Heparin Nebulization Attenuates Bleomycin-Induced Pulmonary Fibrosis through the Coagulation Activation Pathway: A Metabolomics Study

Heparin Nebulization Attenuates Bleomycin-Induced Pulmonary Fibrosis through the Coagulation Activation Pathway: A Metabolomics Study | 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 Heparin Nebulization Attenuates Bleomycin-Induced Pulmonary Fibrosis through the Coagulation Activation Pathway: A Metabolomics Study Dong Lai, Changfu Ji, Rui Zhang, Jeng-Yuan Yao, Zhaozhong Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7571251/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Objective: Pulmonary fibrosis represents a severe interstitial lung disease pathologically characterized by excessive collagen deposition. This study aimed to investigate the effects of heparin nebulization on bleomycin-induced pulmonary fibrosis in rats and elucidate the underlying metabolic mechanisms. Methods: Thirty-six Sprague-Dawley rats were randomly assigned to three groups via simple randomization:the sham group, the saline group and the heparin group. The pulmonary fibrosis rats was induced through intratracheal bleomycin instillation, and confirmed by computed tomography (CT). Commencing on day 7 post-induction, the heparin group received 7-day heparin nebulization therapy, while the saline group received saline nebulization therapy. The sham group underwent neither bleomycin instillation nor nebulization therapy.The study evaluated pulmonary imaging examinations, pathological analyses,blood biomarkers (D-dimer,Hydroxyproline (Hyp),Interleukin-6(IL-6), α2-plasmin inhibitor-plasmin complex(PAP),thrombin-antithrombin complex(TAT),Tumor Tecrosis Factor(TNF-α)) and metabolomic characteristics.Results: Upon the seventh day, both the saline and heparin groups exhibited notably heightened blood biomarkers in contrast to the sham group. By the fourteenth day, the heparin group had significantly lower levels of D-dimer,Hyp, IL-6, TAT and TNF-α than the saline group. Additionally, the lung imaging and pathology scores in the heparin group were significantly improved relative to those in the saline group. Metabolomic analysis demonstrated distinct metabolic profiles among the groups, In the heparin group, there were 21 up-regulated and 50 down-regulated in the positive ion mode, and 77 up-regulated and 87 down-regulated in the negative ion mode. The key metabolic pathways involved included linoleic acid metabolism,taurine and hypotaurine metabolism,purine metabolism,arachidonic acid metabolism. Conclusion: Nebulized heparin significantly attenuated pulmonary fibrosis in this rat model. Metabolomic analysis highlighted the involvement of coagulation-associated metabolic pathways, proposing novel therapeutic targets for pulmonary fibrosis. Heparin Nebulization Pulmonary Fibrosis Metabolomics Coagulation Activation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Pulmonary fibrosis (PF) is a progressive interstitial lung disease characterized by persistent fibroblast activation, excessive deposition of the extracellular matrix, and distortion of the alveolar architecture [1] . Idiopathic interstitial pneumonia most frequently manifests as PF and results in the progressive impairment of lung function [2] . As the condition deteriorates, patients experience a continuous decline in respiratory capacity, with a median survival period of merely 3 to 5 years [3] . Consequently, its prognosis poorer than that of numerous malignanciess, leading to its classification as a “tumor-like disease” [4] . Mounting evidence currently implicates that the uncontrolled activation of the tissue-factor-driven extrinsic coagulation cascade plays a role in amplifying inflammation, fibrin deposition, and matrix remodeling during the progression of PF [ 5, 6] . In mouse models, heparin sulfate with o-sulfate residues has been demonstrated to impede allergic asthma inflammation, underscoring the potential of novel glycosaminoglycans or mimetics in ameliorating pulmonary fibrosis [7] .Targeting coagulation mediators is thus mechanistically appealing. Unfractionated heparin (UFH) accelerates antithrombin-mediated inhibition of thrombin and simultaneously attenuates inflammation. Systemic or inhaled UFH has alleviated bleomycin-induced fibrosis and acute lung injury in multiple animal models [ 8 –1 1 ] .However, systemic administration is restricted by the risk of bleeding.Nebulization represents a clinically feasible strategy for the direct delivery of UFH to the lung parenchyma, optimizing local efficacy while minimizing systemic exposure. Pulmonary fibrosis is a complex pathological process involving multiple cells, molecules, and signaling pathways [12] , Yet the associated metabolic adaptations during fibrogenesis have not been comprehensively characterized. Considering the intricate crosstalk between coagulation and cellular metabolism [13] , untargeted metabolomics [1 4 ] provides a potent tool for captureing UFH-induced metabolic reprogramming and identifying additional antifibrotic pathways. Accordingly, we hypothesized that a brief 7-day course of nebulized UFH initiated subsequent to the onset of fibrogenesis would down-regulate coagulation-induced inflammation, alleviate lung injury, and prompt a distinct alteration in coagulation-associated metabolic pathways. To verify this hypothesis, high-resolution computed tomography(CT), histopathology, coagulation and inflammatory biomarkers,and liquid chromatography-tandem mass spectrometry (LC-MS/MS) based metabolomics were integrated in a rat model of bleomycin-induced PF. Methods 2.1. Animal study Thirty-six male adult Sprague Dawley (SD) rats(250-350g) were procured from Hangzhou Medical College (license SCXK-(Zhe)-2024-0002). All experimental animals were housed in a specific-pathogen-free (SPF) environment and underwent for a 7-day acclimation period prior to the experiment. we provided with the standard conditions(20-26ºC temperature,50%-55% humidity,and unrestricted access to standard rodent chow and water). All experimental procedures conformed to the ARRIVE 2.0 guidelines and were approved by the Institutional Animal Care. Following an acclimation period, the rats pulmonary fibrosis model were constructed via intratracheal administration of bleomycin at a dosage of 5mg/kg,as depicted in Graphical Abstract. Rats were randomized assigned into three groups(n=12 per group):the sham group,the saline group,and the heparin group.Under isoflurane anesthesia, rats in the saline group and the heparin group were administered intratracheal bleomycin(5 mg kg⁻¹ dissolved in 50 µL of saline,Yiseen Biologics, 60216ES60), while those in the sham received only saline. The success of the pulmonary fibrosis model was confirmed via high-resolution CT(64-slice CT scanner, GE Healthcare Systems,Optima CT680 Expert) on day 7. Beginning from day 7, rats in the heparin group were subjected to nebulized heparin treatment(15 mg kg⁻¹, administered twice daily, with a particle size ranging from 3 to 5 µm, using a PARIBOYSX nebulizer, Changshan Pharmaceutical, batch F201231208) for a duration of 7 days. In contrast, rats in the sham and the saline group inhaled saline.CT imaging, cardiac blood sampling(conducted on Day 0, Day 7, and Day 14), and lung harvest(performed on Day 14) were carried out. 2.2.B lood Biomarkers Plasma levels of TAT, PAP,IL-6, TNF-α, Hyp, and D-dimer were quantified by multifunctional microplate reader (Synergy LX, USA),plate washing machine (Shanghai Kehua, ST-36W),paraffin Microtome (Jinhua Kelatay, CR-603);waters Xevo G2-XS QTof quadrupole time-of-flight mass spectrometer (Waters Corporation, Xevo G2-XS QTof),ensuring the highest level of accuracy in the measurements.The evaluation of TAT,PAP,IL-6, TNF-α, Hyp, and D-dimer was performed by CUSABIO/Huamei Bio and Qisong Bio. 2.3.Pulmonary Imaging examination and Pathological Analyses On days 0, 7, and 14 of the experimental protocol, the assigned rats underwent Computed tomography(CT) imaging to evaluate the experimental model. The rats were anesthetized with 3% isoflurane via inhalation and secured on an acrylic plate. The imaging area was carefully positioned, axial slices were acquired, and the images were reconstructed. The severity of pulmonary fibrosis was independently graded by Imaging physician blinded to the experimental groups using a 0-5 scoring within CT Imaging feature [15] (Table 1),and the mean scores were subsequently analyzed. To assess pulmonary histopathological changes and collagen deposition, lung sections 8μm thick will be prepared on day 14 and stained with hematoxylin and eosin (H&E). The presence of pulmonary fibrosis will be determined by microscopic examination.The severity of pulmonary fibrosis was independently graded by two pathologists blinded to the experimental groups. Table 1.CT Scoring Criteria for rat pulmonary fibrosis. Score CT Imaging Features [15] 0 Normal lung architecture : Clear lung fields with regular, delicate, and evenly distributed bronchovascular markings. Natural course of bronchi and vessels. No abnormal opacities. Uniform parenchymal density. Normal lobar morphology without thickening or distortion. Indicates intact pulmonary structure/function without inflammation or fibrosis. 1 Focal ground-glass opacity (GGO) : Slightly thickened/blurred lung markings with scattered ground-glass opacities (<10% unilateral lung area), predominantly peripheral or in lower lobes. Preserved visibility of vascular and bronchial structures through hazy areas. 2 Diffuse GGO with micronodules : Markedly thickened/disordered lung markings. GGO involvement expands (10-30% unilateral lung). Emerging fine reticular opacities indicating early fibrosis from interstitial fibroblast proliferation. 3 Reticular changes with subpleural lines : Mixed GGO and reticular opacities (30-50% unilateral lung). Early honeycombing (mainly basal/peripheral). Interlobular septal thickening and subpleural curvilinear lines (1-2mm thick). 4 Honeycombing with traction bronchiectasis : Predominant reticular/honeycomb changes with reduced GGO (50-70% unilateral lung). Mild volume loss (diminished lung fields). Distorted bronchovascular bundles. 5 Extensive consolidation and architectural destruction : Advanced honeycombing (>70% unilateral lung). Severe volume loss with elevated lung density. Markedly distorted bronchovascular bundles. Traction bronchiectasis with irregular bronchial dilation/path. 2.4. Metabolomics S ample P reparation Plasma was collected into heparinized tubes, subjected to centrifugation(1500g, 10 min, 4ºC), and stored at -80ºC. Samples were thawed on ice.Subsequently,50 µL of plasma was mixed with 200 µL of methanol, vortexed, and sonicated (5 min, 4ºC). After incubation at 4ºC for 1 h and centrifugation at15,000 g for 10 min, the supernatant was dried under a nitrogen stream and re-dissolved in 100 µL of an acetonitrile:water (1:1,v/v) solution for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. 2.5. LC–MS/MS A cquisition and D ata P rocessing Chromatographic separation was carried out on an ACQUITY UPLC(Waters)at a temperature of 40ºC. Solvent A, consisting of 0.1% formic acid in water, and solvent B (acetonitrile) were employed at a flow rate of 0.4 mL min⁻¹. The gradient elution program was as follows: from 0 to 1 min, the proportion of solvent B was maintained at 10%, from 1 to 7.5 min, the proportion of solvent B increased linearly from 10% to 65%; from 7.5 to 10.5 min, the proportion of solvent B further increased from 65% to 100%; from 10.5 to 12 min, the proportion of solvent B decreased from 100% back to 10%. The injection volume was set at 2 µL.The QToF mass spectrometer was operated in both positive ion(ESI+) and negative ion (ESI-) modes, with a mass range of 50–1,200 m/z. The source parameters were as follows: the capillary voltage was set at 3.0 kV, the cone voltage was 40V, the desolvation gas flow rate was 800 L h⁻¹at a temperature of 450ºC, and the source temperature was maintained at 100ºC. Leucine-enkephalin (m/z 556.2771+/554.2615-) was used for lock-mass calibration.The raw data were centroided using MassLynx 4.1 software. Subsequently, the data were processed in Progenesis QI 2.1 for alignment, deconvolution, and total-ion-current normalization. Statistical analysis and pathway analysis were conducted using MetaboAnalyst 6.0 and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. 3. Statistical A nalysis Data were analyzed using SPSS 26.0. Continuous variables are presented as mean ± SD or median (IQR) after Shapiro-Wilk normality testing. For comparisons between two groups, either the Mann–Whitney U test or Student's t-test was appropriately employed; for comparisons involving three or more groups, one - way analysis of variance (ANOVA) or the Kruskal-Wallis test with Bonferroni correction was utilized.Statistical significance was denoted by a two-tailed P<0.05. Metabolomic differential features were identified at a false discovery rate (FDR) less<than 0.05 (Benjamini-Hochberg). Pathway enrichment and impact scores were generated in MetaboAnalyst based on the Kyoto Encyclopedia of Gene s and Genomes (KEGG) topology. Results 4 . 1. Establish Successful Pulmonary Fibrosis Model and Comprehensive Assessment of Therapeutic Interventions The pulmonary fibrosis rats was induced through intratracheal bleomycin instillation, and confirmed by CT. After heparin nebulization treatment, the degree of pulmonary fibrosis was significantly improved(Fig 1). On the seventh day, CT imaging scores for pulmonary fibrosis in both the saline group and the heparin group were significantly higher than those in the sham group(P0.05). By the fourteenth day, both the saline group and the heparin group presented significantly higher fibrosis scores compared to the sham group(P<0.01). Nevertheless,the scores of the heparin group were significantly lower than those of the saline group(p<0.01) . (Table 2). On the fourteenth day, hematoxylin and eosin (H&E) staining results revealed that in the sham group, fine collagen fibers were present in the bronchial walls of rats, and a small amount of collagen fiber deposition was observed between the alveolar walls. In the saline group, alveolar wall collapse was noted, accompanied by significant pulmonary interstitial fibrosis, consolidation, and compensatory dilation of the remaining alveoli. However, in the heparin group, pulmonary interstitial fibrosis was markedly reduced, the pulmonary septum was widened, and only very mild fibrosis was detected(Fig 2). Table 2 CT scan scores in rats at day 7 and day 14 internationalist assessment CT scan scores on Day7 Day7 Z P the sham group 1.0 (0.5, 1.5) 4.231* 0.00 the saline group 3.5 (3.0, 4.0) 0.631** 0.528 the heparin group 2.5 (1.5, 3.0) 4.250*** 0.00 Intergroup comparisons 24.817 0.00 CT scan scores on Day 14 Day 14 Z P the sham group 1.0 (0.5, 1.5) 4.213* 0.00 the saline group 3.5 (3.0, 4.0) 3.483** 0.00 the heparin group 2.5 (1.5, 3.0) 3.788*** 0.00 Intergroup comparisons 27.453 0.00 “*” signifies a comparison between the sham group and the saline group. “**” signifies a comparison between the saline group and the heparin group. “***” signifies a comparison between the sham group and the heparin group. 4.2. Blood Biomarkers On Day 0, no significant differences were observed among the groups. On Day 7,the levels of D-dimer,Hyp, IL-6, PAP,TAT and TNF-α in both the saline group and the heparin group were significantly higher than those in the sham group(P<0.05 or P 0.05).On Day 14, the saline group exhibited significantly higher levels of D-dimer,Hyp, IL-6, PAP,TAT and TNF-α compared to the sham group(P<0.05 or P<0.01). Conversely, the heparin group showed no significant differences from the sham group, with the exception of TAT. When compared with the saline group, the heparin group had significantly lower levels of D-dimer,Hyp, IL-6,TAT and TNF-α (P<0.05 or P<0.01)(Fig 3). 4.3 Metabolomics 4.3.1 Partial Least Squares Discriminant Analysis (PLS-DA) In this study, a total of 614 and 622 endogenous metabolites were identified based on positive and negative ion patterns, respectively. Multivariate statistical analysis utilizing Partial Least Squares Discriminant Analysis (PLS-DA) revealed a significant segregation of metabolic profiles among the three groups(Fig 4). 4.3.2 Orthogonal P artial L east S quares- D iscriminant A nalysis ( O PLS-DA) and Volcano map analysis When orthogonal PLS-DA was applied to compare the heparin and saline groups, 21 metabolites were observed to be significantly up-regulated, whereas 50 metabolites were down-regulated in the positive ion mode(fold change >1.5,p<0.05). In the negative ion mode, 77 metabolites were significantly up-regulated and 87 were down-regulated. These findings suggest that heparin intervention notably modified the body's metabolic profile, which involves intricate regulatory mechanisms(Fig 5). 4.3.2 Major differential metabolites Differential metabolites were extracted from volcanic maps and subsequently compared with the Human Metabolome Database (HMDB). Based on precise mass numbers and MS/MS fragment information, a series of specific differential metabolites were identified. These metabolites can be classified into several categories, including organic acids (such as 1,2,3-propyl-tricarboxylic acid, 5-thymidine acid, 2-furanoic acid, etc.), nucleosides and nucleotides (e.g, AICAR, flavone mononucleotide, N2-methylguanosine), bile acids and their conjugates (taurocholic acid, deoxycholate, stone cholic acid, glycinate conjugate, etc.), lipids (lysophosphatidylcholine (16:1(9Z)/0:0), PC(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(22:4(7Z,10Z,13Z,16Z)/P-18:1(11Z)), etc.), and other compounds (acetyl-n-formyl-5-methoxyquinolamine nicotinic acid ribonucleoside, tetrahydrocortisone palmitoleate, 4,6-dihydroxyquinoline, etc.). A comprehensive list of these metabolites is presented in Table 3. Table 3 A comprehensive list of these metabolites is systematically presented. 4.3.3 Enrichment analysis to identify the influenced pathways Enrichment analysis revealed that several metabolic pathways, including linoleic acid metabolism,taurine and hypotaurine metabolism,purine metabolism,pyrimidine metabolism,arachidonic acid metabolism, were significantly influenced. The alterations in these key metabolites and pathways imply that heparin might regulate the body’s metabolic network via multiple pathways and nodes, thus exerting potential protective or interventional effects. This discovery provides a valuable avenue for further exploration of the regulatory mechanisms of heparin in disease processes(Fig 6). Discussion Pulmonary fibrosis (PF), a fatal interstitial lung disease, is characterized by structural remodeling and dysfunction of lung tissue, which ultimately results in respiratory failure [16] . Although the pathogenesis of idiopathic pulmonary fibrosis (IPF) remains not fully elucidated, inflammation, oxidative stress, and abnormal extracellular matrix deposition are acknowledged contributing factors [17] . Emerging evidence highlights the pivotal role of the coagulation system in the advancement of pulmonary fibrosis [18] . Individuals diagnosed with IPF present abnormal coagulation parameters, characterized by heightened coagulant activity and diminished fibrinolytic activity.These alterations can potentially result in pulmonary microvascular embolism, thereby aggravating tissue damage and fibrosis. Moreover, metabolic dysregulation, encompassing pathways related to energy, amino acid, and lipid metabolism, is intricately associated with PF [19] . These metabolic alterations engage in interactions with the coagulation system, exerting an impact on clotting factors and metabolite generation, which highlights their potential as diagnostic and therapeutic targets. Our bleomycin-induced rat model (5 mg·kg⁻¹, intratracheal) effectively replicated the crucial characteristics of pulmonary fibrosis, including elevated hydroxyproline levels, heightened inflammatory markers(IL-6,TNF-α), and dysregulated coagulation parameters (TAT, D-dimer) [20] .These findings are consistent with previous research,which has demonstrated that inflammation and coagulation activation act synergistically to promote fibroblast proliferation, collagen deposition, and fibrinolysis imbalance in pulmonary fibrosis [21,22] . The concurrent elevation of coagulation and inflammatory markers emphasizes the interaction between these systems during disease progression. In comparison with the saline group, heparin nebulization notably diminished the levels of Hyp,a crucial indicator of collagen deposition, and simultaneously inhibited the production of pro-inflammatory cytokines IL-6 and TNF-α,along with the TAT, thus reducing the degree of coagulation activation.Heparin exerts its effects via a dual- mechanism approach:first,by binding to antithrombin to mediate its anticoagulant function,thereby inhibiting thrombin generation and fibrin formation;second, through its anti-inflammatory action, which suppresses inflammatory cell infiltration,cytokine release,and oxidative stress. The synergistic interplay of these two mechanisms may jointly alleviate fibrosis and coagulation abnormalities.These findings are in line with previous research, which suggests that heparin can effectively mitigate extracellular matrix accumulation and fibrotic remodeling in a bleomycin-induced model [10] ,highlighting its therapeutic potential that extends beyond traditional anticoagulant applications to cover chronic inflammatory and fibrotic diseases such as pulmonary fibrosis. Further exploration of optimal dosing regimens and long-term efficacy will contribute to enhancing its clinical translational value. The rat model of pulmonary fibrosis was assessed via CT imaging and pathological grading criteria. After model establishment, the scores of the saline group and the heparin group were significantly higher than those of the sham group. However, there was no significant difference between the saline group and the heparin group, which verified the successful establishment of the bleomycin - induced pulmonary fibrosis model. Following nebulization intervention, the scores of the heparin group were significantly lower than those of the saline group, indicating that heparin nebulization intervention could ameliorate the imaging and pathological manifestations of pulmonary fibrosis. These scoring results were in accordance with the results of the enzyme-linked immunosorbent assay (ELISA), further validating the therapeutic effect of heparin intervention on pulmonary fibrosis. This consistency corroborates the conclusion reported in the literature that heparin can improve pulmonary fibrosis in the model [23.24] ,and supports its role in alleviating Broussonetia papyrifera injury.CT and histopathological scoring confirmed the successful establishment of the model, with the bleomycin - exposed group having higher fibrosis scores. Heparin intervention significantly improved the post - treatment imaging and pathological scores, which was correlated with the observed reduction in fibrosis burden detected by ELISA. Metabolomic analysis reveals that heparin nebulization can regulate crucial metabolic pathways involved in the pathogenesis of pulmonary fibrosis. The antifibrotic effects of heparin are associated with the following aspects:1. Arachidonic acid metabolism: The suppression of this pathway leads to a reduction in the production of inflammatory mediators (such as prostaglandins and leukotriens) [25] .2. Glycerophospholipid metabolism:Altered membrane dynamics and signaling, potentially alleviate fibroblast activation.3. Linoleic acid metabolism:The attenuation of oxidative stress and inflammation.4.Purine metabolism: The modulation of energy metabolism and inflammatory signaling. Coagulation mechanisms further contributed to fibrosis, as thrombin-induced PAR-1 activation promotes fibroblast proliferation and collagen synthesis [26] . The dual targeting of metabolic and coagulation pathways by heparin highlights its therapeutic potential.Specific metabolites associated with coagulation dysregulation in pulmonary fibrosisare as follows:Bile acids (taurine, deoxycholic acid): Disrupted metabolism was correlated with imbalances in coagulation factors, potentially via FXR signaling [27] .Fatty acids (stearic acid, behenic acid): Altered membrane properties influenced platelet function and coagulation factor activity.Phospholipids (lysophosphatidylcholine, phosphatidylcholine): Dysregulated assembly of prothrombin complexes was observed.AICAR: It was linked to purine metabolism, affecting inflammatory and coagulation pathways.These findings imply that metabolic reprogramming exacerbates fibrin deposition and fibroblast activation, establishing a self-perpetuating cycle of fibrosis. The outcomes of this experiment demonstrated consistency between metabolomics and functional analysis,specifically showing alignment between the metabolomics data and ELISA assay results:Heparin reduced the levels of IL-6, TNF-α, and TAT levels, which was concomitant with its regulation of the arachidonic acid, glycerophospholipid, and purine pathways.Partial least squares-discriminant analysis (PLS-DA) and volcano plots verified distinct clustering among groups, with the arachidonic acid and glycerophospholipid pathways being pivotal to the antifibrotic effects of heparin.This coherence emphasizes the multi-target action of heparin, which links metabolic, inflammatory, and coagulation mechanisms in pulmonary fibrosis. Although this study has achieved certain results, it is subject to limitations. It only monitored the 7 -day impact of heparin nebulization, and the long-term effects necessitate further investigation. Moreover, it only explored the influence of heparin on coagulation and inflammatory markers, and the therapeutic mechanism of heparin in pulmonary fibrosis remains ambiguous.In future studies， the sample size should be enlarged,the observation period should be extended, and detection techniques should be enhanced to conduct a more comprehensive assessment of the efficacy of heparin. Integrating molecular biology techniques to investigate the impact of heparin on signaling pathways related to pulmonary fibrosis can offer a more profound theoretical foundation for treatment. Conclusions This paper focuses on the coagulation mechanism, metabolic pathways, and the innovative heparin nebulization intervention in pulmonary fibrosis. A rat model of pulmonary fibrosis was established through intratracheal bleomycin injection. In the saline group and the heparin group, markers of pulmonary fibrosis, inflammation, and coagulation underwent significant alterations, indicating the involvement of the inflammatory and coagulation systems in the pathogenesis of the disease.The pivotal finding is that heparin nebulization can effectively impede the progression of pulmonary fibrosis,alleviate inflammation, and suppress the activation of the coagulation system. Its anticoagulant and anti-inflammatory properties present a novel therapeutic approach for pulmonary fibrosis when compared with traditional methods. Metabolomic analysis reveals that various metabolites, such as bile acids, fatty acids, phospholipid metabolites, and AICAR, are closely associated with the coagulation mechanism of pulmonary fibrosis. The mutual verification of metabolomic analysis and ELISA results emphasizes the strong correlation between the coagulation mechanism and multiple metabolic pathways. Intervening in these metabolic links, particularly considering the impact of heparin nebulization on them, represents a promising anti-fibrosis strategy.In summary, this study emphasizes the innovative application of heparin nebulization and the significance of metabolomic analysis in comprehending the coagulation processes of pulmonary fibrosis, thereby opening up new therapeutic prospects. Declarations Ethics Approval This study was approved by the Medical Ethics Committee of Xiamen Medical College Affiliated Second Hospital(Approval ID: 2022001). Data Availability All data generated or analysed during this study are included in this published article. Competing Interests The authors declare that they have no competing interests. Funding Statement The research was supported by the Natural Science Foundation of Fujian Province (Grant No. 2022J0112) and the Second Affiliated Hospital of Xiamen Medical College (Grant No. XYEY2022006). CRediT authorship contribution statement Dong Lai: Investigation,Writing-original draft; Changfu Ji:Data curation, Formal analysis; Rui Zhang,Zhaozhong Li,Siliang Tan, Xinlian Lai,and Jeng-Yuan Yao:Experimental operation,Investigation, Data curation; Yaw-Syan Fu: Formal analysis;Yan Tian :Writing-review &editing, Supervision. Acknowledgements We would like to thank Dr. Yaw-Syan Fu and Dr. Jeng-Yuan Yao for their methodological advice, as well as the technical team for their analytical support. References Zhu J, Liu L, Ma X, Cao X, Chen Y, Qu X, et al. The Role of DNA Damage and Repair in Idiopathic Pulmonary Fibrosis. Antioxidants (Basel, Switzerland). 2022;11(11):2292. doi:10.3390/antiox11112292. Lee SY, Park SY, Lee SH, Kim H, Kwon JH, Yoo JY, et al. The deubiquitinase UCHL3 mediates p300 - dependent chemokine signaling in alveolar type II cells to promote pulmonary fibrosis. Exp Mol Med. 2023;55(8):1795–1805. doi:10.1038/s12276-023-01066-1. 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Astragaloside trigger autophagy: Implication a potential therapeutic strategy for pulmonary fibrosis. Biomed Pharmacother. 2022;154:113603. doi:10.1016/j.biopha.2022.113603. D’Alessandro E, Scaf B, Munts C, van Hunnik A, Trevelyan CJ, Verheule S, et al. Coagulation Factor Xa Induces Proinflammatory Responses in Cardiac Fibroblasts via Activation of Protease - Activated Receptor - 1. Cells. 2021;10(11):2958. doi:10.3390/cells10112958. Fuchs PÖ, Calitz C, Pavlović N, Binet F, Solbak SMØ, Danielson UH, et al. Fibrin fragment E potentiates TGF - β - induced myofibroblast activation and recruitment. Cell Signal. 2020;72:109661. doi:10.1016/j.cellsig.2020.109661 Sritharan SS, Kølner-Augustson L, Kronborg-White S, Prior TS, Møller J, Bendstrup E. [Antifibrotic therapy in other fibrotic interstitial lung diseases than idiopathic pulmonary fibrosis]. Ugeskr Laeger. 2021 Dec 6;183(49):V04210348. Danish. Liu J, Wang X, Wang F, Teng L, Cao J. 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Int J Biol Markers. 2024;39(2):130–140. doi:10.1177/03936155241229454. Table Table 3 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files table3.xlsx Table 3 A comprehensive list of these metabolites is systematically presented. Graphicabstract.tif Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 11 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers invited by journal 09 Dec, 2025 Editor invited by journal 11 Nov, 2025 Editor assigned by journal 10 Sep, 2025 Submission checks completed at journal 10 Sep, 2025 First submitted to journal 09 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Medical College","correspondingAuthor":false,"prefix":"","firstName":"Changfu","middleName":"","lastName":"Ji","suffix":""},{"id":558892411,"identity":"3fdb2477-cb09-4173-93d4-a170d0d96c36","order_by":2,"name":"Rui Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Xiamen Medical College","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Zhang","suffix":""},{"id":558892412,"identity":"1d921be5-fb84-4406-ba09-b8bec547949a","order_by":3,"name":"Jeng-Yuan Yao","email":"","orcid":"","institution":"Xiamen Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jeng-Yuan","middleName":"","lastName":"Yao","suffix":""},{"id":558892419,"identity":"f67fb8c0-24b2-4dfa-b541-23fecda655c9","order_by":4,"name":"Zhaozhong Li","email":"","orcid":"","institution":"The Second Affiliated Hospital of Xiamen Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhaozhong","middleName":"","lastName":"Li","suffix":""},{"id":558892421,"identity":"38a8d61e-6d71-4a79-844c-84216e16c750","order_by":5,"name":"Siliang Tan","email":"","orcid":"","institution":"the Third Affiliated Hospital of Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Siliang","middleName":"","lastName":"Tan","suffix":""},{"id":558892422,"identity":"7d3ab317-b1b9-45e5-875f-d9ae6a0dfc85","order_by":6,"name":"Xinlian Lai","email":"","orcid":"","institution":"Shanghang County Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinlian","middleName":"","lastName":"Lai","suffix":""},{"id":558892423,"identity":"c35e0cde-9188-4576-9e4c-316a4edbf4dc","order_by":7,"name":"Yaw-Syan Fu","email":"","orcid":"","institution":"Xiamen Medical 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08:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7571251/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7571251/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98431097,"identity":"b6cd4a32-ddc6-4e24-a086-95885a765f61","added_by":"auto","created_at":"2025-12-17 16:47:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243107,"visible":true,"origin":"","legend":"\u003cp\u003eCT images obtained of the three groups;(a)the sham group;(b)the saline group;(c)the heparin group;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/5084e4d513a3be5d43de91bd.png"},{"id":98135718,"identity":"a22d1de4-c101-41f2-942e-98ef3f21f6be","added_by":"auto","created_at":"2025-12-13 16:21:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":434687,"visible":true,"origin":"","legend":"\u003cp\u003eLung histopathology of the three groups;(A)the sham group;(B)the saline group;(C)the heparin group;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/5857e2b113600119f7741803.png"},{"id":98431550,"identity":"b4d3ece9-c3d7-419b-8d14-873978fdd4b2","added_by":"auto","created_at":"2025-12-17 16:47:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62668,"visible":true,"origin":"","legend":"\u003cp\u003eBaseline Group:on day 0; Induction of PF-model:on day 7; Therapeutic Intervention:on day 14;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/9de3db596f0932c1295bb051.png"},{"id":98135721,"identity":"7dc6fe0b-e0f2-49f4-a1ed-a39840ac4dcf","added_by":"auto","created_at":"2025-12-13 16:21:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169179,"visible":true,"origin":"","legend":"\u003cp\u003ePartial Least Squares-discriminant analysis (PLS-DA) and heatmap analysis indicated distinct clustering patterns of major metabolites among the three groups.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/509cb4c5bf322f7d3f1d5e87.png"},{"id":98431228,"identity":"f1d1d4e1-de1a-47d5-9c43-d97138c0f1cd","added_by":"auto","created_at":"2025-12-17 16:47:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182556,"visible":true,"origin":"","legend":"\u003cp\u003eOrthogonal Partial Least Squares-discriminant analysis (OPLS-DA) employed for comparing the saline group and the heparin group, revealed a significant compositional difference between groups. The Volcano map shown in the positive ion mode showed 21 significantly upregulated and 50 significantly downregulated metabolites in the Heparin group. In the negative ion mode, the Heparin group exhibited 77 significantly upregulated and 87 significantly downregulated metabolites(fold change\u0026gt;1.5,p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/d92a1464ad46befd08f41ba4.png"},{"id":98431221,"identity":"8dba6217-9e15-4f08-ae6e-d80d71c5ddef","added_by":"auto","created_at":"2025-12-17 16:47:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159066,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment analysis was conducted to identify the influenced pathways.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/ae04a14886b8bbe484dc8bbe.png"},{"id":98444817,"identity":"00bbb44b-6f23-4b0c-823f-1085da1c6206","added_by":"auto","created_at":"2025-12-17 17:17:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2027570,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/fc6371fd-e377-472b-97a3-bf1ef955a576.pdf"},{"id":98135724,"identity":"db3d48a7-2a5a-4f7e-930d-594ec23b724e","added_by":"auto","created_at":"2025-12-13 16:21:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14003,"visible":true,"origin":"","legend":"\u003cp\u003eTable 3 A comprehensive list of these metabolites is systematically presented.\u003c/p\u003e","description":"","filename":"table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/4977dadb54a0410fe2998be7.xlsx"},{"id":98430546,"identity":"d6bb1f2b-5dec-4ad6-b588-8c3933fb1a9b","added_by":"auto","created_at":"2025-12-17 16:45:41","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1329393,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7571251/v1/45c2944d8d63e8ff501f96b2.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Heparin Nebulization Attenuates Bleomycin-Induced Pulmonary Fibrosis through the Coagulation Activation Pathway: A Metabolomics Study","fulltext":[{"header":"Background","content":"\u003cp\u003ePulmonary fibrosis (PF) is a progressive interstitial lung disease characterized by persistent fibroblast activation, excessive deposition of the extracellular matrix, and distortion of the alveolar architecture \u003csup\u003e[1]\u003c/sup\u003e. Idiopathic interstitial pneumonia\u0026nbsp;most frequently manifests as PF and\u0026nbsp;results in the progressive impairment of lung function\u003csup\u003e[2]\u003c/sup\u003e. As the condition deteriorates, patients experience a continuous decline in respiratory capacity, with a median survival period of merely 3 to 5 years \u003csup\u003e[3]\u003c/sup\u003e.\u0026nbsp;Consequently, its prognosis poorer than that of numerous malignanciess, leading to its classification as a “tumor-like disease” \u003csup\u003e[4]\u003c/sup\u003e.\u0026nbsp;Mounting evidence currently implicates that the uncontrolled activation of the tissue-factor-driven extrinsic coagulation cascade plays a role in amplifying inflammation, fibrin deposition, and matrix remodeling during the progression of PF\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e5,\u0026nbsp;\u003c/sup\u003e\u003csup\u003e6]\u003c/sup\u003e.\u0026nbsp;In mouse models, heparin sulfate with o-sulfate residues has been demonstrated to impede allergic asthma inflammation, underscoring the potential of novel glycosaminoglycans or mimetics in ameliorating pulmonary fibrosis \u003csup\u003e[7]\u003c/sup\u003e.Targeting coagulation mediators is\u0026nbsp;thus\u0026nbsp;mechanistically\u0026nbsp;appealing. Unfractionated heparin (UFH) accelerates antithrombin-mediated inhibition of thrombin\u0026nbsp;and simultaneously\u0026nbsp;attenuates\u0026nbsp;inflammation. Systemic or inhaled UFH has\u0026nbsp;alleviated\u0026nbsp;bleomycin-induced fibrosis and acute lung injury in multiple animal models \u003csup\u003e[\u003c/sup\u003e\u003csup\u003e8\u003c/sup\u003e\u003csup\u003e–1\u003c/sup\u003e\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e.However, systemic administration is\u0026nbsp;restricted\u0026nbsp;by\u0026nbsp;the risk of\u0026nbsp;bleeding.Nebulization represents a clinically feasible strategy\u0026nbsp;for the direct\u0026nbsp;delivery\u0026nbsp;of\u0026nbsp;UFH to the lung parenchyma,\u0026nbsp;optimizing\u0026nbsp;local efficacy while minimizing systemic exposure.\u0026nbsp;Pulmonary fibrosis is a complex pathological process involving multiple cells, molecules, and signaling pathways\u003csup\u003e[12]\u003c/sup\u003e,\u0026nbsp;Yet the\u0026nbsp;associated\u0026nbsp;metabolic adaptations during fibrogenesis have not been\u0026nbsp;comprehensively\u0026nbsp;characterized.\u0026nbsp;Considering\u0026nbsp;the\u0026nbsp;intricate\u0026nbsp;crosstalk between coagulation and cellular metabolism\u003csup\u003e[13]\u003c/sup\u003e, untargeted metabolomics\u003csup\u003e[1\u003c/sup\u003e\u003csup\u003e4\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e provides\u0026nbsp;a\u0026nbsp;potent\u0026nbsp;tool\u0026nbsp;for\u0026nbsp;captureing\u0026nbsp;UFH-induced metabolic reprogramming and\u0026nbsp;identifying\u0026nbsp;additional antifibrotic pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccordingly, we hypothesized that a brief 7-day course of nebulized UFH initiated subsequent to the onset of fibrogenesis would down-regulate coagulation-induced inflammation, alleviate lung injury, and prompt a distinct alteration in coagulation-associated metabolic pathways. To verify this hypothesis, high-resolution computed tomography(CT), histopathology, coagulation and inflammatory biomarkers,and liquid chromatography-tandem mass spectrometry (LC-MS/MS) based metabolomics were integrated in a rat model of bleomycin-induced PF.\u003c/p\u003e"},{"header":"Methods ","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u003c/strong\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty-six male adult Sprague Dawley (SD) rats(250-350g) were procured from Hangzhou Medical College (license SCXK-(Zhe)-2024-0002). All experimental animals were housed in a specific-pathogen-free (SPF) environment and underwent for a 7-day acclimation period prior to the experiment.\u0026nbsp;we\u0026nbsp;provided with the standard conditions(20-26\u0026ordm;C\u0026nbsp;temperature,50%-55%\u0026nbsp;humidity,and\u0026nbsp;unrestricted access to standard rodent chow and water). All\u0026nbsp;experimental\u0026nbsp;procedures conformed to the ARRIVE 2.0 guidelines and were approved by the Institutional Animal Care.\u003c/p\u003e\n\u003cp\u003eFollowing an acclimation period,\u0026nbsp;the rats pulmonary fibrosis model were constructed via intratracheal administration of bleomycin at a dosage of 5mg/kg,as depicted in Graphical Abstract. Rats were randomized\u0026nbsp;assigned\u0026nbsp;into three groups(n=12\u0026nbsp;per group):the sham group,the saline group,and the heparin group.Under isoflurane anesthesia, rats in the saline group and the heparin group were administered intratracheal bleomycin(5 mg kg⁻\u0026sup1;\u0026nbsp;dissolved in 50 \u0026micro;L of saline,Yiseen Biologics, 60216ES60), while those in the sham received only saline. The success of the pulmonary fibrosis model was confirmed via high-resolution CT(64-slice CT scanner, GE Healthcare Systems,Optima CT680 Expert) on day 7. Beginning\u0026nbsp;from\u0026nbsp;day\u0026nbsp;7, rats in the heparin group were subjected to nebulized heparin treatment(15 mg kg⁻\u0026sup1;, administered twice daily, with a particle size ranging from 3 to 5 \u0026micro;m, using a\u0026nbsp;PARIBOYSX nebulizer,\u0026nbsp;Changshan Pharmaceutical, batch F201231208) for a duration of 7 days. In contrast, rats in the sham and the saline group inhaled saline.CT imaging, cardiac blood sampling(conducted on Day 0, Day 7, and Day 14), and lung harvest(performed on Day 14) were carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.B\u003c/strong\u003e\u003cstrong\u003elood Biomarkers\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlasma levels of TAT, PAP,IL-6, TNF-\u0026alpha;, Hyp, and D-dimer were quantified by multifunctional microplate reader (Synergy LX, USA),plate washing machine (Shanghai Kehua, ST-36W),paraffin Microtome (Jinhua Kelatay, CR-603);waters Xevo G2-XS QTof quadrupole time-of-flight mass spectrometer (Waters Corporation, Xevo G2-XS QTof),ensuring the highest level of accuracy in the measurements.The evaluation of\u0026nbsp;TAT,PAP,IL-6, TNF-\u0026alpha;, Hyp, and D-dimer was performed by\u0026nbsp;CUSABIO/Huamei Bio and Qisong Bio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.Pulmonary Imaging examination and Pathological Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn days 0, 7, and 14 of the experimental protocol, the assigned rats underwent Computed tomography(CT) imaging to evaluate the experimental model. The rats were anesthetized with 3% isoflurane via inhalation and secured on an acrylic plate. The imaging area was carefully positioned, axial slices were acquired, and the images were reconstructed. The severity of pulmonary fibrosis was independently graded by Imaging physician blinded to the experimental groups using a 0-5 scoring within CT Imaging feature\u003csup\u003e[15]\u003c/sup\u003e(Table 1),and the mean scores were subsequently analyzed.\u003c/p\u003e\n\u003cp\u003eTo assess pulmonary histopathological changes and collagen deposition, lung sections 8\u0026mu;m thick will be prepared on day 14 and stained with hematoxylin and eosin (H\u0026amp;E). The presence of pulmonary fibrosis will be determined by microscopic examination.The severity of pulmonary fibrosis was independently graded by two pathologists blinded to the experimental groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1.CT Scoring Criteria for rat pulmonary fibrosis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003eScore\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCT Imaging Features\u003c/strong\u003e\u003csup\u003e[15]\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal lung architecture\u003c/strong\u003e: Clear lung fields with regular, delicate, and evenly distributed bronchovascular markings. Natural course of bronchi and vessels. No abnormal opacities. Uniform parenchymal density. Normal lobar morphology without thickening or distortion. Indicates intact pulmonary structure/function without inflammation or fibrosis.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFocal ground-glass opacity (GGO)\u003c/strong\u003e: Slightly thickened/blurred lung markings with scattered ground-glass opacities (\u0026lt;10% unilateral lung area), predominantly peripheral or in lower lobes. Preserved visibility of vascular and bronchial structures through hazy areas.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDiffuse GGO with micronodules\u003c/strong\u003e: Markedly thickened/disordered lung markings. GGO involvement expands (10-30% unilateral lung). Emerging fine reticular opacities indicating early fibrosis from interstitial fibroblast proliferation.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReticular changes with subpleural lines\u003c/strong\u003e: Mixed GGO and reticular opacities (30-50% unilateral lung). Early honeycombing (mainly basal/peripheral). Interlobular septal thickening and subpleural curvilinear lines (1-2mm thick).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHoneycombing with traction bronchiectasis\u003c/strong\u003e: Predominant reticular/honeycomb changes with reduced GGO (50-70% unilateral lung). Mild volume loss (diminished lung fields). Distorted bronchovascular bundles.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 519px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtensive consolidation and architectural destruction\u003c/strong\u003e: Advanced honeycombing (\u0026gt;70% unilateral lung). Severe volume loss with elevated lung density. Markedly distorted bronchovascular bundles. Traction bronchiectasis with irregular bronchial dilation/path.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Metabolomics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003eample\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003ereparation\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasma was collected into heparinized tubes, subjected to centrifugation(1500g, 10 min, 4\u0026ordm;C), and stored at\u0026nbsp;-80\u0026ordm;C. Samples were thawed on ice.Subsequently,50 \u0026micro;L of plasma was mixed with 200 \u0026micro;L of methanol, vortexed, and sonicated (5 min, 4\u0026ordm;C). After incubation at 4\u0026ordm;C\u0026nbsp;for 1 h and centrifugation at15,000 g for 10 min, the supernatant was dried under a nitrogen stream and re-dissolved in 100 \u0026micro;L of an acetonitrile:water (1:1,v/v) solution for liquid chromatography\u0026ndash;tandem mass spectrometry (LC-MS/MS) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;LC\u0026ndash;MS/MS\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003ecquisition and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eata\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003erocessing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatographic separation was carried out on an ACQUITY UPLC(Waters)at a temperature of 40\u0026ordm;C. Solvent A, consisting of 0.1% formic acid in water, and solvent B (acetonitrile) were employed at a flow rate of 0.4 mL min⁻\u0026sup1;. The gradient elution program was as follows: from 0 to 1 min, the proportion of solvent B was maintained at 10%, from 1 to 7.5 min, the proportion of solvent B increased linearly from 10% to 65%; from 7.5 to 10.5 min, the proportion of solvent B further increased from 65% to 100%; from 10.5 to 12 min, the proportion of solvent B decreased from 100% back to 10%. The injection volume was set at 2 \u0026micro;L.The QToF mass spectrometer was operated in both positive ion(ESI+) and negative ion (ESI-) modes, with a mass range of 50\u0026ndash;1,200 m/z. The source parameters were as follows: the capillary voltage was set at 3.0 kV, the cone voltage was 40V, the desolvation gas flow rate was 800 L h⁻\u0026sup1;at a temperature of 450\u0026ordm;C, and the source temperature was maintained at 100\u0026ordm;C. Leucine-enkephalin (m/z 556.2771+/554.2615-) was used for lock-mass calibration.The raw data were centroided using MassLynx 4.1 software. Subsequently, the data were processed in Progenesis QI 2.1 for alignment, deconvolution, and total-ion-current normalization. Statistical analysis and pathway analysis were conducted using MetaboAnalyst 6.0 and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Statistical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003enalysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS 26.0. Continuous variables are presented as mean \u0026plusmn; SD or median (IQR) after Shapiro-Wilk normality testing. For comparisons between two groups, either the Mann\u0026ndash;Whitney U test or Student\u0026apos;s t-test was appropriately employed; for comparisons involving three or more groups, one - way analysis of variance (ANOVA) or the Kruskal-Wallis test with Bonferroni correction was utilized.Statistical significance was denoted by a two-tailed P\u0026lt;0.05. Metabolomic differential features were identified at a false discovery rate (FDR) less\u0026lt;than 0.05 (Benjamini-Hochberg). Pathway enrichment and impact scores were generated in MetaboAnalyst based on the Kyoto Encyclopedia of Gene s and Genomes (KEGG) topology.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e1.\u003c/strong\u003e Establish Successful Pulmonary Fibrosis Model and Comprehensive Assessment of Therapeutic Interventions\u003c/p\u003e\n\u003cp\u003eThe pulmonary fibrosis rats was induced through intratracheal bleomycin instillation, and confirmed by CT. After heparin nebulization treatment, the degree of pulmonary fibrosis was significantly improved(Fig 1).\u0026nbsp;On the seventh day, CT imaging scores for pulmonary fibrosis in both the saline group and the heparin group were significantly higher than those in the sham group(P\u0026lt;0.01),yet no substantial distinction was observed between the saline and heparin groups(P\u0026gt;0.05). By the fourteenth day, both the saline group and the heparin group presented significantly higher fibrosis scores compared to the sham group(P\u0026lt;0.01). Nevertheless,the scores of the heparin group were significantly lower than those of the saline group(p\u0026lt;0.01) . (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the fourteenth day, hematoxylin and eosin (H\u0026amp;E) staining results revealed that in the sham group, fine collagen fibers were present in the bronchial walls of rats, and a small amount of collagen fiber deposition was observed between the alveolar walls. In the saline group, alveolar wall collapse was noted, accompanied by significant pulmonary interstitial fibrosis, consolidation, and compensatory dilation of the remaining alveoli. However, in the heparin group, pulmonary interstitial fibrosis was markedly reduced, the pulmonary septum was widened, and only very mild fibrosis was detected(Fig 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2\u0026nbsp;CT\u0026nbsp;scan\u0026nbsp;scores in rats at day\u0026nbsp;7 and\u0026nbsp;day\u0026nbsp;14\u0026nbsp;internationalist assessment\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003eCT\u0026nbsp;scan\u0026nbsp;scores\u0026nbsp;on Day7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003eDay7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003ethe sham group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e1.0 (0.5, 1.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e4.231*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003ethe saline group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e3.5 (3.0, 4.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e0.631**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.528\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003ethe heparin group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e2.5 (1.5, 3.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e4.250***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003eIntergroup comparisons\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e24.817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003eCT scan scores on Day 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003eDay 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003ethe sham group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e1.0 (0.5, 1.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e4.213*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003ethe saline group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e3.5 (3.0, 4.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e3.483**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003ethe heparin group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e2.5 (1.5, 3.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e3.788***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 269px;\"\u003e\n \u003cp\u003eIntergroup comparisons\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e27.453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026ldquo;*\u0026rdquo;\u0026nbsp;signifies a comparison between\u0026nbsp;the sham group\u0026nbsp;and\u0026nbsp;the saline group. \u0026ldquo;**\u0026rdquo;\u0026nbsp;signifies a comparison between\u0026nbsp;the saline group\u0026nbsp;and\u0026nbsp;the heparin group. \u0026ldquo;***\u0026rdquo;\u0026nbsp;signifies a comparison between\u0026nbsp;the sham group\u0026nbsp;and\u0026nbsp;the heparin group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Blood Biomarkers\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn Day 0, no significant differences were observed among the groups. On Day 7,the levels of D-dimer,Hyp, IL-6, PAP,TAT and TNF-\u0026alpha; in both the saline group and the heparin group were significantly higher than those in the sham group(P\u0026lt;0.05 or P\u0026lt;0.01),whereas no significant difference was detected between the saline group and the heparin group(P\u0026gt; 0.05).On Day 14, the saline group exhibited significantly higher levels of D-dimer,Hyp, IL-6, PAP,TAT and TNF-\u0026alpha; compared to the sham group(P\u0026lt;0.05 or P\u0026lt;0.01). Conversely, the heparin group showed no significant differences from the sham group, with the exception of TAT. When compared with the saline group, the heparin group had significantly lower levels of D-dimer,Hyp, IL-6,TAT and TNF-\u0026alpha; (P\u0026lt;0.05 or P\u0026lt;0.01)(Fig 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMetabolomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePartial Least Squares Discriminant Analysis (PLS-DA)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, a total of 614 and 622 endogenous metabolites were identified based on positive and negative ion patterns, respectively. Multivariate statistical analysis utilizing Partial Least Squares Discriminant Analysis (PLS-DA) revealed a significant segregation of metabolic profiles among the three groups(Fig 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eOrthogonal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003eartial\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003cstrong\u003eeast\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003equares-\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eiscriminant\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003enalysis (\u003c/strong\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003cstrong\u003ePLS-DA)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eVolcano map analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen orthogonal PLS-DA was applied to compare the heparin and saline groups, 21 metabolites were observed to be significantly up-regulated, whereas 50 metabolites were down-regulated in the positive ion mode(fold change \u0026gt;1.5,p\u0026lt;0.05). In the negative ion mode, 77 metabolites were significantly up-regulated and 87 were down-regulated. These findings suggest that heparin intervention notably modified the body\u0026apos;s metabolic profile, which involves intricate regulatory mechanisms(Fig 5).\u003c/p\u003e\n\u003ch4\u003e4.3.2 Major differential metabolites\u003c/h4\u003e\n\u003cp\u003eDifferential metabolites were extracted from volcanic maps and subsequently compared with the Human Metabolome Database (HMDB). Based on precise mass numbers and MS/MS fragment information, a series of specific differential metabolites were identified. These metabolites can be classified into several categories, including organic acids (such as 1,2,3-propyl-tricarboxylic acid, 5-thymidine acid, 2-furanoic acid, etc.), nucleosides and nucleotides (e.g, AICAR, flavone mononucleotide, N2-methylguanosine), bile acids and their conjugates (taurocholic acid, deoxycholate, stone cholic acid, glycinate conjugate, etc.), lipids (lysophosphatidylcholine (16:1(9Z)/0:0), PC(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PE(22:4(7Z,10Z,13Z,16Z)/P-18:1(11Z)), etc.), and other compounds (acetyl-n-formyl-5-methoxyquinolamine nicotinic acid ribonucleoside, tetrahydrocortisone palmitoleate, 4,6-dihydroxyquinoline, etc.). A comprehensive list of these metabolites is presented in Table 3.\u003c/p\u003e\n\u003cp\u003eTable 3 A comprehensive list of these metabolites is systematically presented.\u003c/p\u003e\n\u003ch4\u003e4.3.3 Enrichment analysis to identify the influenced pathways\u003c/h4\u003e\n\u003cp\u003eEnrichment analysis revealed that several metabolic pathways, including linoleic acid metabolism,taurine and hypotaurine metabolism,purine metabolism,pyrimidine metabolism,arachidonic acid metabolism, were significantly influenced. The alterations in these key metabolites and pathways imply that heparin might regulate the body\u0026rsquo;s metabolic network via multiple pathways and nodes, thus exerting potential protective or interventional effects. This discovery provides a valuable avenue for further exploration of the regulatory mechanisms of heparin in disease processes(Fig 6).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePulmonary fibrosis (PF), a fatal interstitial lung disease, is characterized by structural remodeling and dysfunction of lung tissue, which ultimately results in respiratory failure\u003csup\u003e[16]\u003c/sup\u003e. Although the pathogenesis of idiopathic pulmonary fibrosis (IPF) remains not fully elucidated, inflammation, oxidative stress, and abnormal extracellular matrix deposition are acknowledged contributing factors\u003csup\u003e[17]\u003c/sup\u003e. Emerging evidence highlights the pivotal role of the coagulation system in the advancement of pulmonary fibrosis\u003csup\u003e[18]\u003c/sup\u003e. Individuals diagnosed with IPF present abnormal coagulation parameters, characterized by heightened coagulant activity and diminished fibrinolytic activity.These alterations can potentially result in pulmonary microvascular embolism, thereby aggravating tissue damage and fibrosis. Moreover, metabolic dysregulation, encompassing pathways related to energy, amino acid, and lipid metabolism, is intricately associated with PF\u003csup\u003e[19]\u003c/sup\u003e. These metabolic alterations engage in interactions with the coagulation system, exerting an impact on clotting factors and metabolite generation, which highlights their potential as diagnostic and therapeutic targets.\u003c/p\u003e\n\u003cp\u003eOur bleomycin-induced rat model (5 mg·kg⁻¹, intratracheal) effectively replicated the crucial characteristics of pulmonary fibrosis, including elevated hydroxyproline levels, heightened inflammatory markers(IL-6,TNF-α), and dysregulated coagulation parameters (TAT, D-dimer) \u003csup\u003e[20]\u003c/sup\u003e .These findings are consistent with previous research,which has demonstrated that inflammation and coagulation activation act synergistically to promote fibroblast proliferation, collagen deposition, and fibrinolysis imbalance in pulmonary fibrosis \u003csup\u003e[21,22]\u003c/sup\u003e. The concurrent elevation of coagulation and inflammatory markers emphasizes the interaction between these systems during disease progression.\u003c/p\u003e\n\u003cp\u003eIn comparison with the saline group, heparin nebulization notably diminished the levels of Hyp,a crucial indicator of collagen deposition, and simultaneously inhibited the production of pro-inflammatory cytokines IL-6 and TNF-α,along with the TAT, thus reducing the degree of coagulation activation.Heparin exerts its effects via a dual- mechanism approach:first,by binding to antithrombin to mediate its anticoagulant function,thereby inhibiting thrombin generation and fibrin formation;second, through its anti-inflammatory action, which suppresses inflammatory cell infiltration,cytokine release,and oxidative stress. The synergistic interplay of these two mechanisms may jointly alleviate fibrosis and coagulation abnormalities.These findings are in line with previous research, which suggests that heparin can effectively mitigate extracellular matrix accumulation and fibrotic remodeling in a bleomycin-induced model\u003csup\u003e[10]\u003c/sup\u003e,highlighting its therapeutic potential that extends beyond traditional anticoagulant applications to cover chronic inflammatory and fibrotic diseases such as pulmonary fibrosis. Further exploration of optimal dosing regimens and long-term efficacy will contribute to enhancing its clinical translational value.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe rat model of pulmonary fibrosis was assessed via CT imaging and pathological grading criteria. After model establishment, the scores of the saline group and the heparin group were significantly higher than those of the sham group. However, there was no significant difference between the saline group and the heparin group, which verified the successful establishment of the bleomycin - induced pulmonary fibrosis model.\u003c/p\u003e\n\u003cp\u003eFollowing nebulization intervention, the scores of the heparin group were significantly lower than those of the saline group, indicating that heparin nebulization intervention could ameliorate the imaging and pathological manifestations of pulmonary fibrosis. These scoring results were in accordance with\u0026nbsp;the results of the enzyme-linked immunosorbent assay\u0026nbsp;(ELISA),\u0026nbsp;further validating the therapeutic effect of heparin intervention on pulmonary fibrosis. This consistency corroborates the conclusion reported in the literature that heparin can improve pulmonary fibrosis in the model\u003csup\u003e[23.24]\u003c/sup\u003e,and supports its\u0026nbsp;role in alleviating Broussonetia papyrifera injury.CT and histopathological scoring confirmed the successful establishment of the model, with the bleomycin - exposed group having higher fibrosis scores. Heparin intervention significantly improved the post - treatment imaging and pathological scores, which was correlated with the observed reduction in fibrosis burden detected by ELISA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMetabolomic analysis reveals that heparin nebulization can regulate crucial metabolic pathways involved in the pathogenesis of pulmonary fibrosis. The antifibrotic effects of heparin are associated with the following aspects:1. Arachidonic acid metabolism: The suppression of this pathway leads to a reduction in the production of inflammatory mediators (such as prostaglandins and leukotriens)\u003csup\u003e[25]\u003c/sup\u003e.2. Glycerophospholipid metabolism:Altered membrane dynamics and signaling, potentially alleviate fibroblast activation.3. Linoleic acid metabolism:The attenuation of oxidative stress and inflammation.4.Purine metabolism: The modulation of energy metabolism and inflammatory signaling.\u003c/p\u003e\n\u003cp\u003eCoagulation mechanisms further contributed to fibrosis, as thrombin-induced PAR-1 activation promotes fibroblast proliferation and collagen synthesis \u003csup\u003e[26]\u003c/sup\u003e. The dual targeting of metabolic\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eand coagulation pathways by heparin highlights its therapeutic potential.Specific metabolites associated with coagulation dysregulation in pulmonary fibrosisare as follows:Bile acids (taurine, deoxycholic acid): Disrupted metabolism was correlated with imbalances in coagulation factors, potentially via FXR signaling \u003csup\u003e[27]\u003c/sup\u003e.Fatty acids (stearic acid, behenic acid): Altered membrane properties influenced platelet function and coagulation factor activity.Phospholipids (lysophosphatidylcholine, phosphatidylcholine): Dysregulated assembly of prothrombin complexes was observed.AICAR: It was linked to purine metabolism, affecting inflammatory and coagulation pathways.These findings imply that metabolic reprogramming exacerbates fibrin deposition and fibroblast activation, establishing a self-perpetuating cycle of fibrosis.\u003c/p\u003e\n\u003cp\u003eThe outcomes of this experiment demonstrated\u0026nbsp;consistency\u0026nbsp;between metabolomics and functional analysis,specifically showing alignment between the metabolomics data and ELISA assay results:Heparin reduced the levels of IL-6, TNF-α, and TAT levels, which was concomitant with its regulation of the arachidonic acid, glycerophospholipid, and purine pathways.Partial least squares-discriminant analysis (PLS-DA) and volcano plots verified distinct clustering among groups, with the arachidonic acid and glycerophospholipid pathways being pivotal to the antifibrotic effects of heparin.This coherence emphasizes the multi-target action of heparin, which links metabolic, inflammatory, and coagulation mechanisms in pulmonary fibrosis.\u003c/p\u003e\n\u003cp\u003eAlthough this study has achieved certain results, it is subject to limitations. It only monitored the 7 -day impact of heparin nebulization, and the long-term effects necessitate further investigation. Moreover, it only explored the influence of heparin on coagulation and inflammatory markers, and the therapeutic mechanism of heparin in pulmonary fibrosis remains ambiguous.In future studies， the sample size should be enlarged,the observation period should be extended, and detection techniques should be enhanced to conduct a more comprehensive assessment of the efficacy of heparin. Integrating molecular biology techniques to investigate the impact of heparin on signaling pathways related to pulmonary fibrosis can offer a more profound theoretical foundation for treatment.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis paper focuses on the coagulation mechanism, metabolic pathways, and the innovative heparin nebulization intervention in pulmonary fibrosis. A rat model of pulmonary fibrosis was established through intratracheal bleomycin injection. In the saline group and the heparin group, markers of pulmonary fibrosis, inflammation, and coagulation underwent significant alterations, indicating the involvement of the inflammatory and coagulation systems in the pathogenesis of the disease.The pivotal finding is that heparin nebulization can effectively impede the progression of pulmonary fibrosis,alleviate inflammation, and suppress the activation of the coagulation system. Its anticoagulant and anti-inflammatory properties present a novel therapeutic approach for pulmonary fibrosis when compared with traditional methods. Metabolomic analysis reveals that various metabolites, such as bile acids, fatty acids, phospholipid metabolites, and AICAR, are closely associated with the coagulation mechanism of pulmonary fibrosis. The mutual verification of metabolomic analysis and ELISA results emphasizes the strong correlation between the coagulation mechanism and multiple metabolic pathways. Intervening in these metabolic links, particularly considering the impact of heparin nebulization on them, represents a promising anti-fibrosis strategy.In summary, this study emphasizes the innovative application of heparin nebulization and the significance of metabolomic analysis in comprehending the coagulation processes of pulmonary fibrosis, thereby opening up new therapeutic prospects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of\u0026nbsp;Xiamen Medical College Affiliated Second Hospital(Approval ID: 2022001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research\u0026nbsp;was supported by the Natural Science Foundation of Fujian Province (Grant No. 2022J0112) and\u0026nbsp;the Second Affiliated Hospital of Xiamen Medical College\u0026nbsp;(Grant No. XYEY2022006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDong Lai: Investigation,Writing-original draft; Changfu Ji:Data curation, Formal analysis;\u003c/p\u003e\n\u003cp\u003eRui Zhang,Zhaozhong Li,Siliang Tan, Xinlian Lai,and Jeng-Yuan Yao:Experimental operation,Investigation, Data curation; Yaw-Syan Fu: Formal analysis;Yan Tian :Writing-review \u0026amp;editing, Supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Yaw-Syan Fu and Dr. Jeng-Yuan Yao for their methodological advice, as well as the technical team for their analytical support.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhu J, Liu L, Ma X, Cao X, Chen Y, Qu X, et al. 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Cell Signal. 2020;72:109661. doi:10.1016/j.cellsig.2020.109661\u003c/li\u003e\n\u003cli\u003eSritharan SS, K\u0026oslash;lner-Augustson L, Kronborg-White S, Prior TS, M\u0026oslash;ller J, Bendstrup E. [Antifibrotic therapy in other fibrotic interstitial lung diseases than idiopathic pulmonary fibrosis]. Ugeskr Laeger. 2021 Dec 6;183(49):V04210348. Danish.\u003c/li\u003e\n\u003cli\u003eLiu J, Wang X, Wang F, Teng L, Cao J. Attenuation effects of heparin - superoxide dismutase conjugate on bleomycin - induced lung fibrosis in vivo and radiation - induced inflammatory cytokine expression in vitro. Biomed Pharmacother. 2009;63(7):484\u0026ndash;491. doi:10.1016/j.biopha.2008.04.009.\u003c/li\u003e\n\u003cli\u003ePereira M, Liang J, Edwards - Hicks J, Meadows AM, Hinz C, Liggi S, et al. Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti - inflammatory effects of fasting. 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Int J Biol Markers. 2024;39(2):130\u0026ndash;140. doi:10.1177/03936155241229454.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 3 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Heparin Nebulization, Pulmonary Fibrosis, Metabolomics, Coagulation Activation","lastPublishedDoi":"10.21203/rs.3.rs-7571251/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7571251/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Pulmonary fibrosis represents a severe interstitial lung disease pathologically characterized by excessive collagen deposition. This study aimed to investigate the effects of heparin nebulization on bleomycin-induced pulmonary fibrosis in rats and elucidate the underlying metabolic mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Thirty-six Sprague-Dawley rats were randomly assigned to three groups via simple randomization:the sham group, the saline group and the heparin group. The pulmonary fibrosis rats was induced through intratracheal bleomycin instillation, and confirmed by computed tomography (CT). Commencing on day 7 post-induction, the heparin group received 7-day heparin nebulization therapy, while the saline group received saline nebulization therapy. The sham group underwent neither bleomycin instillation nor nebulization therapy.The study evaluated pulmonary imaging examinations, pathological analyses,blood biomarkers (D-dimer,Hydroxyproline (Hyp),Interleukin-6(IL-6), α2-plasmin inhibitor-plasmin complex(PAP),thrombin-antithrombin complex(TAT),Tumor Tecrosis Factor(TNF-α)) and metabolomic characteristics.Results: Upon the seventh day, both the saline and heparin groups exhibited notably heightened blood biomarkers in contrast to the sham group. By the fourteenth day, the heparin group had significantly lower levels of D-dimer,Hyp, IL-6, TAT and TNF-α than the saline group. Additionally, the lung imaging and pathology scores in the heparin group were significantly improved relative to those in the saline group. Metabolomic analysis demonstrated distinct metabolic profiles among the groups, In the heparin group, there were 21 up-regulated and 50 down-regulated in the positive ion mode, and 77 up-regulated and 87 down-regulated in the negative ion mode. The key metabolic pathways involved included linoleic acid metabolism,taurine and hypotaurine metabolism,purine metabolism,arachidonic acid metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eNebulized heparin significantly attenuated pulmonary fibrosis in this rat model. Metabolomic analysis highlighted the involvement of coagulation-associated metabolic pathways, proposing novel therapeutic targets for pulmonary fibrosis.\u003c/p\u003e","manuscriptTitle":"Heparin Nebulization Attenuates Bleomycin-Induced Pulmonary Fibrosis through the Coagulation Activation Pathway: A Metabolomics Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-13 16:21:04","doi":"10.21203/rs.3.rs-7571251/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-12-11T12:48:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41321761146092649131428348694617552636","date":"2025-12-09T09:45:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T09:03:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-11T06:47:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T01:43:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T01:42:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2025-09-09T08:09:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84e942ac-72d9-4fa8-be74-e9b70aa248cd","owner":[],"postedDate":"December 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-13T16:21:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-13 16:21:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7571251","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7571251","identity":"rs-7571251","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}