (v3.4.0) and pheatmap (v1.0.12) ( http://www.phenoscanner.medschl.cam.ac.uk/ ). All code and analysis pipelines are available upon request to ensure reproducibility. 2.9 Ethical Considerations and Data Availability Ethical approval and informed consent were not required for this study as it is based on secondary analysis of publicly available aggregated summary statistics. All original GWAS and transcriptomic studies included in this analysis had obtained appropriate ethical approval and informed consent from their respective participants. 3. Results 3.1 Dysregulation of the NDUFS1/BRK1 axis in COPD and its clinical relevance Molecular analysis of lung tissues revealed significant alterations in the expression of key regulatory genes. NDUFS1 expression was markedly elevated in COPD samples compared to healthy controls (p < 0.001; Fig. 1 A). Conversely, BRK1 transcript levels showed significant reduction in the same patient cohort (p < 0.001; Fig. 1 B). This reciprocal expression pattern suggests potential counter-regulatory interactions between these genes in COPD pathogenesis. To assess the clinical relevance of these molecular changes, we examined BRK1 expression across disease severity stages. Progressive reduction in BRK1 mRNA levels was observed with increasing GOLD stages, with statistically significant differences between specific stages (Fig. 1 C). Furthermore, BRK1 expression demonstrated a significant positive correlation with forced vital capacity (R = 0.31, p = 0.031), a key pulmonary function parameter (Fig. 1 D). Analysis of the relationship between NDUFS1 and BRK1 revealed a significant negative correlation across all samples (R = -0.32, p = 0.00016), supporting potential regulatory interactions (Fig. 1 E). Gene set analysis focusing on cellular structural components revealed significant suppression of the actin cytoskeleton pathway in COPD tissues compared to controls (p < 0.001; Fig. 1 F). This finding suggests that the dysregulation of the NDUFS1/BRK1 axis may be functionally linked to alterations in cellular structural integrity, providing a potential mechanistic basis for the observed clinical correlations. 3.2 Integrating Computational Pharmacology and Transcriptomics Reveals a NDUFS1-Targeting Strategy for Restoring actin cytoskeleton pathway in COPD Unsupervised hierarchical clustering analysis of genome-wide expression data revealed clear separation between COPD and control samples, indicating disease-specific transcriptional reprogramming (Fig. 2 A). Further investigation of the actin cytoskeleton pathway, a critical component of cellular structure and function, demonstrated systematic downregulation of related genes in COPD tissues compared to controls (Fig. 2 B). Gene Set Enrichment Analysis confirmed significant suppression of the actin cytoskeleton pathway in COPD. The GSEA plot showed negative enrichment (NES = -1.68, FDR < 0.001), indicating coordinated downregulation of cytoskeleton-related genes (Fig. 2 C). In contrast, control samples exhibited significant positive enrichment of the same pathway (NES = 1.67, FDR < 0.001), confirming pathway integrity in healthy tissues (Fig. 2 D). To explore potential regulators of cytoskeletal function, we examined relationships between key genes and pathway activity. NDUFS1 expression showed a significant negative correlation with actin cytoskeleton pathway scores (R = -0.51, p = 1.9×10–10) (Fig. 2 E). Conversely, BRK1 expression demonstrated a strong positive correlation with the same pathway (R = 0.64, p < 2.2×10–16) (Fig. 2 F). These opposing correlations suggest that NDUFS1 and BRK1 may exert counter-regulatory effects on cytoskeletal maintenance. The coordinated downregulation of actin cytoskeleton genes in COPD, combined with the divergent associations of NDUFS1 and BRK1 with this pathway, provides a coherent molecular framework. The actin cytoskeleton is essential for maintaining alveolar architecture and epithelial barrier function, and its disruption may contribute to the characteristic tissue destruction in COPD. The opposing correlations of NDUFS1; negative and BRK1 ;positive with cytoskeletal integrity suggest a potential regulatory axis where NDUFS1-mediated suppression of BRK1 could lead to cytoskeletal dysfunction, offering a novel mechanism for COPD pathogenesis. 3.3 Functional Pathway Enrichment Analysis Functional enrichment analysis based on differentially expressed genes associated with NDUFS1 and BRK1 revealed distinct biological pathways (Fig. 3 ). For genes positively associated with BRK1 expression (Fig. 3 A), significant enrichment was observed in pathways related to actin cytoskeleton regulation​ (adjusted p-value < 0.001) and bacterial invasion of epithelial cells​ (adjusted p-value < 0.01). These findings align with BRK1's established role in cytoskeletal dynamics and cellular defense mechanisms. The color gradient (from dark blue to light yellow) indicates the significance level, with darker colors representing more significant p-values. For genes negatively associated with NDUFS1 expression (Fig. 3 B), the most significantly enriched pathway was oxidative phosphorylation (adjusted p-value < 0.001), consistent with the function of NDUFS1 as a mitochondrial complex I subunit. Additional pathways including carbon metabolism and biosynthesis of amino acids​ were also significantly enriched (adjusted p-value < 0.01). The dot size represents gene count within each pathway. Statistical significance and interpretation: Pathway enrichment was determined using hypergeometric testing with Benjamini-Hochberg correction for multiple comparisons. The strong enrichment of oxidative phosphorylation pathways for NDUFS1-associated genes supports the hypothesis that NDUFS1 dysregulation affects mitochondrial energy metabolism in COPD. Conversely, the enrichment of cytoskeletal and cellular defense pathways for BRK1-associated genes provides mechanistic insight into how BRK1 downregulation may contribute to impaired structural integrity and increased susceptibility to environmental insults in COPD pathogenesis. 3.4 MR Analysis Reveals a Causal Protective Role of BRK1 against COPD Risk To investigate the potential causal relationship between genetically predicted BRK1 expression and COPD risk, we conducted a two-sample MR analysis using nine SNPs as instrumental variables. As demonstrated in Fig. 4 A, analysis using the IVW method revealed a significant inverse causal association between genetically elevated BRK1 expression and COPD risk, yielding an odds ratio of 0.949 with a 95 percent confidence interval from 0.910 to 0.990 and attaining statistical significance at p = 0.016. This protective association was corroborated by complementary analytical approaches, with the Weighted Median method producing an odds ratio of 0.953 and the Weighted Mode method generating an odds ratio of 0.952, both achieving statistical significance at p equals 0.008 and 0.034, respectively. Although estimates from MR-Egger and Simple Mode analyses did not attain statistical significance, with p-values of 0.193 and 0.405 respectively, their consistent directional alignment with the primary findings reinforces the robustness of the observed protective effect. The forest plot presented in Fig. 4 B illustrates individual variant effect estimates and corresponding 95% confidence intervals for all nine instrumental single nucleotide polymorphisms. Each genetic variant demonstrated effect directions concordant with the overall protective estimate. Furthermore, visual examination of the MR scatter plot in Fig. 4 C supports the proposed the negative association, evidenced by the uniformly downward trending regression slopes generated through Inverse Variance Weighted, MR-Egger, and Weighted Median methodologies. Collectively, this MR analysis provides compelling genetic evidence supporting a causal relationship wherein elevated genetically predicted BRK1 expression associates with reduced COPD susceptibility, establishing BRK1 as a biologically plausible protective factor in COPD pathogenesis. 3.5 Sensitivity analyses for the causal effect of genetically predicted BRK1 expression on COPD risk To validate the reliability of our MR findings regarding the protective effect of BRK1 against COPD risk, we conducted a comprehensive set of sensitivity analyses(Fig. 5 ). These analyses were specifically designed to evaluate potential horizontal pleiotropy and to ensure that no single genetic variant disproportionately influenced our results. As shown in Fig. 5 A, using funnel plot analysis and the MR-Egger intercept test to assess directional pleiotropy, we observed a non-significant MR-Egger intercept = -0.002, p = 0.712, indicating no substantial evidence that the instrumental variables influenced COPD risk through pathways independent of BRK1 expression. This finding supports the validity of the core MR assumption. Visual inspection of the funnel plot revealed a generally symmetric distribution of individual single nucleotide polymorphism estimates around the inverse variance weighted summary estimate, with most points clustering near the null value. Notably, a single variant, rs10258, exhibited divergent characteristics with an effect direction opposite to the majority of instrumental variables and exceptionally high precision, measured as the inverse of its standard error at forty-eight point three, potentially suggesting pleiotropic properties that warrant further investigation. To evaluate the stability of our causal estimates, we performed a leave-one-out sensitivity analysis. As shown in Fig. 5 B, this approach systematically excluded each instrumental variable in turn and recalculated the inverse variance weighted derived causal estimate. The results demonstrated remarkable stability, as all leave-one-out estimates remained statistically significant and their confidence intervals exhibited substantial overlap with the overall estimate obtained using all nine SNPs. This consistency supports the proposed that our primary finding is robust and not unduly influenced by any individual genetic variant. Collectively, our sensitivity analyses provide strong additional support for the causal interpretation of the main MR results. The absence of significant horizontal pleiotropy combined with the stability of effect estimates across multiple analytical approaches strengthens the genetic evidence that elevated genetically predicted BRK1 expression confers protection against COPD development. 3.6 MR Analysis indicates a Causal Effect of NDUFS1 on BRK1 Expression To investigate the potential causal influence of NDUFS1 expression on BRK1 levels, we performed a two-sample MR analysis using genetic variants as instrumental variables. The results are presented in Fig. 6 . The primary analysis using the Inverse Variance Weighted method yielded a statistically significant causal estimate (OR = 0.873; 95% CI: 0.769–0.991; p = 0.036), indicating that a genetically predicted one-unit increase in NDUFS1 expression is associated with a significant reduction in BRK1 expression. The point estimates from complementary MR methods, including the Weighted Median, Simple Mode, and Weighted Mode, were all less than 1.0, suggesting a consistent direction of the negative causal effect, although they did not reach conventional statistical significance thresholds (p = 0.094, 0.131, and 0.128, respectively) (Fig. 6 A). The MR Egger estimate was not significant (p = 0.452). The individual Wald ratio estimates for each instrumental variable SNP are detailed in Fig. 6 B. The combined MR Egger and Inverse Variance Weighted summary estimates align with the primary analysis, indicating a negative overall effect. The scatter plot (Fig. 6 C) visually supports the proposed the negative trend, as evidenced by the downward slopes of the regression lines from all four MR methods. The close alignment of these lines, particularly between the Inverse Variance Weighted and MR Egger methods, suggests the absence of substantial directional horizontal pleiotropy that would invalidate the MR assumptions. Collectively, this MR analysis provides genetic evidence supporting a causal, negative regulatory relationship whereby elevated NDUFS1 expression leads to decreased BRK1 expression. This finding establishes a potential upstream molecular mechanism for the observed dysregulation of the NDUFS1/BRK1 axis in COPD pathogenesis. 3.7 Network Pharmacology Analysis Identifies Potential Bioactive Compounds Targeting NDUFS1 Construction and Analysis of the Compound-Target Network To identify potential therapeutic agents capable of modulating the NDUFS1/BRK1 axis, we performed network pharmacology analysis. First, we constructed an interaction network comprising predicted bioactive compounds and their potential protein targets relevant to COPD pathogenesis (Fig. 7 A). The network revealed β-sitosterol as a central node, exhibiting connections with multiple key targets including CYP1A1, UGT1A1, and AKR1C1, all of which are implicated in oxidative stress response and xenobiotic metabolism—processes critically dysregulated in COPD. The high degree of connectivity suggests that β-sitosterol may exert multi-target effects, potentially enhancing its therapeutic efficacy. Molecular Docking Validates High-Affinity Binding to NDUFS1 To validate and quantify the interaction between the identified central compound and our primary target, we performed molecular docking simulations against NDUFS1. As shown in Fig. 7 B, β-sitosterol demonstrated the strongest predicted binding affinity to NDUFS1, with a docking score of -9.70 kcal/mol. Other related phytosterols and flavonoids, including sitosterol (-9.40 kcal/mol), quercetin (-9.00 kcal/mol), kaempferol (-8.40 kcal/mol), and stigmasterol (-8.20 kcal/mol), also showed favorable binding energies below the conventional threshold of -7.0 kcal/mol, indicating stable binding interactions. The superior score of β-sitosterol suggests it is the most promising candidate for direct NDUFS1 modulation. Integration of Network and Docking Results The integration of network topology (identifying β-sitosterol as a key hub) and molecular docking (confirming its high-affinity binding to NDUFS1) provides a two-tiered validation. This approach not only identifies a potential lead compound but also proposes a mechanistic basis for its action—through direct interaction with NDUFS1, a central regulator of mitochondrial function and redox balance implicated in our previous analyses. Conclusion of the Analysis This combined network pharmacology and molecular docking strategy successfully identifies β-sitosterol as a top-ranking, multi-target bioactive compound with high predicted affinity for NDUFS1. These computational findings provide a strong rationale for the subsequent experimental investigation of β-sitosterol (and its source, Sea Buckthorn) as a modulator of the NDUFS1/BRK1 axis and actin cytoskeleton pathway in COPD models. 4. Discussion Chronic obstructive pulmonary disease (COPD) remains a formidable global health challenge, affecting approximately 300 million individuals worldwide and ranking as the third leading cause of mortality( 8 ). COPD management employs a stepwise strategy tailored to disease severity and patient phenotypes. For moderate-to-severe cases, particularly those with exacerbation risks, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines prioritize triple therapy—combining inhaled corticosteroids (ICS), long-acting β2-agonists (LABA), and long-acting muscarinic antagonists (LAMA)—to alleviate symptoms and reduce acute exacerbations( 8 , 19 , 20 ). However, approximately 50% of severe patients continue to experience frequent exacerbations post-treatment, culminating in accelerated lung function decline, elevated hospitalization rates, and increased mortality. For individuals refractory to dual or triple therapy, novel targeted biologics like dupilumab—the first biologic approved for COPD—offer promise for type 2-inflammatory phenotypes (e.g., eosinophil counts ≥ 300 cells/µL) via IL-4/IL-13 pathway inhibition( 4 , 21 ). Yet, its efficacy remains limited in non–type 2 phenotypes, while high costs constrain accessibility. In contrast, Traditional Chinese Medicine (TCM) emphasizes holistic, multi-target regulation, aligning with COPD’s complex pathogenesis( 13 , 22 ). Network pharmacology enables systematic dissection of herbal medicine’s multi-component, multi-target, and multi-pathway interactions, addressing gaps in mechanistic understanding. By elucidating specific targets and pathways of active compounds,such as, the NDUFS1/BRK1 axis and associated oxidative stress cytoskeletal pathways, this approach may scientifically support TCM’s empirical efficacy scientifically, fosters international recognition, and proposes innovative solutions for therapeutic bottlenecks. Multi-target interventions, guided by network pharmacology, may yield broader efficacy by concurrently modulating key disease nodes, potentially improving long-term outcomes, slowing progression, and reducing resistance risks from single-pathway suppression. Furthermore, precise identification of herbal targets informs personalized integrative strategies—combining TCM with conventional drugs—tailored to clinical or TCM-defined subtypes, optimizing COPD management through synergistic, evidence-based combinations.This progressive respiratory disorder, characterized by persistent airflow limitation and chronic inflammation, continues to impose substantial morbidity and economic burden on healthcare systems globally. Current therapeutic approaches, predominantly centered on bronchodilators and corticosteroids, primarily address symptom management rather than modifying the underlying disease progression or reversing established lung damage. The persistent limitations of these interventions underscore the critical need to identify novel therapeutic targets and elucidate fundamental mechanisms driving COPD pathogenesis. This study, through an integrative multi-omics approach, systematically uncovers for the first time the critical role of the NDUFS1/BRK1 regulatory axis in COPD. Key findings include significantly upregulated expression of NDUFS1 and concomitantly downregulated expression of BRK1 in COPD lung tissues, with a strong negative correlation observed between the two. BRK1 expression progressively declines with increasing disease severity (as stratified by GOLD stages) and shows a significant positive correlation with FVC, a key pulmonary function parameter. Functional enrichment analyses reveal substantial suppression of the actin cytoskeleton pathway in COPD, with its activity negatively correlated with NDUFS1 expression and positively correlated with BRK1 expression. MR analysis provides genetic evidence supporting a potential protective role of genetically predicted BRK1 against COPD risk and further suggests a potential causal, negative effect of NDUFS1 on BRK1 expression, assuming the validity of instrumental variable assumptions. Leveraging this axis, network pharmacology identifies β-sitosterol as a lead compound with potential inhibitory activity against NDUFS1. This work represents the first report of coordinated dysregulation of NDUFS1 and BRK1 in COPD. NDUFS1, a core subunit of mitochondrial complex I, is upregulated in COPD—a finding consistent with established paradigms implicating mitochondrial dysfunction and oxidative stress in COPD pathogenesis (e.g., increased mitochondrial reactive oxygen species ROS production leading to cellular damage)( 17 , 23 ). Conversely, BRK1, a critical regulator of the actin cytoskeleton and component of the WAVE regulatory complex, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This offers a novel molecular explanation for emphysematous destruction and epithelial barrier impairment observed in COPD. Previous studies have demonstrated that mitochondrial ROS can modulate transcription factors such as Nrf2 and FoxO, which are known to regulate oxidative stress responses and cytoskeletal dynamics( 13 ). It is plausible that elevated NDUFS1 expression enhances mitochondrial ROS generation, which in turn influences BRK1 expression through these transcription factors. Moreover, hypoxia-inducible factor 1-alpha, activated under COPD-related hypoxic conditions, may also contribute to transcriptional repression of BRK1. Epigenetic mechanisms, including DNA methylation and histone modifications, have been shown to regulate gene expression in COPD and may further mediate BRK1 downregulation. Future studies employing chromatin immunoprecipitation, methylation profiling, and reporter assays are warranted to validate these regulatory pathways. Regarding NDUFS1, prior reports indicate its altered expression in COPD lung tissue, consistent with our findings, though the direct link to BRK1 has not been previously described. BRK1’s role in respiratory diseases remains underexplored; this study is the first to implicate BRK1 as a potentially protective factor in COPD pathogenesis. Cross-talk with established COPD-related signaling pathways such as TGF-β, Wnt, and Notch may exist, given their known involvement in cytoskeletal remodeling and fibrosis. BRK1 and the WAVE complex have been implicated in Wnt signaling modulation, suggesting potential integrative mechanisms that warrant further exploration. Conversely, BRK1, a regulator of the actin cytoskeleton, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This provides a novel molecular explanation for emphysematous destruction and epithelial barrier impairment in COPD. In contrast to prior studies predominantly focused on inflammation or protease–antiprotease imbalance, our findings bridge mitochondrial dysfunction and loss of cellular structural integrity via the NDUFS1/BRK1 axis, proposing an integrative pathophysiological framework. Regarding the mechanism by which NDUFS1 regulates BRK1, our data suggest transcriptional or post-transcriptional control( 24 , 25 ). The robust inverse expression correlation, coupled with MR evidence supporting a potential causal effect of NDUFS1 on BRK1, leads us to hypothesize that NDUFS1 overexpression may enhance mitochondrial reactive oxygen species (ROS) production, thereby modulating specific transcription factors (e.g., Nrf2, FoxO, or HIF-1α) or inducing epigenetic alterations that ultimately suppress BRK1 transcription. This hypothesis transforms correlative observations into testable molecular pathways, warranting future validation through chromatin immunoprecipitation, reporter assays, and related functional experiments. A major strength of this study lies in its multidimensional integration: transcriptomics delineated expression profiles and pathway alterations, MR provided genetic-level causal inference, and network pharmacology pointed toward therapeutic opportunities—collectively enhancing the robustness and translational relevance of our findings. Nevertheless, several limitations must be acknowledged. First, key findings are based on transcriptomic data and require validation at the protein level. Second, although MR supports a directional causal relationship, definitive confirmation necessitates experimental validation in cellular and animal models and 4. Discussion Chronic obstructive pulmonary disease (COPD) remains a formidable global health challenge, affecting approximately 300 million individuals worldwide and ranking as the third leading cause of mortality( 8 ). COPD management employs a stepwise strategy tailored to disease severity and patient phenotypes. For moderate-to-severe cases, particularly those with exacerbation risks, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines prioritize triple therapy—combining inhaled corticosteroids (ICS), long-acting β2-agonists (LABA), and long-acting muscarinic antagonists (LAMA)—to alleviate symptoms and reduce acute exacerbations( 8 , 19 , 20 ). However, approximately 50% of severe patients continue to experience frequent exacerbations post-treatment, culminating in accelerated lung function decline, elevated hospitalization rates, and increased mortality. For individuals refractory to dual or triple therapy, novel targeted biologics like dupilumab—the first biologic approved for COPD—offer promise for type 2-inflammatory phenotypes (e.g., eosinophil counts ≥ 300 cells/µL) via IL-4/IL-13 pathway inhibition( 4 , 21 ). Yet, its efficacy remains limited in non–type 2 phenotypes, while high costs constrain accessibility. In contrast, Traditional Chinese Medicine (TCM) emphasizes holistic, multi-target regulation, aligning with COPD’s complex pathogenesis( 13 , 22 ). Network pharmacology enables systematic dissection of herbal medicine’s multi-component, multi-target, and multi-pathway interactions, addressing gaps in mechanistic understanding. By elucidating specific targets and pathways of active compounds,such as, the NDUFS1/BRK1 axis and associated oxidative stress cytoskeletal pathways, this approach validates TCM’s empirical efficacy scientifically, fosters international recognition, and proposes innovative solutions for therapeutic bottlenecks. Multi-target interventions, guided by network pharmacology, may yield broader efficacy by concurrently modulating key disease nodes, potentially improving long-term outcomes, slowing progression, and reducing resistance risks from single-pathway suppression. Furthermore, precise identification of herbal targets informs personalized integrative strategies—combining TCM with conventional drugs—tailored to clinical or TCM-defined subtypes, optimizing COPD management through synergistic, evidence-based combinations.This progressive respiratory disorder, characterized by persistent airflow limitation and chronic inflammation, continues to impose substantial morbidity and economic burden on healthcare systems globally. Current therapeutic approaches, predominantly centered on bronchodilators and corticosteroids, primarily address symptom management rather than modifying the underlying disease progression or reversing established lung damage. The persistent limitations of these interventions underscore the critical need to identify novel therapeutic targets and elucidate fundamental mechanisms driving COPD pathogenesis. Through integrative bioinformatics analysis, we propose the NDUFS1/BRK1 axis as a potential pivotal regulator in COPD, warranting further experimental validation. Key findings include significantly upregulated expression of NDUFS1 and concomitantly downregulated expression of BRK1 in COPD lung tissues, with a strong negative correlation observed between the two. BRK1 expression progressively declines with increasing disease severity (as stratified by GOLD stages) and shows a significant positive correlation with FVC, a key pulmonary function parameter. Functional enrichment analyses reveal substantial suppression of the actin cytoskeleton pathway in COPD, with its activity negatively correlated with NDUFS1 expression and positively correlated with BRK1 expression. MR analysis provides genetic evidence supporting a protective role of BRK1 against COPD risk and further suggests a potential causal, negative effect of NDUFS1 on BRK1 expression. Leveraging this axis, network pharmacology identifies β-sitosterol as a lead compound with potential inhibitory activity against NDUFS1. This work represents the first report of coordinated dysregulation of NDUFS1 and BRK1 in COPD. NDUFS1, a core subunit of mitochondrial complex I, is upregulated in COPD—a finding consistent with established paradigms implicating mitochondrial dysfunction and oxidative stress in COPD pathogenesis, increased mitochondrial reactive oxygen species ROS production leading to cellular damage). Conversely, BRK1, a critical regulator of the actin cytoskeleton and component of the WAVE regulatory complex, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This provides a novel molecular explanation for emphysematous destruction and epithelial barrier impairment observed in COPD. Previous studies have demonstrated that mitochondrial ROS can modulate transcription factors such as Nrf2 and FoxO, which are known to regulate oxidative stress responses and cytoskeletal dynamics( 13 ). It is plausible that elevated NDUFS1 expression enhances mitochondrial ROS generation, which in turn influences BRK1 expression through these transcription factors. Moreover, hypoxia-inducible factor 1-alpha, activated under COPD-related hypoxic conditions, may also contribute to transcriptional repression of BRK1. Epigenetic mechanisms, including DNA methylation and histone modifications, have been shown to regulate gene expression in COPD and may further mediate BRK1 downregulation. Future studies employing chromatin immunoprecipitation, methylation profiling, and reporter assays are warranted to validate these regulatory pathways. Regarding NDUFS1, prior reports indicate its altered expression in COPD lung tissue, consistent with our findings, though the direct link to BRK1 has not been previously described. BRK1’s role in respiratory diseases remains underexplored; this study is the first to implicate BRK1 as a potentially protective factor in COPD pathogenesis. Cross-talk with established COPD-related signaling pathways such as TGF-β, Wnt, and Notch may exist, given their known involvement in cytoskeletal remodeling and fibrosis. BRK1 and the WAVE complex have been implicated in Wnt signaling modulation, suggesting potential integrative mechanisms that warrant further exploration.This work represents the first report of coordinated dysregulation of NDUFS1 and BRK1 in COPD. NDUFS1, a core subunit of mitochondrial complex I, is upregulated in COPD—a finding consistent with established paradigms implicating mitochondrial dysfunction and oxidative stress in COPD pathogenesis( 17 , 23 ). Conversely, BRK1, a regulator of the actin cytoskeleton, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This provides a novel molecular explanation for emphysematous destruction and epithelial barrier impairment in COPD. In contrast to prior studies predominantly focused on inflammation or protease–antiprotease imbalance, our findings bridge mitochondrial dysfunction and loss of cellular structural integrity via the NDUFS1/BRK1 axis, proposing an integrative pathophysiological framework. Regarding the mechanism by which NDUFS1 regulates BRK1, our data suggest transcriptional or post-transcriptional control( 24 , 25 ). The robust inverse expression correlation, coupled with MR evidence supporting a potential causal effect of NDUFS1 on BRK1, leads us to hypothesize that NDUFS1 overexpression may enhance mitochondrial reactive oxygen species (ROS) production, thereby modulating specific transcription factors (e.g., Nrf2, FoxO, or HIF-1α) or inducing epigenetic alterations that ultimately suppress BRK1 transcription. This hypothesis transforms correlative observations into testable molecular pathways, warranting future validation through chromatin immunoprecipitation, reporter assays, and related functional experiments. A major strength of this study lies in its multidimensional integration: transcriptomics delineated expression profiles and pathway alterations, MR provided genetic-level causal inference, and network pharmacology pointed toward therapeutic opportunities—collectively enhancing the robustness and translational relevance of our findings. Nevertheless, several limitations must be acknowledged. First, key findings are based on transcriptomic data and require validation at the protein level. Second, although MR supports a directional causal relationship, definitive confirmation necessitates experimental validation in cellular and animal models. Third, bulk RNA-seq data cannot resolve cell-type-specific contributions (e.g., from epithelial cells, fibroblasts, or immune cells) to the dysregulation of this axis. Finally, while BRK1 expression correlates significantly with FVC, the moderate correlation coefficient (R = 0.31) suggests limited utility as a standalone biomarker, potentially requiring combination with other indicators for clinical application. Caution should be exercised when extrapolating these results to other groups, as our study cohort was predominantly composed of European. This study holds translational promise. BRK1’s association with disease severity and lung function positions it as a candidate biomarker for assessing COPD progression. More importantly, the NDUFS1/BRK1 axis itself represents a novel therapeutic target. Strategies aimed at restoring BRK1 function or inhibiting excessive NDUFS1 activity are thus plausible interventions. Notably, β-sitosterol—identified via network pharmacology—emerges as a promising lead compound, with computational modeling predicting high binding affinity for NDUFS1. However, translation faces challenges: as a plant-derived phytosterol, β-sitosterol requires rigorous evaluation of pulmonary-specific delivery, bioavailability, and long-term safety. Moreover, targeting mitochondrial complex I (via NDUFS1 inhibition) carries potential off-target risks to systemic energy metabolism, demanding careful therapeutic window assessment. Future preclinical studies in COPD animal models are essential to validate both efficacy and safety of interventions targeting this axis. In summary, we predict NDUFS1/BRK1 as a previously underappreciated yet pivotal regulatory axis in COPD, linking upstream mitochondrial dysfunction to downstream cytoskeletal collapse and offering a new mechanistic perspective on structural lung damage( 26 ). Genetic evidence supports a protective role for BRK1 and positions NDUFS1 as a potential upstream regulator. Computational screening highlights β-sitosterol as a starting point for therapeutic development. Future work should prioritize: ( 1 ) experimental validation of the molecular mechanisms underlying NDUFS1-mediated BRK1 regulation in cellular and animal models; ( 2 ) evaluation of β-sitosterol or alternative NDUFS1 inhibitors in preclinical COPD models; and ( 3 ) prospective validation of BRK1 as a prognostic biomarker in larger clinical cohorts. Deepening our understanding of this novel axis may pave the way for disease-modifying therapies in COPD. . Third, bulk RNA-seq data cannot resolve cell-type-specific contributions (e.g., from epithelial cells, fibroblasts, or immune cells) to the dysregulation of this axis. Finally, while BRK1 expression correlates significantly with FVC, the moderate correlation coefficient (R = 0.31) suggests limited utility as a standalone biomarker, potentially requiring combination with other indicators for clinical application. This study holds clear translational promise. BRK1’s association with disease severity and lung function positions it as a candidate biomarker for assessing COPD progression. More importantly, the NDUFS1/BRK1 axis itself represents a novel therapeutic target. Strategies aimed at restoring BRK1 function or inhibiting excessive NDUFS1 activity are thus plausible interventions. Notably, β-sitosterol—identified via network pharmacology—emerges as a promising lead compound, with computational modeling predicting high binding affinity for NDUFS1. However, translation faces challenges: as a plant-derived phytosterol, β-sitosterol requires rigorous evaluation of pulmonary-specific delivery, bioavailability, and long-term safety. Moreover, targeting mitochondrial complex I (via NDUFS1 inhibition) carries potential off-target risks to systemic energy metabolism, demanding careful therapeutic window assessment. Future preclinical studies in COPD animal models are essential to validate both efficacy and safety of interventions targeting this axis. In summary, we identify NDUFS1/BRK1 as a previously underappreciated yet pivotal regulatory axis in COPD, linking upstream mitochondrial dysfunction to downstream cytoskeletal collapse and offering a new mechanistic perspective on structural lung damage( 26 ). Genetic evidence supports a protective role for BRK1 and positions NDUFS1 as a potential upstream regulator. Computational screening highlights β-sitosterol as a starting point for therapeutic development. Future work should prioritize: ( 1 ) experimental validation of the molecular mechanisms underlying NDUFS1-mediated BRK1 regulation in cellular and animal models; ( 2 ) evaluation of β-sitosterol or alternative NDUFS1 inhibitors in preclinical COPD models; and ( 3 ) prospective validation of BRK1 as a prognostic biomarker in larger clinical cohorts. Deepening our understanding of this novel axis may pave the way for disease-modifying therapies in COPD. 5. Conclusion This study elucidates a novel pathogenic mechanism in COPD driven by the dysregulation of the NDUFS1/BRK1 axis, which bridges mitochondrial dysfunction and actin cytoskeleton disruption. Through an integrative multi-omics approach, we provide robust genetic evidence via MR that elevated NDUFS1 causally suppresses BRK1, contributing to disease progression and impaired pulmonary function. Furthermore, our network pharmacology and molecular docking analyses identify β-sitosterol as a promising therapeutic candidate capable of targeting NDUFS1. These findings not only advance the understanding of COPD pathophysiology but also highlight the potential of modulating the mitochondrial-cytoskeletal interface as a new strategy for disease-modifying therapies. Future experimental studies are warranted to validate these computational predictions and explore the clinical efficacy of β-sitosterol or similar agents in COPD management. Declarations Ethics approval and consent to participate Ethical approval and informed consent were not required for this study as it is based on secondary analysis of publicly available aggregated summary statistics. All original GWAS and transcriptomic studies included in this analysis had obtained appropriate ethical approval and informed consent from their respective participants. Consent for publication Not applicable. This manuscript does not contain any individual person’s data, such as personal details, images, or videos. Availability of data and materials The datasets supporting the conclusions of this article are available in public repositories. The transcriptomic datasets analyzed during the current study are available in the GEO repository. The summary-level data for the MR analysis are available from the corresponding GWAS consortia websites . The code and scripts used for the statistical analyses and molecular docking are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Health Commission of Hebei Province, China under Grant number ZF2026417. Authors’ contributions Y. G. and F. M. conceived and designed the study. Y. W. performed the bioinformatics analysis and MR. J.y. conducted the network pharmacology and molecular docking simulations. C. L., A. F. and T. C. interpreted the data and drafted the manuscript. J. Z. , X. Z. and B. L. critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript. Acknowledgements We thank the researchers and participants of the original GWAS and GEO studies for making their data publicly available. We also acknowledge the support of the North China University of Science and Technology Affiliated Hospital for providing the computational resources necessary for this study. References Oh J, et al. Global, regional, and national burden of chronic respiratory diseases and impact of the COVID-19 pandemic, 1990–2023: a Global Burden of Disease study. Nat Med. 2026;32:197–223. Boers E, et al. 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ER-localized ERO1α and caspase-3-mediated cleavage of mitochondrial NDUFS1 drives trichothecene-induced ROS accumulation in liver. Free Radic Biol Med. 2026;244:452–63. Chen T, et al. Loss of NDUFS1 promotes gastric cancer progression by activating the mitochondrial ROS-HIF1α-FBLN5 signaling pathway. Br J Cancer. 2023;129:1261–73. Dunham-Snary KJ, et al. Ndufs2, a Core Subunit of Mitochondrial Complex I, Is Essential for Acute Oxygen-Sensing and Hypoxic Pulmonary Vasoconstriction. Circul Res. 2019;124:1727–46. Eapen MS, Sharma P, Sohal SS. Mitochondrial dysfunction in macrophages: a key to defective bacterial phagocytosis in COPD. Eur Respir J 54, (2019). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8990913","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607554748,"identity":"23a9f9e2-0625-427c-9cda-d6b486cccd03","order_by":0,"name":"Conghui Liu","email":"","orcid":"","institution":"North China University of Science and Technology Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Conghui","middleName":"","lastName":"Liu","suffix":""},{"id":607554749,"identity":"91b3326a-c423-4810-9146-04d97c16665d","order_by":1,"name":"Aishuang Fu","email":"","orcid":"","institution":"North China University of Science and Technology Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Aishuang","middleName":"","lastName":"Fu","suffix":""},{"id":607554750,"identity":"21417f6c-f986-4ed4-a16b-21260c24df00","order_by":2,"name":"Jin Ye","email":"","orcid":"","institution":"North China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Ye","suffix":""},{"id":607554752,"identity":"74d3e6f9-0394-4371-bd63-6e6ee0466656","order_by":3,"name":"Yang Wen","email":"","orcid":"","institution":"North China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Wen","suffix":""},{"id":607554753,"identity":"7725095b-c000-46f2-aa36-6d1684d18016","order_by":4,"name":"Xiaojie Zhang","email":"","orcid":"","institution":"Innovita Biological Technology Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Xiaojie","middleName":"","lastName":"Zhang","suffix":""},{"id":607554754,"identity":"da691f5a-8cfb-4186-b5e3-ee789fb8121f","order_by":5,"name":"Tingyou Chen","email":"","orcid":"","institution":"Innovita Biological Technology Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Tingyou","middleName":"","lastName":"Chen","suffix":""},{"id":607554755,"identity":"ee54a94d-5c28-4999-9814-ee75836fa781","order_by":6,"name":"Bingsheng Lin","email":"","orcid":"","institution":"Innovita Biological Technology Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Bingsheng","middleName":"","lastName":"Lin","suffix":""},{"id":607554756,"identity":"2af17781-c7a9-4d60-9593-87e1965a2998","order_by":7,"name":"Jiakai Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiakai","middleName":"","lastName":"Zhang","suffix":""},{"id":607554757,"identity":"40913e1c-3815-49bd-adb7-b10acbb4d00e","order_by":8,"name":"Yanlei Ge","email":"","orcid":"","institution":"North China University of Science and Technology Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanlei","middleName":"","lastName":"Ge","suffix":""},{"id":607554758,"identity":"b09d76b7-2024-48f7-8c1b-cdb7d416cd71","order_by":9,"name":"Fanyang Mo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYLCCBwZAgvkA44OEihoitSSAtLAlMBs8OHOMWC0MYC1skg9bmAmr5m8/+/BBQsEdOd02HrOKxAY2oEh3Al4tEmfSjQ0SDJ4Zmx3jMbuRuEMGKHJ2A14tBgxpbBIJBocTt93vAWo5w8ZgIJFLQAv/M7CW+m1AWwoS25iJ0CIBsSUB5DAGorRI3HjGDPTLYcNtx9iKJRLOHOMh6Bf+/jTGBx/+HJY3O8a88eOPiho5/vZe/FqQAAcoQhl4iFUOAuwPSFE9CkbBKBgFIwgAAO3RR9n4wQJiAAAAAElFTkSuQmCC","orcid":"","institution":"North China University of Science and Technology Affiliated Hospital","correspondingAuthor":true,"prefix":"","firstName":"Fanyang","middleName":"","lastName":"Mo","suffix":""}],"badges":[],"createdAt":"2026-02-27 18:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8990913/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8990913/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104888043,"identity":"f5b22d76-0776-4347-883a-79ead54b4b4a","added_by":"auto","created_at":"2026-03-18 10:13:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":900717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential Expression and Functional Correlations of the NDUFS1/BRK1 Axis in COPD Pathogenesis. \u003c/strong\u003e(A) NDUFS1 mRNA levels are significantly upregulated in lung tissues from COPD patients compared to healthy controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (B) BRK1 transcript levels are significantly downregulated in COPD samples relative to controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Statistical significance in (A–B) was determined by an unpaired two-tailed t-test. (C) BRK1 expression shows a progressive, stage-dependent decrease across GOLD stages 0–4. Significant differences between specific stages were identified using one-way ANOVA with post-hoc testing (indicated by brackets). (D) Scatter plot demonstrating a significant positive correlation between BRK1 mRNA levels and forced vital capacity (FVC) (Pearson’s R = 0.31, \u003cem\u003ep\u003c/em\u003e= 0.031). The line represents linear regression with a 95% confidence interval. (E) Inverse correlation between NDUFS1 and BRK1 transcript levels across all samples (Pearson’s R = −0.32, \u003cem\u003ep\u003c/em\u003e = 0.00016), shown with linear regression and a 95% confidence band. (F) Gene set enrichment analysis reveals significant suppression of the \"Actin Cytoskeleton\" pathway (GO cellular component) in COPD samples compared to controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Due to non-normal distribution, significance was assessed using the Mann–Whitney U test. Data are presented as mean ± SD unless otherwise noted.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/980cc5486efbb3e2025a4566.png"},{"id":104888041,"identity":"da726bb0-1210-41b6-b411-38e5af233437","added_by":"auto","created_at":"2026-03-18 10:13:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":578862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystematic dysregulation of the actin cytoskeleton pathway in COPD and its associations with NDUFS1 and BRK1 expression. \u003c/strong\u003e(A) Hierarchical clustering of global gene expression profiles.​ Unsupervised clustering reveals distinct transcriptional patterns separating COPD and control samples, with clear segregation evident in the heatmap visualization. (B) Expression patterns of actin cytoskeleton-related genes. Heatmap analysis specifically focusing on genes within the actin cytoskeleton pathway indicates coordinated downregulation in COPD samples (blue) compared to controls (red). Key structural and regulatory genes are annotated on the right. (C) Gene Set Enrichment Analysis of actin cytoskeleton pathway in suppressed state. GSEA plot shows significant negative enrichment of the actin cytoskeleton gene set (NES = -1.68, FDR \u0026lt; 0.001) in the COPD group, indicating systematic downregulation of this cellular structure pathway. (D) Gene Set Enrichment Analysis of actin cytoskeleton pathway in activated state. Complementary GSEA plot indicates significant positive enrichment (NES = 1.67, FDR \u0026lt; 0.001) in the control group, confirming pathway integrity in healthy tissues. (E) Inverse correlation between NDUFS1 expression and actin cytoskeleton pathway activity. Scatter plot shows significant negative relationship (Pearson R = -0.51, p = 1.9×10-10) between NDUFS1 transcript levels and pathway enrichment scores. Linear regression line (blue) with 95% confidence interval (gray shading). (F) Positive correlation between BRK1 expression and actin cytoskeleton pathway activity. Scatter plot indicates strong positive correlation (Pearson R = 0.64, p \u0026lt; 2.2×10-16) between BRK1 expression and pathway activity scores.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/3c5058a8a29107919ff52c6f.png"},{"id":104888080,"identity":"d56c3932-22bd-4a04-b844-7ffe9b2a5618","added_by":"auto","created_at":"2026-03-18 10:13:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":806416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway enrichment analysis of genes associated with BRK1 and NDUFS1 expression.\u003c/strong\u003e (A) Pathways enriched for genes positively correlated with BRK1 expression. The vertical axis lists biological pathways, while the horizontal axis shows the gene count per pathway. Color intensity indicates statistical significance (p-value), with darker colors representing higher significance. Key enriched pathways include those related to actin cytoskeleton regulation and bacterial invasion of epithelial cells. (B) Pathways enriched for genes negatively correlated with NDUFS1 expression. Display format matches panel A. The oxidative phosphorylation pathway shows the strongest enrichment, followed by carbon metabolism and amino acid biosynthesis pathways.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/4edd90a0c42303c5ff6e7d28.png"},{"id":104888150,"identity":"49fb4a9c-55e3-4678-9bcb-505b08bf8294","added_by":"auto","created_at":"2026-03-18 10:13:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMR analysis examining the causal effect of genetically predicted BRK1 expression on COPD risk. \u003c/strong\u003e(A) Forest plot of MR effect estimates using five distinct methods.The plot displays odds ratios represented by red squares with horizontal lines indicating 95 percent confidence intervals for each MR method. The primary inverse variance weighted method IVW estimate shows a significant protective effect with an odds ratio of 0.949 and a p-value of 0.016. The dotted vertical line represents the null effect at an odds ratio of 1.0. All methods show consistent effect directions despite varying significance levels. (B) Combined visualization of MR analysis results. This panel consolidates the point estimates from all five MR methods. The central estimate and confidence interval for the primary IVW method are highlighted in red for emphasis. The consistency in effect direction across complementary methods supports the robustness of the primary finding. (C) Scatter plot of individual genetic variant associations. Each black point represents a single nucleotide polymorphism SNP used as an instrumental variable. The blue trend line illustrates the overall negative causal relationship where genetic variants associated with higher BRK1 expression correlate with lower COPD risk estimates.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/ef4e5ec70994e8a5290cda13.png"},{"id":104888099,"identity":"22573695-7637-4e3e-ac48-d87bf85d3ef5","added_by":"auto","created_at":"2026-03-18 10:13:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":259013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSensitivity analyses for the causal effect of genetically predicted BRK1 expression on COPD risk.\u003c/strong\u003e (A) Funnel plot assessing potential directional pleiotropy. The plot shows the relationship between the β coefficient for each instrumental variable SNP and its precision (1/SE). The close overlap between the Inverse Variance Weighted (light blue) and MR Egger (dark blue) regression lines suggests the absence of significant horizontal pleiotropy. The MR-Egger intercept test yielded a non-significant value of p = 0.712, supporting the validity of the MR assumption. (B) Leave-one-out sensitivity analysis evaluating result robustness. The forest plot displays the IVW-derived causal estimate for BRK1 expression on COPD risk after iteratively removing each individual SNP. The red diamond represents the combined estimate using all SNPs. The consistency of all leave-one-out estimates with the overall estimate, as evidenced by overlapping confidence intervals, supports the proposed that no single genetic variant disproportionately drives the observed protective association. Methods: Horizontal pleiotropy was assessed using the MR-Egger intercept test and visual inspection of the funnel plot. Leave-one-out analysis was performed by systematically excluding each SNP and recalculating the IVW estimate.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/ef7e396c9644c3dde9cee276.png"},{"id":104888091,"identity":"b6a1cdec-b004-44dd-8a10-f538a3673376","added_by":"auto","created_at":"2026-03-18 10:13:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":446679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMR Analysis Demonstrating Causal Effect of NDUFS1 on BRK1 Expression\u003c/strong\u003e (A) Forest plot of MR estimates. The primary analysis using the Inverse Variance Weighted (IVW) method revealed a significant potential causal negative effect of genetically predicted NDUFS1 expression on BRK1 levels (OR = 0.873; 95% CI: 0.769–0.991; p = 0.036). Complementary MR methods, including Weighted Median, Simple Mode, and Weighted Mode, showed consistent direction of effect (OR \u0026lt; 1.0), although they did not reach statistical significance. The MR Egger estimate was not significant (p = 0.452). (B) Individual Wald ratio estimates for each instrumental variable SNP. The forest plot displays the effect size and 95% confidence interval for each genetic variant used as an instrumental variable. The combined MR Egger and IVW summary estimates are shown at the bottom, both indicating a negative overall effect. (C) Scatter plot of SNP effects on NDUFS1 versus BRK1. Each point represents an instrumental variable SNP, with the x-axis showing its effect on NDUFS1 expression and the y-axis showing its effect on BRK1 expression. The downward slopes of the regression lines from all four MR methods (IVW, MR Egger, Weighted Median, and Simple Mode) visually support a potential causal negative effect. The close alignment of the IVW and MR Egger regression lines suggests the absence of substantial directional horizontal pleiotropy.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/ccabba6a52e478dbfd0fbd03.png"},{"id":104888101,"identity":"451c7c3a-d5df-40be-9d18-7dff2f8f2a8b","added_by":"auto","created_at":"2026-03-18 10:13:24","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork pharmacology and molecular docking analysis identifies β-sitosterol as a potential NDUFS1-targeting compound.\u003c/strong\u003e (A) Compound-Target Interaction Network.​ The network depicts predicted interactions between bioactive compounds (central nodes) and potential protein targets. Node size and color represent different entity types (compounds, proteins). β-sitosterol​ (highlighted) emerges as a central hub, connecting to multiple key targets involved in oxidative stress and metabolism, such as CYP1A1, UGT1A1, and AKR1C1. Lines represent predicted interactions. (B) Molecular Docking Scores for Candidate Compounds Binding to NDUFS1. The bar chart displays the predicted binding affinity (docking score in kcal/mol) of the top five candidate compounds against the NDUFS1 protein. β-sitosterol shows the most favorable binding energy (-9.70 kcal/mol), followed by sitosterol, quercetin, kaempferol, and stigmasterol. A more negative score indicates a\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/a6b742c9eca69a21671e6ad8.jpeg"},{"id":107526735,"identity":"7d39f2a3-f92b-4cba-9620-e0b2c4e4b1c0","added_by":"auto","created_at":"2026-04-22 09:43:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3791342,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8990913/v1/c3e299a1-80ce-4e40-af1b-d232590a55f4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The NDUFS1/BRK1 Axis in COPD Pathogenesis: A Multi-Omics Approach Linking Mitochondrial Dysfunction to Actin Cytoskeleton Disruption","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChronic obstructive pulmonary disease (COPD) is a significant global health concern characterized by progressive airflow limitation and chronic inflammation, leading to substantial morbidity and mortality(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). With over 300\u0026nbsp;million affected individuals and ranking as the third leading cause of death worldwide, the burden of this disease is profound, particularly as healthcare costs in the United States surpass \u003cspan\u003e$\u003c/span\u003e50\u0026nbsp;billion annually(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Current therapeutic strategies, including bronchodilators and corticosteroids, predominantly focus on symptom management and do not address the underlying pathophysiological processes that drive disease progression or reverse lung damage, highlighting an urgent need for novel therapeutic targets and mechanisms to combat this debilitating condition(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Traditional Chinese medicine (TCM) has recently demonstrated unique value and potential in the treatment of COPD, with its holistic perspective and multi-target regulatory approach aligning well with the complex pathological network of COPD(\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the complex composition of herbal formulas and the unclear material basis and molecular mechanisms underlying their effects have long limited the modernization and internationalization of TCM(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Network pharmacology, as an emerging research paradigm, systematically elucidates the synergistic mechanisms of multi-component, multi-target, and multi-pathway actions by constructing multidimensional \u0026ldquo;drug\u0026ndash;component\u0026ndash;target\u0026ndash;disease\u0026rdquo; networks, providing a powerful tool to translate traditional empirical knowledge into modern scientific language(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This study takes seabuckthorn, a traditional Chinese herb known for its cough-relieving, expectorant, blood-activating, and stasis-resolving properties, as an example, applying network pharmacology combined with molecular docking techniques to elucidate the potential bioactive compounds and molecular mechanisms underlying its therapeutic effects on COPD at the molecular level, thereby offering new insights for the modernization and clinical translation of TCM(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Recent research efforts have sought to elucidate the molecular mechanisms underlying COPD pathogenesis, revealing critical insights into various regulatory pathways(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Among these, the NDUFS1/BRK1 axis emerges as a notable area of interest. NDUFS1, a mitochondrial complex I subunit, and BRK1, an actin cytoskeleton regulator, have been linked to oxidative stress and cellular integrity in other disease(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePreliminary findings suggest that the reciprocal expression patterns of these two proteins may contribute to tissue destruction associated with COPD, thereby positioning this regulatory axis as a promising target for therapeutic intervention.To investigate the NDUFS1/BRK1 axis, this study employs an integrative approach that combines transcriptomics with Mendelian randomization(MR) and network pharmacology. The use of MR allows researchers to leverage genetic variants as instrumental variables to infer causal relationships, effectively mitigating confounding biases typically present in observational studies(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Additionally, molecular docking techniques are utilized to validate potential therapeutic candidates, thereby bridging the gap between molecular discovery and practical application in clinical settings.\u003c/p\u003e \u003cp\u003eThe primary objectives of this research are threefold: first, to establish the NDUFS1/BRK1 axis as a causal driver of COPD; second, to elucidate the mechanistic link between this axis and actin cytoskeleton dysfunction; and third, to identify small-molecule modulators that could serve as potential therapeutic agents. By elucidating the complex interplay between mitochondrial dysfunction and cytoskeletal integrity, this study aims to contribute significantly to the understanding of COPD pathogenesis and foster the development of targeted therapeutic strategies that could significantly impact patient outcomes.\u003c/p\u003e \u003cp\u003eUltimately, the findings from this study could redefine the molecular landscape of COPD, offering new insights and potential pathways for intervention that address the chronic and progressive nature of this disease. By targeting the NDUFS1/BRK1 axis, we may pave the way for innovative strategies that not only manage symptoms but also potentially halt disease progression and restore lung function in affected individuals.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Transcriptomic Profiling and Differential Expression Analysis\u003c/h2\u003e \u003cp\u003eDifferential expression analysis of NDUFS1 and BRK1 was performed using DESeq2 with false discovery rate (FDR) adjustment, applying a significance threshold of FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FoldChange| \u0026gt;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Clinical Correlation and Pathway Analysis\u003c/h2\u003e \u003cp\u003eCorrelation analyses between gene expression levels and clinical parameters including forced vital capacity (FVC) and GOLD stages were conducted using Pearson's correlation coefficient for normally distributed data and Spearman's rank correlation for non-parametric data. Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler R package to evaluate enrichment of the actin cytoskeleton pathway (GO:0015629). Normalized Enrichment Scores (NES) were calculated with 1000 permutations, and significance was determined at FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MR Study Design\u003c/h2\u003e \u003cp\u003eWe conducted a two-sample MR study to investigate the causal effects of gene expression levels of NDUFS1 and BRK1 on the risk of Chronic Obstructive Pulmonary Disease (COPD), as well as the causal effect of NDUFS1 on BRK1. This study was reported in accordance with the STROBE-MR guidelines (Strengthening the Reporting of Observational Studies in Epidemiology using MR). A completed STROBE-MR checklist is provided in Supplementary File 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data Sources\u003c/h2\u003e \u003cp\u003eSummary-level data for all exposures and outcomes were obtained from publicly available genome-wide association study (GWAS) repositories. No specific ethical approval or individual participant consent was required for this study as only aggregated summary statistics were used.The characteristics of the datasets are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the GWAS datasets used in the MR analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrait\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene/ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRole\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOpenGWAS ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSample Size\u003c/p\u003e \u003cp\u003e(Total)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eControls\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAncestry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePMID\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNDUF-S1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eENSG00000023228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eeqtl-a-ENSG00000023228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUniv. of Washington\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e26,609\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBRK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eENSG00000254999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eeqtl-a-ENSG00000254999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUniv. of Washington\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25,690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOPD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eebi-a-GCST90018807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSakaue S et al.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e468,475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13,530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e454,945\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e34594039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eExposure 1 (NDUFS1): Summary statistics for NDUFS1 (ENSG00000023228) gene expression were sourced from the University of Washington (OpenGWAS ID: eqtl-a-ENSG00000023228). The dataset included 26,609 individuals of European ancestry (Year: 2018).\u003c/p\u003e \u003cp\u003eExposure 2 (BRK1): Summary statistics for BRK1 (ENSG00000254999) gene expression were obtained from the University of Washington (OpenGWAS ID: eqtl-a-ENSG00000254999), comprising 25,690 individuals of European ancestry (Year: 2018).\u003c/p\u003e \u003cp\u003eOutcome (COPD): Summary statistics for COPD were derived from the Sakaue S et al. study (OpenGWAS ID: ebi-a-GCST90018807). This study included a total of 468,475 participants, consisting of 13,530 cases and 454,945 controls of European ancestry. Citation: Sakaue S, et al. Nature Genetics. 2021; PMID: 34594039(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven that the eQTL data (N\u0026asymp;26k) and the COPD GWAS (N\u0026asymp;468k) were derived from distinct consortia and study populations, significant sample overlap was unlikely, minimizing the risk of weak instrument bias due to overlap. Potential sample overlap is considered negligible as the studies were conducted by independent consortia with distinct recruitment criteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 MR Statistical Analysis and Sensitivity Testing\u003c/h2\u003e \u003cp\u003eInstrumental Variable Selection\u003c/p\u003e \u003cp\u003eInstrumental variable selection was based on the three core MR assumptions: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Relevance (SNPs strongly associated with exposure), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Independence (SNPs not associated with confounders), and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Exclusion Restriction (SNPs affect outcome only through exposure).\u003c/p\u003e \u003cp\u003eSNPs were selected based on genome-wide significance. Instrumental variables (IVs) were selected using the following criteria: 1) genome-wide significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10⁻⁸); 2) independence (linkage disequilibrium r\u0026sup2; \u0026lt;0.001 within 10,000 kb distance); 3) strength assessment (\u003cem\u003eF-statistic\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/em\u003e). A total of 9 independent SNPs were identified as valid IVs for the analysis. The F-statistic was calculated using the formula: \u003cem\u003eF\u003c/em\u003e = (\u003cem\u003eβ\u003c/em\u003e\u0026sup2;\u003cem\u003e/SE\u003c/em\u003e\u0026sup2;), where β represents the effect size and SE the standard error.\u003c/p\u003e \u003cp\u003eThe inverse variance weighted (IVW) method served as the primary analysis approach. Complementary methods including weighted median, MR-Egger, weighted mode, and simple mode were employed for sensitivity analysis. Heterogeneity was assessed using Cochran's Q statistic, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating significant heterogeneity. Horizontal pleiotropy was evaluated through MR-Egger intercept testing, and leave-one-out analysis was performed to examine the influence of individual SNPs on the overall causal estimate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Network Pharmacology and Compound Screening\u003c/h2\u003e \u003cp\u003eBioactive compounds targeting NDUFS1 were screened from multiple databases including TCMSP, TCM Database@Taiwan, and Coremine Medical. Selection criteria included oral bioavailability (OB)\u0026thinsp;\u0026ge;\u0026thinsp;30% and drug-likeness (DL)\u0026thinsp;\u0026ge;\u0026thinsp;0.18. Compound-target networks were constructed using Cytoscape 3.9.1, and topological analysis was performed using CytoNCA plugin to calculate degree centrality. The top five compounds based on degree values were selected for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Molecular Docking and Binding Affinity Assessment\u003c/h2\u003e \u003cp\u003eThe three-dimensional structure of NDUFS1 (UniProt ID: P28331) was retrieved from the Protein Data Bank (PDB ID: 5LDX). Ligand structures were obtained from PubChem database and optimized using ChemBio3D Ultra 14.0. Molecular docking simulations were performed using AutoDock Vina with the following parameters: grid box size 60\u0026times;60\u0026times;60 A, exhaustiveness setting of 20. Each ligand-receptor pair underwent 20 independent docking runs, and the conformation with the lowest binding energy was selected for interaction analysis. Hydrogen bonding, hydrophobic interactions, and binding energies were analyzed using Discovery Studio 2016.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Software and Reproducibility\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using R version 4.2.1. Specific packages included DESeq2 (v1.36.0) for differential expression, clusterProfiler (v4.4.4) for enrichment analysis, and Two Sample MR (v0.5.6) for MR. Visualization was performed using gplot2 (v3.4.0) and pheatmap (v1.0.12) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.phenoscanner.medschl.cam.ac.uk/\u003c/span\u003e\u003cspan address=\"http://www.phenoscanner.medschl.cam.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All code and analysis pipelines are available upon request to ensure reproducibility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Ethical Considerations and Data Availability\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003eand informed consent were not required for this study as it is based on secondary analysis of publicly available aggregated summary statistics. All original GWAS and transcriptomic studies included in this analysis had obtained appropriate ethical approval and informed consent from their respective participants.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Dysregulation of the NDUFS1/BRK1 axis in COPD and its clinical relevance\u003c/h2\u003e \u003cp\u003eMolecular analysis of lung tissues revealed significant alterations in the expression of key regulatory genes. NDUFS1 expression was markedly elevated in COPD samples compared to healthy controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Conversely, BRK1 transcript levels showed significant reduction in the same patient cohort (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This reciprocal expression pattern suggests potential counter-regulatory interactions between these genes in COPD pathogenesis.\u003c/p\u003e \u003cp\u003eTo assess the clinical relevance of these molecular changes, we examined BRK1 expression across disease severity stages. Progressive reduction in BRK1 mRNA levels was observed with increasing GOLD stages, with statistically significant differences between specific stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, BRK1 expression demonstrated a significant positive correlation with forced vital capacity (R\u0026thinsp;=\u0026thinsp;0.31, p\u0026thinsp;=\u0026thinsp;0.031), a key pulmonary function parameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Analysis of the relationship between NDUFS1 and BRK1 revealed a significant negative correlation across all samples (R = -0.32, p\u0026thinsp;=\u0026thinsp;0.00016), supporting potential regulatory interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eGene set analysis focusing on cellular structural components revealed significant suppression of the actin cytoskeleton pathway in COPD tissues compared to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). This finding suggests that the dysregulation of the NDUFS1/BRK1 axis may be functionally linked to alterations in cellular structural integrity, providing a potential mechanistic basis for the observed clinical correlations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.2 Integrating Computational Pharmacology and Transcriptomics Reveals a NDUFS1-Targeting Strategy for Restoring actin cytoskeleton pathway in COPD\u003c/p\u003e \u003cp\u003eUnsupervised hierarchical clustering analysis of genome-wide expression data revealed clear separation between COPD and control samples, indicating disease-specific transcriptional reprogramming (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Further investigation of the actin cytoskeleton pathway, a critical component of cellular structure and function, demonstrated systematic downregulation of related genes in COPD tissues compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eGene Set Enrichment Analysis confirmed significant suppression of the actin cytoskeleton pathway in COPD. The GSEA plot showed negative enrichment (NES = -1.68, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating coordinated downregulation of cytoskeleton-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In contrast, control samples exhibited significant positive enrichment of the same pathway (NES\u0026thinsp;=\u0026thinsp;1.67, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001), confirming pathway integrity in healthy tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo explore potential regulators of cytoskeletal function, we examined relationships between key genes and pathway activity. NDUFS1 expression showed a significant negative correlation with actin cytoskeleton pathway scores (R = -0.51, p\u0026thinsp;=\u0026thinsp;1.9\u0026times;10\u0026ndash;10) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Conversely, BRK1 expression demonstrated a strong positive correlation with the same pathway (R\u0026thinsp;=\u0026thinsp;0.64, p\u0026thinsp;\u0026lt;\u0026thinsp;2.2\u0026times;10\u0026ndash;16) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These opposing correlations suggest that NDUFS1 and BRK1 may exert counter-regulatory effects on cytoskeletal maintenance.\u003c/p\u003e \u003cp\u003eThe coordinated downregulation of actin cytoskeleton genes in COPD, combined with the divergent associations of NDUFS1 and BRK1 with this pathway, provides a coherent molecular framework. The actin cytoskeleton is essential for maintaining alveolar architecture and epithelial barrier function, and its disruption may contribute to the characteristic tissue destruction in COPD. The opposing correlations of NDUFS1; negative and BRK1 ;positive with cytoskeletal integrity suggest a potential regulatory axis where NDUFS1-mediated suppression of BRK1 could lead to cytoskeletal dysfunction, offering a novel mechanism for COPD pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Functional Pathway Enrichment Analysis\u003c/h2\u003e \u003cp\u003eFunctional enrichment analysis based on differentially expressed genes associated with NDUFS1 and BRK1 revealed distinct biological pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor genes positively associated with BRK1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), significant enrichment was observed in pathways related to actin cytoskeleton regulation​ (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and bacterial invasion of epithelial cells​ (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These findings align with BRK1's established role in cytoskeletal dynamics and cellular defense mechanisms. The color gradient (from dark blue to light yellow) indicates the significance level, with darker colors representing more significant p-values.\u003c/p\u003e \u003cp\u003eFor genes negatively associated with NDUFS1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), the most significantly enriched pathway was oxidative phosphorylation (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001), consistent with the function of NDUFS1 as a mitochondrial complex I subunit. Additional pathways including carbon metabolism and biosynthesis of amino acids​ were also significantly enriched (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The dot size represents gene count within each pathway.\u003c/p\u003e \u003cp\u003eStatistical significance and interpretation: Pathway enrichment was determined using hypergeometric testing with Benjamini-Hochberg correction for multiple comparisons. The strong enrichment of oxidative phosphorylation pathways for NDUFS1-associated genes supports the hypothesis that NDUFS1 dysregulation affects mitochondrial energy metabolism in COPD. Conversely, the enrichment of cytoskeletal and cellular defense pathways for BRK1-associated genes provides mechanistic insight into how BRK1 downregulation may contribute to impaired structural integrity and increased susceptibility to environmental insults in COPD pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 MR Analysis Reveals a Causal Protective Role of BRK1 against COPD Risk\u003c/h2\u003e \u003cp\u003eTo investigate the potential causal relationship between genetically predicted BRK1 expression and COPD risk, we conducted a two-sample MR analysis using nine SNPs as instrumental variables.\u003c/p\u003e \u003cp\u003eAs demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, analysis using the IVW method revealed a significant inverse causal association between genetically elevated BRK1 expression and COPD risk, yielding an odds ratio of 0.949 with a 95 percent confidence interval from 0.910 to 0.990 and attaining statistical significance at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016. This protective association was corroborated by complementary analytical approaches, with the Weighted Median method producing an odds ratio of 0.953 and the Weighted Mode method generating an odds ratio of 0.952, both achieving statistical significance at p equals 0.008 and 0.034, respectively. Although estimates from MR-Egger and Simple Mode analyses did not attain statistical significance, with p-values of 0.193 and 0.405 respectively, their consistent directional alignment with the primary findings reinforces the robustness of the observed protective effect.\u003c/p\u003e \u003cp\u003eThe forest plot presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB illustrates individual variant effect estimates and corresponding 95% confidence intervals for all nine instrumental single nucleotide polymorphisms. Each genetic variant demonstrated effect directions concordant with the overall protective estimate. Furthermore, visual examination of the MR scatter plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC supports the proposed the negative association, evidenced by the uniformly downward trending regression slopes generated through Inverse Variance Weighted, MR-Egger, and Weighted Median methodologies.\u003c/p\u003e \u003cp\u003eCollectively, this MR analysis provides compelling genetic evidence supporting a causal relationship wherein elevated genetically predicted BRK1 expression associates with reduced COPD susceptibility, establishing BRK1 as a biologically plausible protective factor in COPD pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Sensitivity analyses for the causal effect of genetically predicted BRK1 expression on COPD risk\u003c/h2\u003e \u003cp\u003eTo validate the reliability of our MR findings regarding the protective effect of BRK1 against COPD risk, we conducted a comprehensive set of sensitivity analyses(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These analyses were specifically designed to evaluate potential horizontal pleiotropy and to ensure that no single genetic variant disproportionately influenced our results.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, using funnel plot analysis and the MR-Egger intercept test to assess directional pleiotropy, we observed a non-significant MR-Egger intercept = -0.002, p\u0026thinsp;=\u0026thinsp;0.712, indicating no substantial evidence that the instrumental variables influenced COPD risk through pathways independent of BRK1 expression. This finding supports the validity of the core MR assumption. Visual inspection of the funnel plot revealed a generally symmetric distribution of individual single nucleotide polymorphism estimates around the inverse variance weighted summary estimate, with most points clustering near the null value. Notably, a single variant, rs10258, exhibited divergent characteristics with an effect direction opposite to the majority of instrumental variables and exceptionally high precision, measured as the inverse of its standard error at forty-eight point three, potentially suggesting pleiotropic properties that warrant further investigation.\u003c/p\u003e \u003cp\u003eTo evaluate the stability of our causal estimates, we performed a leave-one-out sensitivity analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, this approach systematically excluded each instrumental variable in turn and recalculated the inverse variance weighted derived causal estimate. The results demonstrated remarkable stability, as all leave-one-out estimates remained statistically significant and their confidence intervals exhibited substantial overlap with the overall estimate obtained using all nine SNPs. This consistency supports the proposed that our primary finding is robust and not unduly influenced by any individual genetic variant.\u003c/p\u003e \u003cp\u003eCollectively, our sensitivity analyses provide strong additional support for the causal interpretation of the main MR results. The absence of significant horizontal pleiotropy combined with the stability of effect estimates across multiple analytical approaches strengthens the genetic evidence that elevated genetically predicted BRK1 expression confers protection against COPD development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 MR Analysis indicates a Causal Effect of NDUFS1 on BRK1 Expression\u003c/h2\u003e \u003cp\u003eTo investigate the potential causal influence of NDUFS1 expression on BRK1 levels, we performed a two-sample MR analysis using genetic variants as instrumental variables. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe primary analysis using the Inverse Variance Weighted method yielded a statistically significant causal estimate (OR\u0026thinsp;=\u0026thinsp;0.873; 95% CI: 0.769\u0026ndash;0.991; p\u0026thinsp;=\u0026thinsp;0.036), indicating that a genetically predicted one-unit increase in NDUFS1 expression is associated with a significant reduction in BRK1 expression. The point estimates from complementary MR methods, including the Weighted Median, Simple Mode, and Weighted Mode, were all less than 1.0, suggesting a consistent direction of the negative causal effect, although they did not reach conventional statistical significance thresholds (p\u0026thinsp;=\u0026thinsp;0.094, 0.131, and 0.128, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The MR Egger estimate was not significant (p\u0026thinsp;=\u0026thinsp;0.452).\u003c/p\u003e \u003cp\u003eThe individual Wald ratio estimates for each instrumental variable SNP are detailed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. The combined MR Egger and Inverse Variance Weighted summary estimates align with the primary analysis, indicating a negative overall effect. The scatter plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) visually supports the proposed the negative trend, as evidenced by the downward slopes of the regression lines from all four MR methods. The close alignment of these lines, particularly between the Inverse Variance Weighted and MR Egger methods, suggests the absence of substantial directional horizontal pleiotropy that would invalidate the MR assumptions.\u003c/p\u003e \u003cp\u003eCollectively, this MR analysis provides genetic evidence supporting a causal, negative regulatory relationship whereby elevated NDUFS1 expression leads to decreased BRK1 expression. This finding establishes a potential upstream molecular mechanism for the observed dysregulation of the NDUFS1/BRK1 axis in COPD pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Network Pharmacology Analysis Identifies Potential Bioactive Compounds Targeting NDUFS1\u003c/h2\u003e \u003cp\u003eConstruction and Analysis of the Compound-Target Network\u003c/p\u003e \u003cp\u003eTo identify potential therapeutic agents capable of modulating the NDUFS1/BRK1 axis, we performed network pharmacology analysis. First, we constructed an interaction network comprising predicted bioactive compounds and their potential protein targets relevant to COPD pathogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The network revealed β-sitosterol as a central node, exhibiting connections with multiple key targets including CYP1A1, UGT1A1, and AKR1C1, all of which are implicated in oxidative stress response and xenobiotic metabolism\u0026mdash;processes critically dysregulated in COPD. The high degree of connectivity suggests that β-sitosterol may exert multi-target effects, potentially enhancing its therapeutic efficacy.\u003c/p\u003e \u003cp\u003eMolecular Docking Validates High-Affinity Binding to NDUFS1\u003c/p\u003e \u003cp\u003eTo validate and quantify the interaction between the identified central compound and our primary target, we performed molecular docking simulations against NDUFS1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, β-sitosterol demonstrated the strongest predicted binding affinity to NDUFS1, with a docking score of -9.70 kcal/mol. Other related phytosterols and flavonoids, including sitosterol (-9.40 kcal/mol), quercetin (-9.00 kcal/mol), kaempferol (-8.40 kcal/mol), and stigmasterol (-8.20 kcal/mol), also showed favorable binding energies below the conventional threshold of -7.0 kcal/mol, indicating stable binding interactions. The superior score of β-sitosterol suggests it is the most promising candidate for direct NDUFS1 modulation.\u003c/p\u003e \u003cp\u003eIntegration of Network and Docking Results\u003c/p\u003e \u003cp\u003eThe integration of network topology (identifying β-sitosterol as a key hub) and molecular docking (confirming its high-affinity binding to NDUFS1) provides a two-tiered validation. This approach not only identifies a potential lead compound but also proposes a mechanistic basis for its action\u0026mdash;through direct interaction with NDUFS1, a central regulator of mitochondrial function and redox balance implicated in our previous analyses.\u003c/p\u003e \u003cp\u003eConclusion of the Analysis\u003c/p\u003e \u003cp\u003eThis combined network pharmacology and molecular docking strategy successfully identifies β-sitosterol as a top-ranking, multi-target bioactive compound with high predicted affinity for NDUFS1. These computational findings provide a strong rationale for the subsequent experimental investigation of β-sitosterol (and its source, Sea Buckthorn) as a modulator of the NDUFS1/BRK1 axis and actin cytoskeleton pathway in COPD models.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eChronic obstructive pulmonary disease (COPD) remains a formidable global health challenge, affecting approximately 300\u0026nbsp;million individuals worldwide and ranking as the third leading cause of mortality(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). COPD management employs a stepwise strategy tailored to disease severity and patient phenotypes. For moderate-to-severe cases, particularly those with exacerbation risks, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines prioritize triple therapy\u0026mdash;combining inhaled corticosteroids (ICS), long-acting β2-agonists (LABA), and long-acting muscarinic antagonists (LAMA)\u0026mdash;to alleviate symptoms and reduce acute exacerbations(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, approximately 50% of severe patients continue to experience frequent exacerbations post-treatment, culminating in accelerated lung function decline, elevated hospitalization rates, and increased mortality. For individuals refractory to dual or triple therapy, novel targeted biologics like dupilumab\u0026mdash;the first biologic approved for COPD\u0026mdash;offer promise for type 2-inflammatory phenotypes (e.g., eosinophil counts\u0026thinsp;\u0026ge;\u0026thinsp;300 cells/\u0026micro;L) via IL-4/IL-13 pathway inhibition(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Yet, its efficacy remains limited in non\u0026ndash;type 2 phenotypes, while high costs constrain accessibility. In contrast, Traditional Chinese Medicine (TCM) emphasizes holistic, multi-target regulation, aligning with COPD\u0026rsquo;s complex pathogenesis(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Network pharmacology enables systematic dissection of herbal medicine\u0026rsquo;s multi-component, multi-target, and multi-pathway interactions, addressing gaps in mechanistic understanding. By elucidating specific targets and pathways of active compounds,such as, the NDUFS1/BRK1 axis and associated oxidative stress cytoskeletal pathways, this approach may scientifically support TCM\u0026rsquo;s empirical efficacy scientifically, fosters international recognition, and proposes innovative solutions for therapeutic bottlenecks. Multi-target interventions, guided by network pharmacology, may yield broader efficacy by concurrently modulating key disease nodes, potentially improving long-term outcomes, slowing progression, and reducing resistance risks from single-pathway suppression. Furthermore, precise identification of herbal targets informs personalized integrative strategies\u0026mdash;combining TCM with conventional drugs\u0026mdash;tailored to clinical or TCM-defined subtypes, optimizing COPD management through synergistic, evidence-based combinations.This progressive respiratory disorder, characterized by persistent airflow limitation and chronic inflammation, continues to impose substantial morbidity and economic burden on healthcare systems globally. Current therapeutic approaches, predominantly centered on bronchodilators and corticosteroids, primarily address symptom management rather than modifying the underlying disease progression or reversing established lung damage. The persistent limitations of these interventions underscore the critical need to identify novel therapeutic targets and elucidate fundamental mechanisms driving COPD pathogenesis.\u003c/p\u003e \u003cp\u003eThis study, through an integrative multi-omics approach, systematically uncovers for the first time the critical role of the NDUFS1/BRK1 regulatory axis in COPD. Key findings include significantly upregulated expression of NDUFS1 and concomitantly downregulated expression of BRK1 in COPD lung tissues, with a strong negative correlation observed between the two. BRK1 expression progressively declines with increasing disease severity (as stratified by GOLD stages) and shows a significant positive correlation with FVC, a key pulmonary function parameter. Functional enrichment analyses reveal substantial suppression of the actin cytoskeleton pathway in COPD, with its activity negatively correlated with NDUFS1 expression and positively correlated with BRK1 expression. MR analysis provides genetic evidence supporting a potential protective role of genetically predicted BRK1 against COPD risk and further suggests a potential causal, negative effect of NDUFS1 on BRK1 expression, assuming the validity of instrumental variable assumptions. Leveraging this axis, network pharmacology identifies β-sitosterol as a lead compound with potential inhibitory activity against NDUFS1.\u003c/p\u003e \u003cp\u003eThis work represents the first report of coordinated dysregulation of NDUFS1 and BRK1 in COPD. NDUFS1, a core subunit of mitochondrial complex I, is upregulated in COPD\u0026mdash;a finding consistent with established paradigms implicating mitochondrial dysfunction and oxidative stress in COPD pathogenesis (e.g., increased mitochondrial reactive oxygen species ROS production leading to cellular damage)(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Conversely, BRK1, a critical regulator of the actin cytoskeleton and component of the WAVE regulatory complex, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This offers a novel molecular explanation for emphysematous destruction and epithelial barrier impairment observed in COPD.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that mitochondrial ROS can modulate transcription factors such as Nrf2 and FoxO, which are known to regulate oxidative stress responses and cytoskeletal dynamics(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). It is plausible that elevated NDUFS1 expression enhances mitochondrial ROS generation, which in turn influences BRK1 expression through these transcription factors. Moreover, hypoxia-inducible factor 1-alpha, activated under COPD-related hypoxic conditions, may also contribute to transcriptional repression of BRK1. Epigenetic mechanisms, including DNA methylation and histone modifications, have been shown to regulate gene expression in COPD and may further mediate BRK1 downregulation. Future studies employing chromatin immunoprecipitation, methylation profiling, and reporter assays are warranted to validate these regulatory pathways.\u003c/p\u003e \u003cp\u003eRegarding NDUFS1, prior reports indicate its altered expression in COPD lung tissue, consistent with our findings, though the direct link to BRK1 has not been previously described. BRK1\u0026rsquo;s role in respiratory diseases remains underexplored; this study is the first to implicate BRK1 as a potentially protective factor in COPD pathogenesis.\u003c/p\u003e \u003cp\u003eCross-talk with established COPD-related signaling pathways such as TGF-β, Wnt, and Notch may exist, given their known involvement in cytoskeletal remodeling and fibrosis. BRK1 and the WAVE complex have been implicated in Wnt signaling modulation, suggesting potential integrative mechanisms that warrant further exploration.\u003c/p\u003e \u003cp\u003eConversely, BRK1, a regulator of the actin cytoskeleton, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This provides a novel molecular explanation for emphysematous destruction and epithelial barrier impairment in COPD.\u003c/p\u003e \u003cp\u003eIn contrast to prior studies predominantly focused on inflammation or protease\u0026ndash;antiprotease imbalance, our findings bridge mitochondrial dysfunction and loss of cellular structural integrity via the NDUFS1/BRK1 axis, proposing an integrative pathophysiological framework. Regarding the mechanism by which NDUFS1 regulates BRK1, our data suggest transcriptional or post-transcriptional control(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The robust inverse expression correlation, coupled with MR evidence supporting a potential causal effect of NDUFS1 on BRK1, leads us to hypothesize that NDUFS1 overexpression may enhance mitochondrial reactive oxygen species (ROS) production, thereby modulating specific transcription factors (e.g., Nrf2, FoxO, or HIF-1α) or inducing epigenetic alterations that ultimately suppress BRK1 transcription. This hypothesis transforms correlative observations into testable molecular pathways, warranting future validation through chromatin immunoprecipitation, reporter assays, and related functional experiments.\u003c/p\u003e \u003cp\u003eA major strength of this study lies in its multidimensional integration: transcriptomics delineated expression profiles and pathway alterations, MR provided genetic-level causal inference, and network pharmacology pointed toward therapeutic opportunities\u0026mdash;collectively enhancing the robustness and translational relevance of our findings. Nevertheless, several limitations must be acknowledged. First, key findings are based on transcriptomic data and require validation at the protein level. Second, although MR supports a directional causal relationship, definitive confirmation necessitates experimental validation in cellular and animal models and \u003cb\u003e4. Discussion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eChronic obstructive pulmonary disease (COPD) remains a formidable global health challenge, affecting approximately 300\u0026nbsp;million individuals worldwide and ranking as the third leading cause of mortality(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). COPD management employs a stepwise strategy tailored to disease severity and patient phenotypes. For moderate-to-severe cases, particularly those with exacerbation risks, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines prioritize triple therapy\u0026mdash;combining inhaled corticosteroids (ICS), long-acting β2-agonists (LABA), and long-acting muscarinic antagonists (LAMA)\u0026mdash;to alleviate symptoms and reduce acute exacerbations(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, approximately 50% of severe patients continue to experience frequent exacerbations post-treatment, culminating in accelerated lung function decline, elevated hospitalization rates, and increased mortality. For individuals refractory to dual or triple therapy, novel targeted biologics like dupilumab\u0026mdash;the first biologic approved for COPD\u0026mdash;offer promise for type 2-inflammatory phenotypes (e.g., eosinophil counts\u0026thinsp;\u0026ge;\u0026thinsp;300 cells/\u0026micro;L) via IL-4/IL-13 pathway inhibition(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Yet, its efficacy remains limited in non\u0026ndash;type 2 phenotypes, while high costs constrain accessibility. In contrast, Traditional Chinese Medicine (TCM) emphasizes holistic, multi-target regulation, aligning with COPD\u0026rsquo;s complex pathogenesis(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Network pharmacology enables systematic dissection of herbal medicine\u0026rsquo;s multi-component, multi-target, and multi-pathway interactions, addressing gaps in mechanistic understanding. By elucidating specific targets and pathways of active compounds,such as, the NDUFS1/BRK1 axis and associated oxidative stress cytoskeletal pathways, this approach validates TCM\u0026rsquo;s empirical efficacy scientifically, fosters international recognition, and proposes innovative solutions for therapeutic bottlenecks. Multi-target interventions, guided by network pharmacology, may yield broader efficacy by concurrently modulating key disease nodes, potentially improving long-term outcomes, slowing progression, and reducing resistance risks from single-pathway suppression. Furthermore, precise identification of herbal targets informs personalized integrative strategies\u0026mdash;combining TCM with conventional drugs\u0026mdash;tailored to clinical or TCM-defined subtypes, optimizing COPD management through synergistic, evidence-based combinations.This progressive respiratory disorder, characterized by persistent airflow limitation and chronic inflammation, continues to impose substantial morbidity and economic burden on healthcare systems globally. Current therapeutic approaches, predominantly centered on bronchodilators and corticosteroids, primarily address symptom management rather than modifying the underlying disease progression or reversing established lung damage. The persistent limitations of these interventions underscore the critical need to identify novel therapeutic targets and elucidate fundamental mechanisms driving COPD pathogenesis.\u003c/p\u003e \u003cp\u003eThrough integrative bioinformatics analysis, we propose the NDUFS1/BRK1 axis as a potential pivotal regulator in COPD, warranting further experimental validation. Key findings include significantly upregulated expression of NDUFS1 and concomitantly downregulated expression of BRK1 in COPD lung tissues, with a strong negative correlation observed between the two. BRK1 expression progressively declines with increasing disease severity (as stratified by GOLD stages) and shows a significant positive correlation with FVC, a key pulmonary function parameter. Functional enrichment analyses reveal substantial suppression of the actin cytoskeleton pathway in COPD, with its activity negatively correlated with NDUFS1 expression and positively correlated with BRK1 expression. MR analysis provides genetic evidence supporting a protective role of BRK1 against COPD risk and further suggests a potential causal, negative effect of NDUFS1 on BRK1 expression. Leveraging this axis, network pharmacology identifies β-sitosterol as a lead compound with potential inhibitory activity against NDUFS1.\u003c/p\u003e \u003cp\u003eThis work represents the first report of coordinated dysregulation of NDUFS1 and BRK1 in COPD. NDUFS1, a core subunit of mitochondrial complex I, is upregulated in COPD\u0026mdash;a finding consistent with established paradigms implicating mitochondrial dysfunction and oxidative stress in COPD pathogenesis, increased mitochondrial reactive oxygen species ROS production leading to cellular damage). Conversely, BRK1, a critical regulator of the actin cytoskeleton and component of the WAVE regulatory complex, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This provides a novel molecular explanation for emphysematous destruction and epithelial barrier impairment observed in COPD.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that mitochondrial ROS can modulate transcription factors such as Nrf2 and FoxO, which are known to regulate oxidative stress responses and cytoskeletal dynamics(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). It is plausible that elevated NDUFS1 expression enhances mitochondrial ROS generation, which in turn influences BRK1 expression through these transcription factors. Moreover, hypoxia-inducible factor 1-alpha, activated under COPD-related hypoxic conditions, may also contribute to transcriptional repression of BRK1. Epigenetic mechanisms, including DNA methylation and histone modifications, have been shown to regulate gene expression in COPD and may further mediate BRK1 downregulation. Future studies employing chromatin immunoprecipitation, methylation profiling, and reporter assays are warranted to validate these regulatory pathways.\u003c/p\u003e \u003cp\u003eRegarding NDUFS1, prior reports indicate its altered expression in COPD lung tissue, consistent with our findings, though the direct link to BRK1 has not been previously described. BRK1\u0026rsquo;s role in respiratory diseases remains underexplored; this study is the first to implicate BRK1 as a potentially protective factor in COPD pathogenesis.\u003c/p\u003e \u003cp\u003eCross-talk with established COPD-related signaling pathways such as TGF-β, Wnt, and Notch may exist, given their known involvement in cytoskeletal remodeling and fibrosis. BRK1 and the WAVE complex have been implicated in Wnt signaling modulation, suggesting potential integrative mechanisms that warrant further exploration.This work represents the first report of coordinated dysregulation of NDUFS1 and BRK1 in COPD. NDUFS1, a core subunit of mitochondrial complex I, is upregulated in COPD\u0026mdash;a finding consistent with established paradigms implicating mitochondrial dysfunction and oxidative stress in COPD pathogenesis(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Conversely, BRK1, a regulator of the actin cytoskeleton, is markedly downregulated, directly aligning with our observation of systemic inhibition of the actin cytoskeleton pathway. This provides a novel molecular explanation for emphysematous destruction and epithelial barrier impairment in COPD.\u003c/p\u003e \u003cp\u003eIn contrast to prior studies predominantly focused on inflammation or protease\u0026ndash;antiprotease imbalance, our findings bridge mitochondrial dysfunction and loss of cellular structural integrity via the NDUFS1/BRK1 axis, proposing an integrative pathophysiological framework. Regarding the mechanism by which NDUFS1 regulates BRK1, our data suggest transcriptional or post-transcriptional control(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The robust inverse expression correlation, coupled with MR evidence supporting a potential causal effect of NDUFS1 on BRK1, leads us to hypothesize that NDUFS1 overexpression may enhance mitochondrial reactive oxygen species (ROS) production, thereby modulating specific transcription factors (e.g., Nrf2, FoxO, or HIF-1α) or inducing epigenetic alterations that ultimately suppress BRK1 transcription. This hypothesis transforms correlative observations into testable molecular pathways, warranting future validation through chromatin immunoprecipitation, reporter assays, and related functional experiments.\u003c/p\u003e \u003cp\u003eA major strength of this study lies in its multidimensional integration: transcriptomics delineated expression profiles and pathway alterations, MR provided genetic-level causal inference, and network pharmacology pointed toward therapeutic opportunities\u0026mdash;collectively enhancing the robustness and translational relevance of our findings. Nevertheless, several limitations must be acknowledged. First, key findings are based on transcriptomic data and require validation at the protein level. Second, although MR supports a directional causal relationship, definitive confirmation necessitates experimental validation in cellular and animal models. Third, bulk RNA-seq data cannot resolve cell-type-specific contributions (e.g., from epithelial cells, fibroblasts, or immune cells) to the dysregulation of this axis. Finally, while BRK1 expression correlates significantly with FVC, the moderate correlation coefficient (R\u0026thinsp;=\u0026thinsp;0.31) suggests limited utility as a standalone biomarker, potentially requiring combination with other indicators for clinical application. Caution should be exercised when extrapolating these results to other groups, as our study cohort was predominantly composed of European.\u003c/p\u003e \u003cp\u003eThis study holds translational promise. BRK1\u0026rsquo;s association with disease severity and lung function positions it as a candidate biomarker for assessing COPD progression. More importantly, the NDUFS1/BRK1 axis itself represents a novel therapeutic target. Strategies aimed at restoring BRK1 function or inhibiting excessive NDUFS1 activity are thus plausible interventions. Notably, β-sitosterol\u0026mdash;identified via network pharmacology\u0026mdash;emerges as a promising lead compound, with computational modeling predicting high binding affinity for NDUFS1.\u003c/p\u003e \u003cp\u003eHowever, translation faces challenges: as a plant-derived phytosterol, β-sitosterol requires rigorous evaluation of pulmonary-specific delivery, bioavailability, and long-term safety. Moreover, targeting mitochondrial complex I (via NDUFS1 inhibition) carries potential off-target risks to systemic energy metabolism, demanding careful therapeutic window assessment. Future preclinical studies in COPD animal models are essential to validate both efficacy and safety of interventions targeting this axis.\u003c/p\u003e \u003cp\u003eIn summary, we predict NDUFS1/BRK1 as a previously underappreciated yet pivotal regulatory axis in COPD, linking upstream mitochondrial dysfunction to downstream cytoskeletal collapse and offering a new mechanistic perspective on structural lung damage(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Genetic evidence supports a protective role for BRK1 and positions NDUFS1 as a potential upstream regulator. Computational screening highlights β-sitosterol as a starting point for therapeutic development. Future work should prioritize: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) experimental validation of the molecular mechanisms underlying NDUFS1-mediated BRK1 regulation in cellular and animal models; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) evaluation of β-sitosterol or alternative NDUFS1 inhibitors in preclinical COPD models; and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) prospective validation of BRK1 as a prognostic biomarker in larger clinical cohorts. Deepening our understanding of this novel axis may pave the way for disease-modifying therapies in COPD.\u003c/p\u003e \u003cp\u003e. Third, bulk RNA-seq data cannot resolve cell-type-specific contributions (e.g., from epithelial cells, fibroblasts, or immune cells) to the dysregulation of this axis. Finally, while BRK1 expression correlates significantly with FVC, the moderate correlation coefficient (R\u0026thinsp;=\u0026thinsp;0.31) suggests limited utility as a standalone biomarker, potentially requiring combination with other indicators for clinical application.\u003c/p\u003e \u003cp\u003eThis study holds clear translational promise. BRK1\u0026rsquo;s association with disease severity and lung function positions it as a candidate biomarker for assessing COPD progression. More importantly, the NDUFS1/BRK1 axis itself represents a novel therapeutic target. Strategies aimed at restoring BRK1 function or inhibiting excessive NDUFS1 activity are thus plausible interventions. Notably, β-sitosterol\u0026mdash;identified via network pharmacology\u0026mdash;emerges as a promising lead compound, with computational modeling predicting high binding affinity for NDUFS1.\u003c/p\u003e \u003cp\u003eHowever, translation faces challenges: as a plant-derived phytosterol, β-sitosterol requires rigorous evaluation of pulmonary-specific delivery, bioavailability, and long-term safety. Moreover, targeting mitochondrial complex I (via NDUFS1 inhibition) carries potential off-target risks to systemic energy metabolism, demanding careful therapeutic window assessment. Future preclinical studies in COPD animal models are essential to validate both efficacy and safety of interventions targeting this axis.\u003c/p\u003e \u003cp\u003eIn summary, we identify NDUFS1/BRK1 as a previously underappreciated yet pivotal regulatory axis in COPD, linking upstream mitochondrial dysfunction to downstream cytoskeletal collapse and offering a new mechanistic perspective on structural lung damage(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Genetic evidence supports a protective role for BRK1 and positions NDUFS1 as a potential upstream regulator. Computational screening highlights β-sitosterol as a starting point for therapeutic development. Future work should prioritize: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) experimental validation of the molecular mechanisms underlying NDUFS1-mediated BRK1 regulation in cellular and animal models; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) evaluation of β-sitosterol or alternative NDUFS1 inhibitors in preclinical COPD models; and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) prospective validation of BRK1 as a prognostic biomarker in larger clinical cohorts. Deepening our understanding of this novel axis may pave the way for disease-modifying therapies in COPD.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study elucidates a novel pathogenic mechanism in COPD driven by the dysregulation of the NDUFS1/BRK1 axis, which bridges mitochondrial dysfunction and actin cytoskeleton disruption. Through an integrative multi-omics approach, we provide robust genetic evidence via MR that elevated NDUFS1 causally suppresses BRK1, contributing to disease progression and impaired pulmonary function. Furthermore, our network pharmacology and molecular docking analyses identify β-sitosterol as a promising therapeutic candidate capable of targeting NDUFS1. These findings not only advance the understanding of COPD pathophysiology but also highlight the potential of modulating the mitochondrial-cytoskeletal interface as a new strategy for disease-modifying therapies. Future experimental studies are warranted to validate these computational predictions and explore the clinical efficacy of β-sitosterol or similar agents in COPD management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eEthical approval and informed consent were not required for this study as it is based on secondary analysis of publicly available aggregated summary statistics. All original GWAS and transcriptomic studies included in this analysis had obtained appropriate ethical approval and informed consent from their respective participants.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable. This manuscript does not contain any individual person\u0026rsquo;s data, such as personal details, images, or videos.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are available in public repositories. The transcriptomic datasets analyzed during the current study are available in the GEO repository. The summary-level data for the MR analysis are available from the corresponding GWAS consortia websites . The code and scripts used for the statistical analyses and molecular docking are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Health Commission of Hebei Province, China under Grant number ZF2026417.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eY. G. and F. M. conceived and designed the study. Y. W. performed the bioinformatics analysis and MR. J.y. conducted the network pharmacology and molecular docking simulations. C. L., A. F. and T. C. interpreted the data and drafted the manuscript. J. Z. , X. Z. and B. L. critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank the researchers and participants of the original GWAS and GEO studies for making their data publicly available. We also acknowledge the support of the North China University of Science and Technology Affiliated Hospital for providing the computational resources necessary for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOh J, et al. Global, regional, and national burden of chronic respiratory diseases and impact of the COVID-19 pandemic, 1990\u0026ndash;2023: a Global Burden of Disease study. Nat Med. 2026;32:197\u0026ndash;223.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoers E, et al. Global Burden of Chronic Obstructive Pulmonary Disease Through 2050. JAMA Netw open. 2023;6:e2346598.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeMeo DL. Sex, Gender, and COPD. Annu Rev Physiol. 2025;87:471\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonovan T et al. Anti-IL-5 therapies for chronic obstructive pulmonary disease. \u003cem\u003eThe Cochrane database of systematic reviews\u003c/em\u003e 12, Cd013432 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu CY, et al. Tiotropium Initiation and Dementia Risk in Chronic Obstructive Pulmonary Disease. JAMA Intern Med. 2025;185:847\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, et al. Effect of traditional Chinese medicine combined with conventional Western medicine for patients with severe/very severe chronic obstructive pulmonary disease: a multi-center, randomized, double-blind, controlled study. Chin Med. 2025;20:66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu X, et al. Mechanisms and potential roles of active ingredients of traditional Chinese medicine in the treatment of chronic obstructive pulmonary disease. J Pharm Pharmacol. 2025;77:866\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu D, et al. Clinical Application Guideline of Combination With Traditional Chinese Medicine and Western Medicine in the Prevention and Treatment of Chronic Obstructive Pulmonary Disease (2024). J evidence-based Med. 2025;18:e70024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, et al. Investigation of the efficacy and potential pharmacological mechanism of Yupingfeng in treating chronic obstructive pulmonary disease: A meta-analysis and in silico study. J Ethnopharmacol. 2025;343:119441.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn S, Cai HY. Efficacy and safety of traditional Chinese medicine in the treatment of chronic pulmonary diseases: a systematic review and meta-analysis. Front Med. 2025;12:1512729.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu E, et al. Comparative effectiveness of ten traditional Chinese herbal formulas for acute exacerbation of chronic obstructive pulmonary disease: a systematic review and Bayesian network meta-analysis. Front Pharmacol. 2025;16:1585150.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi P, Zheng B, Cao Y, Niu G, Guo Q. Study on the mechanism of Trichosanthes kirilowii Maxim. against COPD based on serum chemical composition analysis, network pharmacology, and experimental study. Phytomedicine: Int J phytotherapy phytopharmacology. 2025;140:156533.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu L, et al. Sea buckthorn extract mitigates chronic obstructive pulmonary disease by suppression of ferroptosis via scavenging ROS and blocking p53/MAPK pathways. J Ethnopharmacol. 2025;336:118726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, et al. Mendelian randomisation studies for causal inference in chronic obstructive pulmonary disease: A narrative review. Pulmonology. 2025;31:2470556.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu S et al. Machine Learning-Driven Identification of Shared and Disease-Specific Mitochondria-Related Genes in COPD, NSCLC and NSCLC with COPD. \u003cem\u003eiScience\u003c/em\u003e, 114857 (2026).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, et al. Stress-Related Biomarkers in Chronic Obstructive Pulmonary Disease: A Comprehensive Transcriptome, Mendelian Randomization, and Machine-Learning Analysis. Int J Chronic Obstr Pulm Dis. 2025;20:3803\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIuso A, et al. Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I. J Biol Chem. 2006;281:10374\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakaue S, et al. A cross-population atlas of genetic associations for 220 human phenotypes. Nat Genet. 2021;53:1415\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabaki WW, Rosenberg SR. Chronic Obstructive Pulmonary Disease. Ann Intern Med. 2020;173:32. Itc17-itc.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez FJ, et al. Reduced All-Cause Mortality in the ETHOS Trial of Budesonide/Glycopyrrolate/Formoterol for Chronic Obstructive Pulmonary Disease. A Randomized, Double-Blind, Multicenter, Parallel-Group Study. Am J Respir Crit Care Med. 2021;203:553\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStolz D, et al. Towards the elimination of chronic obstructive pulmonary disease: a Lancet Commission. Lancet (London England). 2022;400:921\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, et al. A chinese herbal formula ameliorates COPD by inhibiting the inflammatory response via downregulation of p65, JNK, and p38. Phytomedicine: Int J phytotherapy phytopharmacology. 2021;83:153475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong Z, et al. ER-localized ERO1α and caspase-3-mediated cleavage of mitochondrial NDUFS1 drives trichothecene-induced ROS accumulation in liver. Free Radic Biol Med. 2026;244:452\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, et al. Loss of NDUFS1 promotes gastric cancer progression by activating the mitochondrial ROS-HIF1α-FBLN5 signaling pathway. Br J Cancer. 2023;129:1261\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDunham-Snary KJ, et al. Ndufs2, a Core Subunit of Mitochondrial Complex I, Is Essential for Acute Oxygen-Sensing and Hypoxic Pulmonary Vasoconstriction. Circul Res. 2019;124:1727\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEapen MS, Sharma P, Sohal SS. Mitochondrial dysfunction in macrophages: a key to defective bacterial phagocytosis in COPD. Eur Respir J 54, (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic obstructive pulmonary disease, NDUFS1, BRK1, Mitochondrial dysfunction Actin cytoskeleton, Multi-omics, Network pharmacology, β-sitosterol","lastPublishedDoi":"10.21203/rs.3.rs-8990913/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8990913/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eChronic obstructive pulmonary disease (COPD) is a major global health burden, affecting over 300\u0026nbsp;million people and ranking as the third leading cause of death worldwide. Current therapies do not modify disease progression, and molecular mechanisms linking mitochondrial dysfunction and cytoskeletal disruption remain unclear. Emerging evidence implicates NDUFS1, a subunit of mitochondrial complex I, and BRK1, a key regulator of actin cytoskeleton dynamics, in COPD pathogenesis, potentially through related pathways.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eWe conducted an integrative multi-omics study combining transcriptomic profiling of lung tissues from COPD patients and healthy controls, two-sample Mendelian randomization(MR) using genome-wide association study summary statistics, and network pharmacology coupled with molecular docking. Transcriptomic analyses assessed differential expression of NDUFS1 and BRK1, their correlation with clinical parameters such as forced vital capacity and GOLD stages, and pathway-level changes via gene set enrichment analysis. MR used inverse variance weighted method as primary analysis, supported by MR-Egger, weighted median, and mode-based approaches, with sensitivity tests for pleiotropy. Network pharmacology identified compounds targeting NDUFS1, followed by docking to evaluate binding affinities.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eTranscriptomic results showed NDUFS1 upregulation and BRK1 downregulation in COPD. BRK1 decreased across GOLD stages and correlated positively with forced vital capacity. Gene set enrichment analysis revealed suppression of the actin cytoskeleton pathway, strongly negatively correlated with NDUFS1 and positively with BRK1. MR indicated a protective causal effect of BRK1 on COPD risk, with consistent directional support across methods. Higher NDUFS1 expression causally reduced BRK1 levels, with no significant pleiotropy. β-sitosterol showed the strongest predicted binding affinity to NDUFS1 among screened phytochemicals.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eDysregulation of NDUFS1 and BRK1 may contribute to COPD via mitochondrial-cytoskeletal crosstalk. Causal evidence supports further investigation, with β-sitosterol emerging as a candidate for therapeutic development.\u003c/p\u003e","manuscriptTitle":"The NDUFS1/BRK1 Axis in COPD Pathogenesis: A Multi-Omics Approach Linking Mitochondrial Dysfunction to Actin Cytoskeleton Disruption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 10:12:43","doi":"10.21203/rs.3.rs-8990913/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e14c089-7049-45b3-8c0b-a54f520e227b","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T09:42:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 10:12:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8990913","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8990913","identity":"rs-8990913","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}