The role of age-related genes in idiopathic pulmonary fibrosis and molecular docking analysis of their drug targets

Idiopathic pulmonary fibrosis (IPF) represents a relentlessly progressive interstitial lung disease characterized by pathological extracellular matrix deposition, culminating in irreversible pulmonary architecture distortion and progressive respiratory failure (1). Epidemiologically, this age-related disorder exhibits striking predilection for individuals over 60 years, with a median survival of 3–5 years post-diagnosis, highlighting the critical role of accelerated biological aging processes in disease progression (2). The archetypal usual interstitial pneumonia (UIP) pattern, defined by temporal heterogeneity and fibroblastic foci, has become a diagnostic cornerstone through advances in high-resolution computed tomography (3). While current therapeutic strategies (antifibrotics pirfenidone and nintedanib) demonstrate modest efficacy in slowing forced vital capacity decline (reducing annual loss by ~50%), their inability to reverse established fibrosis or substantially prolong survival underscores an imperative for mechanism-driven therapeutic discovery (4, 5).

The pathobiology of IPF involves a complex interplay of repetitive alveolar epithelial injury, maladaptive repair mechanisms, and dysregulated fibroblast activation, occurring in front of a backdrop of genetic susceptibility and environmental exposures (6, 7). Emerging evidence implicates cellular senescence as a pivotal amplifier of fibrogenic processes, where senescent alveolar epithelial cells and fibroblasts perpetuate tissue injury through senescence-associated secretory phenotype (SASP) mediators including IL-6, TGF-β, and matrix metalloproteinases (8). This pro-fibrotic secretome not only drives myofibroblast differentiation but also reshapes the immune microenvironment through chemokine-mediated recruitment of monocytes and alternatively activated macrophages, establishing a self-sustaining fibro-inflammatory circuit (8 –11). Understanding the molecular mechanisms that connect aging with IPF is essential for identifying potential therapeutic targets.

In summary, this study utilized an integrated multi-omics approach to investigate the role of aging-related genes in the pathogenesis of IPF. Key senescence-associated genes, including CLU and LCN2, were identified and validated as significantly upregulated in both IPF patient samples and bleomycin-induced murine models. Furthermore, through computational drug screening and molecular docking analysis, inulin and meclizine were identified as potential therapeutic candidates for IPF (Figure 1). These findings provide novel insights into the molecular mechanisms linking cellular senescence to pulmonary fibrosis, offering promising avenues for the development of targeted anti-aging therapies in IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal interstitial lung disease characterized by dysregulated fibrogenesis and excessive deposition of extracellular matrix (ECM), ultimately resulting in respiratory failure and a five-year mortality rate exceeding 50% (23 –27). The multifactorial pathogenesis of IPF involves a complex interplay between aging-related genomic instability, including telomere shortening and epigenetic modification, and exposure to environmental stressors such as particulate matter and microbial dysbiosis (28 –30); however, the core mechanistic pathways underlying this interaction remain incompletely understood. Current therapeutic options for IPF are limited. Although antifibrotic agents like pirfenidone and nintedanib can reduce symptom burden and slow disease progression, their impact on long-term survival is modest (31 –33). Given this unmet clinical need, the present study aimed to identify aging-related differentially expressed genes in IPF and to explore potential therapeutic compounds targeting these genes through molecular docking analysis, thereby offering novel insights into future therapeutic strategies.

Our integrated analysis of three independent cohorts delineated a distinct and robust transcriptomic landscape in IPF, defined by 292 differentially expressed genes (176 upregulated and 116 downregulated). These genes are functionally implicated in profound ECM remodelling and the induction of senescence-associated pathways. In particular, a cluster of upregulated genes, including COL1A1, COL14A1, ASPN, CTHRC1 and MMP7, collectively fuels IPF progression by concurrently exacerbating ECM deposition and potentiating cellular senescence. Collectively, these alterations establish and sustain a pro-fibrotic microenvironment in the lungs of individuals with IPF, potentially through a self-reinforcing, senescence-dependent molecular circuit (34 –40).

Cellular senescence, a pathophysiological state marked by irreversible cell-cycle arrest driven by telomere attrition, mitochondrial dysfunction, and genomic instability, emerges as a critical driver of IPF through SASP-mediated extracellular matrix remodeling (41 –43). Nineteen overlapping genes were identified through the intersection of IPF associated DEGs and an aging-related gene set, including representative genes, which substantiates a direct molecular connection between pulmonary fibrosis and biological aging. GO enrichment analysis revealed that these genes are significantly involved in biological processes like glial cell differentiation. Multiple genes have been identified to play critical roles in glial cell function and pathology across different neural contexts. T-box transcription factor 3 (TBX3) is expressed in neural crest-derived glial cells, and its loss leads to reduced enteric glia without affecting neuronal density or gut motility (44). Lipocalin-2 (LCN2) regulates migration, morphology, and reactive astrogliosis in astrocytes and microglia via the Rho-ROCK pathway and upregulates GFAP expression under inflammatory conditions (45, 46). In the retina, SRY-box transcription factor 2 (SOX2) maintains Müller glia in a quiescent progenitor state, and its ablation results in morphological disruption and retinal degeneration (47). Clusterin (CLU) expression in Müller glia is modulated by inflammatory stress such as IL-1β, suggesting a role in photoreceptor suppor (48). Recent studies have revealed that glial cells (including astrocytes, microglia, and Müller cells) contribute to fibrotic processes in various organ systems through diverse mechanisms: mediating glial scar formation in the central nervous system (49), promoting fibronectin deposition via PAD4-dependent citrullination in the retina (50), driving fibrosis as "hepatic neuroglia" through norepinephrine -adrenergic receptor signaling in the liver (51), and participating in fibrotic microenvironment construction via Sox10+ cell expansion in the bone marrow (52). These evidences suggest that pulmonary fibrosis may involve similar neuroglial related mechanisms, such as sympathetic nerve activation regulating the activity of pulmonary fibroblasts through adrenergic receptors (53, 54), neuroglia-immune cell interaction mediated by the NF-κB pathway mediating chronic inflammatory responses (55), and tissue microenvironment remodeling induced by neuroglial- derived factors. These hypotheses await further verification in future experiments.

KEGG enrichment analysis revealed that these differentially expressed genes were significantly enriched in key metabolic pathways, including glycine, serine, and threonine metabolism, as well as cysteine and methionine metabolism. Emerging evidence has highlighted the role of sulfur containing amino acid metabolism in the pathogenesis of pulmonary fibrosis. For instance, STC1 has been shown to exert anti fibrotic effects by stimulating the cysteine glutathione pathway, which upregulates uncoupling protein 2 dependent demethylation of the SMAD7 promoter and promotes its acetylation (56). Meanwhile, methionine metabolism has also garnered attention: methionine synthase reductase (MTRR), a key enzyme in this pathway, has been identified as a potential therapeutic target (57). The drug clioquinol alleviates fibrosis by binding to MTRR, activating the methionine cycle, and increasing the production of methionine and S-adenosylmethionine, thereby suppressing fibroblast activation and extracellular matrix deposition (57). These findings collectively suggest that modulating cysteine and methionine metabolism may offer novel treatment strategies for pulmonary fibrosis through the regulation of cellular redox balance and epigenetic modifications. In contrast, direct evidence linking glycine, serine, and threonine metabolism to pulmonary fibrosis remains limited. Existing studies indicate that the metabolism of these three amino acids is often dysregulated in a coordinated manner in conditions such as metabolic liver disease, wherein supplementation strategies have been shown to ameliorate metabolic imbalance and reduce aberrant collagen synthesis, reflecting a broader role in tissue stress response (58 –60). Thus, although direct mechanistic studies in the lung remain limited, it is plausible that dysregulation of these metabolic pathways may indirectly contribute to pulmonary fibrosis by disrupting collagen turnover and impairing tissue repair processes. This hypothesis, while conceptually compelling, requires rigorous experimental validation.

Our study systematically identified 10 hub genes (IGFBP7, CLU, HMGCR, SOX4, LCN2, TLR4, MDK, MMP7, GDF15, and SOX2) that act as dual-functional regulators of fibrotic remodeling and cellular senescence in IPF. Molecular docking analysis revealed high-affinity binding of the natural polysaccharide inulin to LCN2 and the antihistamine meclizine to CLU, suggesting their potential to modulate these targets for anti-fibrotic therapy. LCN2 plays multifaceted roles in IPF pathogenesis, acting as a biomarker of pulmonary inflammation, a modulator of oxidative stress, and a regulator of alveolar repair. Elevated LCN2 expression in IPF lung tissues and bronchoalveolar lavage fluid (BALF) correlates with reduced respiratory function (61), positioning it as a surrogate biomarker of disease severity. In acute exacerbations (AE-IPF), elevated serum LCN2 levels independently predict poor survival (62), while murine studies reveal its protective role against bleomycin-induced oxidative stress. LCN2^−/− mice exhibit exacerbated lung injury and impaired antioxidant responses (62). Paradoxically, despite its association with neutrophilic inflammation in acute lung injury models, LCN2 deletion does not mitigate fibrosis, suggesting its pathogenic role is secondary to inflammation-driven oxidative stress (61). Furthermore, LCN2 overexpression suppresses alveolar epithelial type 2 (AT2) cell proliferation, a critical repair mechanism, though interleukin-17 (IL-17) partially rescues this deficit, highlighting the therapeutic potential of targeting the LCN2/IL-17 axis (63). LCN2 may also play an important role in the progression of diseases such as Parkinson's, breast cancer, and disc degeneration by regulating cellular senescence (64 –66). CLU exhibits opposing functions in IPF depending on its subcellular localization. Extracellular CLU promotes alveolar epithelial apoptosis, whereas intracellular CLU enhances DNA repair and reduces senescence (67). IPF lungs exhibit a pathological shift toward extracellular CLU dominance, mirroring the fibrotic phenotype of CLU^−/− mice, which display unresolved DNA damage and persistent fibrosis post-bleomycin injury (67). Transcriptomic analyses further link elevated CLU expression to extracellular matrix remodeling and disease progression (68), solidifying its dual roles as a driver of fibrogenesis and a guardian of epithelial homeostasis.

The anti-fibrotic potential of inulin and meclizine is supported by their established roles in mitigating fibrosis across organs. Inulin has been shown to significantly improve liver fibrosis (20) and effectively alleviate colon fibrosis caused by chronic radiation exposure (21), highlighting its therapeutic potential in managing fibrotic conditions across various organs. Similarly, in a mouse adenomyosis model, meclizine administration accelerated endometrial repair and improved endometrial fibrosis in a dose-dependent manner (22). We found that the expression of CLU and LCN2 was significantly higher in IPF lung tissues than in controls using an external validation set and by constructing a mouse IPF model. Given the involvement of CLU and LCN2 in the progression of pulmonary fibrosis and regulation of cellular senescence, it is hypothesized that inulin and meclizine may exert an anti-pulmonary fibrosis effect in the lungs by targeting these genes and modulating cellular senescence, offering a potential new therapeutic approach for IPF. While this study identifies promising therapeutic candidates, several limitations must be acknowledged: (1) The anti-fibrotic effects of inulin and meclizine in IPF remain experimentally unvalidated; (2) The relatively small sample size and the use of only a single time point in our animal experiments may introduce bias and limit the robustness of the findings; future studies with larger cohorts and multiple time points are warranted to strengthen our conclusions; (3) Clinical correlations between hub gene expression and patient outcomes are lacking; (4) Potential batch effects in transcriptomic data could introduce bias. Future work should prioritize in vivo validation of these compounds in IPF models, large-scale multicenter cohorts to confirm biomarker utility, and mechanistic studies to resolve CLU/LCN2's compartment-specific roles.

Furthermore, it is noteworthy that the GSE68239 cohort displayed a distinct clustering pattern in the analysis compared to the other two datasets. This divergence is primarily attributed to technical batch effects inherent in integrating cross-platform microarray data, despite the application of standardized correction algorithms. Additionally, undefined biological heterogeneity across independent cohorts may further contribute to this separation. Importantly, this global-level technical variation does not undermine our core findings. The age-associated key genes, such as CLU and LCN2, identified through differential expression analysis, along with their enriched pathways, were consistently and robustly validated across all three integrated datasets. This consistency confirms that the central biological signals underlying our conclusions are strong and reproducible, effectively transcending the influence of technical artifacts.

In conclusion, our research provides significant insights into the molecular mechanisms underlying IPF by highlighting the role of age-related DEGs and their potential regulatory networks. Through the integration of transcriptomic data and bioinformatics analysis, we have uncovered complex interactions between these genes, identifying promising candidates for therapeutic targets and biomarkers. Furthermore, our findings indicate that inulin and meclizine may slow the progression of pulmonary fibrosis by targeting specific genes, offering potential avenues for treatment. This study lays a foundation for future research to translate these discoveries into effective clinical interventions, contributing to the advancement of IPF management and therapy.