Alamandine Attenuates Signs and Reduces Fibrotic Markers in a Rat Model of Established Pulmonary Fibrosis

Alamandine Attenuates Signs and Reduces Fibrotic Markers in a Rat Model of Established Pulmonary Fibrosis | 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 Alamandine Attenuates Signs and Reduces Fibrotic Markers in a Rat Model of Established Pulmonary Fibrosis Isabel Amaral Martins, Andresa Thomé Silveira, Juliane Flor, Aline Blanco, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9066088/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Introduction: Pulmonary fibrosis (PF) is a challenging interstitial lung disease with limited therapeutic options. This study explores the therapeutic effects of alamandine (ALA), a renin-angiotensin system peptide, in reversing established fibrotic progression in an experimental model. Methods: Male Wistar rats were divided into four groups (n=6): control (CO), ALA-treated (ALA), bleomycin-induced fibrosis (BLM), and bleomycin plus ALA treatment (BA). Fibrosis was induced by intratracheal bleomycin (2.5 mg/kg) on day 0. Subcutaneous ALA treatment (50 µg/kg/day) started on day 10 and continued until day 19. Respiratory mechanics, body weight, Ashcroft score, and lung transforming growth factor-beta (TGF-β) content were evaluated. Plasma RAS peptides were quantified by LC-MS/MS. Results: Bleomycin significantly increased respiratory resistance (0.159±0.047 vs 0.104±0.026 cmH₂O/mL/s in CO, p<0.01) and Ashcroft score, and reduced body weight gain. ALA treatment from day 10–20 markedly improved body weight gain (p<0.001), reduced Ashcroft score (1.21±0.40 vs 2.23±0.35 in BLM, p<0.0001), and decreased lung TGF-β1 content (1.10±0.42 vs 3.70±1.7 pg/mg protein in BLM, p<0.005). In addition to significantly reducing thoracic chamber resistance (p < 0.02), ALA treatment reversed TGF-β elevation (p < 0.005) from 3.70 ± 1.7 in the BLM group to 1.10 ± 0.42 in the BA group and was associated with qualitatively improved respiratory effort, suggesting attenuation of bleomycin-induced fibrogenesis. Conclusion: Despite rapid tissue uptake, late ALA treatment attenuates histological fibrosis and TGF-β1 accumulation and improves clinical well-being in rats with established pulmonary fibrosis. These findings suggest potential therapeutic benefit that warrants further dose- and time-ranging studies. Pulmonary Fibrosis Alamandine TGF-β Respiratory Mechanics Therapeutic Intervention Figures Figure 1 Figure 2 Figure 3 Highlights ● Novel therapeutic approach targets established pulmonary fibrosis. ● Significant improvement in respiratory mechanics with ALA treatment. ● ALA reduces TGF-β levels, underscoring its antifibrotic potential. ● Enhanced body weight gain indicates improved health status with ALA. INTRODUCTION Pulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by relentless progression, with a median survival of approximately 3 to 5 years (Funke-Chambour & Geiser, 2015; Raghu et al., 2011). Despite extensive research efforts, the etiology and pathophysiological mechanisms of PF remain incompletely understood (Raghu et al., 2011). This lack of clarity contributes to the disease's high morbidity and mortality rates, compounded by limited therapeutic options(Dowman et al., 2013). Symptoms such as dyspnea, fatigue, and muscle weakness (Eaton et al., 2005) severely reduce functional capacity and significantly impact quality of life. While traditional therapeutic approaches have primarily focused on prevention, addressing advanced stages of fibrosis is crucial. This gap in research emphasizes the urgent need for innovative therapeutic strategies. Effective therapies that intervene after fibrosis progression are essential for improving patient prognosis. In the bleomycin model, fibrosis is typically established by days 7-14, with peak inflammation resolving and fibrotic remodeling beginning (Hübner et al., 2008; Moeller et al., n.d.). Our chosen therapeutic window (days 10-19) targets this established phase to evaluate reversal potential, though longer models may be needed for chronic effects. Fibrosis is characterized by fibroblast proliferation, extracellular matrix accumulation (Uhal et al., 2012), and oxidative stress (Otoupalova et al., 2020). Understanding these mechanisms and identifying potential therapeutic targets is vital for the development of effective treatments. Alamandine (ALA), a peptide within the renin-angiotensin system, has demonstrated various beneficial effects, particularly in the cardiovascular realm. Its mechanism of action is believed to involve binding to MrgD receptors, which modulate inflammatory responses and oxidative stress. By potentially activating these receptors, ALA may inhibit the production of pro-fibrotic cytokines such as transforming growth factor-beta (TGF-β) in vascular models (Yang et al., 2020), thereby contributing to fibrosis reduction. This dual action of ALA could modulate both inflammation and oxidative stress, presenting a unique therapeutic potential, especially in established fibrosis conditions where tissue remodeling has already compromised lung function. Indeed, our studies have shown that preventive ALA administration significantly attenuates fibrosis (Fernandes et al., 2021) and oxidative stress (Blanco et al., 2025) in a bleomycin-induced model. Notably, plasma ALA concentrations in fibrotic patients are four times lower than in healthy controls, while levels of angiotensin II (Ang II), Ang I, and Ang-(1-7) do not differ significantly (Sipriani et al., 2019). Transforming growth factor-beta plays a pivotal role in disease progression by inducing fibroblast transformation into myofibroblasts, contributing to excessive extracellular matrix deposition (Roberts et al., 1985, 1986). Modulating TGF-β levels is crucial for antifibrotic therapy, as improper regulation can exacerbate tissue injury (Yang et al., 2020). Targeting TGF-β could potentially attenuate fibrosis progression (Lafyatis, 2014). Our previous research indicates that preventive treatment with ALA reduces mesenchymal cells and myofibroblasts, restores total glutathione levels, and enhances antioxidant capacity, thereby supporting antifibrotic therapy through the control of oxidative stress (Blanco et al., 2025). Furthermore, increased airway resistance compromises lung function in fibrotic patients, directly affecting their quality of life (Bachofen & Scherrer, 1967). Improving airway resistance is a key therapeutic goal that may alleviate symptoms and enhance exercise tolerance. Building on our findings from the preventive ALA treatment (Blanco et al., 2025; Fernandes et al., 2021) this study aims to explore the reversal effects of ALA in an experimental pulmonary fibrosis model. Specifically, we will evaluate whether ALA can: (1) modulate TGF-β signaling, (2) reduce airway resistance, (3) decrease oxidative stress, and (4) improve well-being in fibrotic animals. MATERIALS AND METHODS This study utilized five-week-old male Wistar rats from the Animal Facility at the Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). The health of the rats was monitored throughout the experiment by assessing their general behavior and body weight. All procedures adhered to the Guide for the Care and Use of Laboratory Animals (USA -16) and received approval (number 749/21) from the Ethics Committee on Research at UFCSPA. Sample size (n = 5–6 per group) was empirically determined based on previous studies using the same bleomycin-induced pulmonary fibrosis model in our laboratory. Because the primary endpoints (Ashcroft score, thoracic chamber resistance and TGF-β levels in the present study) already showed clear statistical significance (p < 0.01–0.001), the group size provided sufficient power to detect differences while minimizing animal use in accordance with the 3Rs. Bleomycin and ALA Treatment Following a 7-day acclimatization period, animals were ranked by body weight and distributed into four groups in a balanced manner (block distribution) to ensure each group had similar mean initial body weight (n=6 each): control group (CO), ALA-treated group (ALA), bleomycin-only group (BLM), and bleomycin plus ALA treatment group (BA). On day zero, the rats were anesthetized intraperitoneally with ketamine (80 mg/kg) and xylazine (10 mg/kg) and underwent fibrosis induction via intratracheal intubation using a 20G catheter. Through this catheter, a bleomycin solution (2.5 mg/kg, Bonar, Ache) was administered to the BLM and BA groups, while the CO and ALA groups received only a saline solution (0.9% NaCl). The total volume of instillation was 0.2 mL per 200 grams of body weight. In the BA group, animals received bleomycin instillation on day zero and remained without further intervention until ALA treatment (50 mg/kg/day for both ALA and BA groups) starting on day 10, continuing daily until day 19 (Figure 1). This single dose was chosen based on previous preventive studies(Fernandes et al., 2021), but future work should explore dose-response relationships. Immediately following instillation, while still under anesthesia, the animals received a subcutaneous injection of dipyrone (500 mg/mL) diluted in 1 mL of saline to minimize post-procedural pain. From days 10 to 19, the ALA and BA groups received ALA, while the CO and BLM groups continued to receive saline solution administered subcutaneously (Figure 1). On day 20 all rats were euthanized for blood and tissue collection. Body Weight Measurement Body weight was measured (g) at standardized time points throughout the experiment: at the baseline (day 0), and subsequently on days 10 and 20. This systematic monitoring allowed for quantification of the animals' weight progression following the initiation of ALA treatment. Body weight measurements were used to calculate the percentage of weight gain, enabling normalization and precise comparative analysis between experimental groups. The animals were maintained on standard laboratory chow from the animal facility, with no additional nutritional supplements introduced during the experimental period. Respiratory mechanics On the 20 th day, the animals were re-anesthetized with ketamine and xylazine administered intraperitoneally, underwent tracheostomy for the insertion of a 2 mm rigid cannula, which was connected to a mechanical ventilator (FlexiVent, Scireq, Montréal, Canada) to measure respiratory mechanics (pulmonary and the thoracic chamber). Resistance (R), Elastance (E), and Compliance (C) of the lungs and thoracic cavity were evaluated through three sequential series to obtain average values. At the end, the rats were euthanized via an overdose of anesthesia. The organs were immediately harvested and stored at -80°C for further analysis. Ashcroft scale After euthanasia, the left lung was inflated with a 10% formalin-buffered solution and immersed in the same solution for 48 hours. For histological analysis, the lungs were dehydrated, cleared, and embedded in paraffin. Sections of 5 micrometers were prepared using a Histocore Biocut microtome (Leica Microsystems, Wetzlar, Germany) and placed on labeled slides, then stained with Hematoxylin and Eosin. Azan Trichrome staining (TA; isocarmine acid dye) and the Weigert-van Gieson technique were utilized to identify collagen and elastic fibers, respectively. For each lung, 20 fields were examined at 400x magnification. Pulmonary fibrosis was graded according to the Ashcroft Scale(Ashcroft et al., 1988). Histological fibrosis was quantified using the Ashcroft score by two trained investigators who were blinded to the experimental groups. Each slide was scored independently, and the final score for each animal was calculated as the mean of the two evaluations. In cases of discrepant scores (difference > 2 points), a third evaluation was performed by an experienced pulmonary pathologist (also blinded), and the median of the three scores was used. Representative images at low (20x) and high (40x) magnifications are shown in Figure 2 to illustrate morphological changes. TGF-β1 Quantification in Lung Tissue by ELISA Lung tissue samples were collected from all experimental groups and processed for TGF-β1 quantification. Tissues were carefully homogenized using a tissue homogenizer in cold phosphate-buffered saline (PBS) to preserve protein integrity. Homogenates were then centrifuged at 12,000 x g for 15 minutes at 4°C to remove cellular debris. Supernatants were aliquoted and immediately stored at −80°C to prevent protein degradation. Total protein concentration was quantified using bovine serum albumin as a standard. TGF-β1 levels were measured using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kit from Invitrogen Life Sciences (Catalog Number 88-50680; USA), following the manufacturer's recommended protocol. Briefly, samples were thawed on ice and prepared according to the kit instructions. The assay was performed using a microplate reader (SpectraMax M2, EzBiochrom, USA), which allowed for precise quantification of TGF-β1 concentrations. The absorbance was measured at 450 nm, with a reference wavelength of 570 nm for background correction. TGF-β1 levels were expressed as pg/mg of total protein. Determination of plasma concentrations of vasoactive substances by liquid chromatography-tandem mass spectrometry (LC-MS/MS) Samples containing 1 mL of carotid blood were collected in EDTA tubes with the P8340 protease inhibitor cocktail from (Sigma-Aldrich, Merck). Following centrifugation at 3,500 rpm for 10 minutes at refrigerated temperature, the plasma was aliquoted into sterile tubes for further analysis of renin-angiotensin system (RAS) peptides as follows: 950 μL of acetone was added to 50 μL of plasma, and the samples were vortexed for 60 seconds. Subsequently, the samples were centrifuged at 9,000 × g for 6 minutes, and the supernatant was collected, dried under nitrogen, and resuspended in 50 μL of acetonitrile immediately before analysis. The analytical system consisted of a Nexera-i LC-2040C coupled with an LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). Electrospray ionization parameters in positive mode were set as follows: capillary voltage at 4,500 V; desolvation line temperature at 250 °C; heating block temperature at 400 °C; drying gas flow at 10 L/min; and nebulizing gas flow at 3 L/min. Multiple reaction monitoring (MRM) was conducted using the following fragmentation patterns. Ang I : m/z 649.10 → 110.10; 649.10 → 269.10; 649.10 → 426.25; Ang II : m/z 523.80 → 263.20; 523.80 → 70.20; 523.80 → 110.15; Ang A : m/z 501.80 → 70.10; 501.80 → 263.10; 501.80 → 110.05; Ang-(1-7) : m/z 450.30 → 110.10; 450.30 → 70.10; 450.30 → 392.65; Alamandine : m/z 428.45 → 110.10; 428.45 → 211.15; 428.45 → 111.10. Chromatographic separation was performed using an Acquity UPLC® C18 column (2.1 x 50 mm, 1.7 μm particle size) (Waters Corporation, Ireland) in gradient elution mode at a flow rate of 0.3 mL/min. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B), both containing 0.1 % formic acid, programmed as follows: 0–0.5 min, 2% B; 0.5–3.0 min, 2–100% B; 3.0–3.5 min, 100% B; 3.5–3.8 min, 100–2% B; 3.8–8 min, 2% B. The column oven was maintained at 50 °C. Data were processed using LabSolutions software (Shimadzu, Kyoto, Japan). Statistical Analysis Statistical analyses were performed using SPSS version 27.0 (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk normality test was conducted to assess the normality of data distribution. The variables were found to be parametric and are reported as mean ± standard deviation. For the analysis of body weight gain, the Generalized Estimating Equations (GEE) model was used, followed by the Least Significant Difference (LSD) test, which facilitated simultaneous intra- and inter-group comparisons. For normally distributed data, a linear model was employed within the GEE framework, while for non-normally distributed data, the Tweedie model with a logarithmic link function was applied. Additionally, one-way ANOVA with Tukey's post hoc test was conducted for other comparisons, and statistical significance was set at p < 0.05 for all analyses. RESULTS Alamandine Attenuates Weight Loss in Pulmonary Fibrosis. At the beginning of the experiment (day 0), all groups exhibited comparable initial body weights (g): CO at 213 ± 41, ALA at 213 ± 59, BLM at 248 ± 41, and BA at 207 ± 49. During the first nine days, the BLM and BA groups demonstrated significantly (p < 0.001) reduced body weight gain (%) compared to the CO and ALA groups. Specifically, BLM showed a weight gain of 14 ± 8.9 and BA of 8.8 ± 13, in contrast to CO (26 ± 8.5) and ALA (24 ± 4.7). This significant difference suggests that bleomycin-induced fibrosis likely caused respiratory discomfort, which consequently suppressed appetite in the BLM and BA groups during the early stages of the experimental protocol (Fig. 2 , panel A). From day 10 to 19, ALA treatment effectively mitigated body weight loss (%) observed in the initial phase, with the ALA (26.6 ± 12.2) and BA (26.5 ± 9.7) groups achieving body weight gain comparable to the CO group (22.7 ± 8.2). Moreover, the BLM group (15.9 ± 4.7) demonstrated significantly (p < 0.01) reduced body weight gain compared to the ALA and BA groups. As weight gain is directly associated with improved health status(Silva, 2021 ), this outcome indicates that ALA not only halted the progression of established fibrosis but also improved the overall condition of these animals (Fig. 2 , panel B). Impact of Bleomycin on Pulmonary Mechanics: Resistance and Compliance Lung mechanics analysis revealed a significant increase in respiratory resistance (cmH₂O/mL) in the BLM group (0.159 ± 0.047) compared to the CO group (0.104 ± 0.026; p < 0.01). Pulmonary compliance showed a trend toward reduction in the BLM group (0.444 ± 0.146) compared to the CO (0.630 ± 0.057; p < 0.01) and ALA groups (0.102 ± 0.021), but the BA group showed no statistically significant differences compared to other groups, indicating a modest, non-significant improvement (Fig. 3 , panels C and D). Ashcroft score Pulmonary fibrosis was assessed using the Ashcroft scoring system, a standardized histopathological method for quantifying lung fibrosis severity. Our results demonstrated that bleomycin consistently induced significant pulmonary fibrosis. Notably, the BA group (treated with ALA) exhibited a statistically significant lower Ashcroft score (1.21 ± 0.40) compared to the bleomycin-induced group (2.23 ± 0.35; p < 0.0001). This substantial reduction strongly suggests that ALA treatment was highly effective in mitigating lung fibrosis progression and potentially reversing established fibrotic changes (Fig. 3 , panel A). Elevated TGF-β Levels in Bleomycin-Induced Pulmonary Fibrosis TGF-β levels were significantly elevated in the BLM group (3.70 ± 1.7 pg/mg of protein; n = 5) compared to the CO (1.19 ± 0.17; n = 4), ALA (1.24 ± 0.23; n = 5), and BA groups (1.10 ± 0.42; n = 5; p < 0.0015). This marked increase in TGF-β in the BLM group suggests enhanced fibrotic processes characteristic of bleomycin-induced lung injury (Fig. 3 , panel B). Plasma Concentrations of Renin-Angiotensin System Peptides There was a significant difference among treatments for Ang I (p = 0.003), Ang-(1–7) (p = 0.002), and ALA (p = 0.004), indicating an effect of ALA in the fibrosis model. Conversely, Ang II (p = 0.19) and Ang A (p = 0.193) did not show significant differences (Table). Ang I concentration was significantly reduced in the BLM group compared to the CO group, indicating altered peptide regulation. Ang II and Ang A concentrations did not differ significantly among the groups, suggesting stable levels despite the treatment and lung injury. Ang-(1–7) and ALA levels were significantly lower in the BLM group compared to the CO group, supporting the impact of the bleomycin model on peptide levels. However, the BA group showed recovery toward control levels, particularly for Ang-(1–7), indicating a potential protective effect of ALA on these peptides. Results of Tukey's multiple comparison test are presented in Table 1. DISCUSSION ALA treatment significantly reduces TGF-β pathway activation, a critical component in the development of fibrosis (Lafyatis, 2014 ; Roberts et al., 1986 ), and decreases thoracic chamber resistance, a consequence of fibrosis. These effects notably improved feeding behavior, a strong indicator of well-being, which contributed to restored weight gain in the group with established fibrosis that was treated with ALA. Consistent with well-established literature (Liu et al., 2017 ; Wynn, 2011 ), our bleomycin-induced fibrosis model showed significant increases in TGF-β levels, higher Ashcroft scores, and elevated respiratory resistance in the BLM group, confirming effective induction of pulmonary fibrosis. In contrast to prior reports that evaluated ALA exclusively in preventive settings, this study provides the first evidence of therapeutic benefit in a model of established pulmonary fibrosis. This breakthrough is particularly significant because individuals are often unaware of their condition until fibrosis is already present, thus highlighting the relevance and applicability of our findings. Since its discovery, TGF-β has been established as a key fibrotic agent (Roberts et al., 1985 , 1986 ). In the cardiovascular system, ALA has shown promising results in slowing fibrosis progression, evidenced by reductions in arterial pressure and key fibrotic markers (Zhao et al., 2022 ). Research indicates that ALA effectively prevents the increase in collagen I, TGF-β, and connective tissue growth factor in vascular smooth muscle cells under Ang II exposure (Yang et al., 2020 ). Moreover, a comprehensive review has highlighted the potent antifibrotic effects of inhibiting TGF-β signaling across various animal models and organs (Lafyatis, 2014 ), further underscoring its role in fibrosis. This may occur via MrgD receptor activation, which downregulates TGF-β signaling and reduces fibroblast activation (Yang et al., 2020 ). Our study supports these findings, showing that rats with established fibrosis treated with ALA exhibited significant reductions in fibrotic scores and TGF-β levels, demonstrating ALA's potential to suppress fibrotic pathways. By reducing thoracic chamber resistance and inhibiting the TGF-β pathway, ALA alleviated functional impairment in established pulmonary fibrosis. In contrast to previous studies that evaluated ALA exclusively in preventive protocols (Blanco et al., 2025 ; Fernandes et al., 2021 ), the present work demonstrates its therapeutic efficacy in an established disease model, with significant reductions in Ashcroft fibrosis score and improvement in physiological parameters such as body weight recovery and food intake. These findings support ALA as a candidate for repurposing in the treatment of pulmonary fibrosis. Of note, animals in the BA group exhibited markedly faster body weight recovery after day 10 post-bleomycin, reaching pre-induction values by day 20, whereas the untreated bleomycin group remained 12–18% below baseline. This accelerated weight gain paralleled the significant reduction in TGF-β levels and is consistent with the concept that body weight trajectory is a sensitive overall status (Silva, 2021 ). Such easily monitored parameters may therefore serve as valuable complementary endpoints in preclinical evaluation of anti-fibrotic drug candidates. In our study, animals treated with both BLM and ALA experienced significantly less respiratory distress compared to those treated with BLM alone. Notably, ALA treatment resulted in a remarkable 26% weight gain in the BA group between days 10 and 19, comparable to that of the ALA and CO groups. This increase represents a 10% greater weight gain than the BLM group, which showed only a 15.9% increase during the same period. Additionally, our findings demonstrated that ALA treatment not only reduced resistance within the pulmonary chamber in the BA group but also restored it to levels comparable to those of the CO and ALA groups. This is consistent with the literature, which reports that lung tissue resistance in individuals with pulmonary fibrosis is, on average, approximately four times greater than that in age-matched healthy individuals (Bachofen & Scherrer, 1967 ). Notably, while no significant differences in airway resistance were observed among the CO, ALA, and BA groups, ALA treatment showed a modest trend toward improvement in thoracic chamber compliance; this modest effect could be physiologically meaningful but requires confirmation in larger studies. Since the thoracic chamber, unlike the lung, is not directly affected by bleomycin, any observed improvements in the combined lung and thoracic system suggest an even greater impact on lung tissue alone. While our findings demonstrate the potential efficacy of ALA in models of established pulmonary fibrosis, certain limitations must be acknowledged. First, the treatment duration was relatively short, and the dose may not have been sufficient to observe the full effects of ALA therapy. Future studies should explore longer treatment regimens, multiple doses for dose-response curves, and inclusion of standard treatments like pirfenidone to compare efficacy. Although our animal model provides valuable insights, translating these results to humans requires caution. Clinical trials are necessary to confirm the safety and efficacy of ALA in patients with pulmonary fibrosis. Surprisingly, plasma ALA concentrations did not increase significantly in treated animals despite clear antifibrotic efficacy in lung tissue. This observation, also reported for other short-half-life therapeutic peptides, suggests rapid tissue uptake and/or local metabolism rather than systemic accumulation — a potentially advantageous pharmacokinetic profile that limits off-target effects while preserving activity at the site of injury. In addition, plasma ALA levels may have remained similar across groups because, according to pathophysiological demands, circulating ALA can be rapidly converted into the bioactive pentapeptide ALA-(1–5), potentially masking detectable increases in its precursor even after exogenous administration (Santos et al., 2026 ). Furthermore, bleomycin administration alone markedly reduced circulating levels of Ang I, Ang-(1–7), and ALA. Late ALA treatment prevented these reductions, resulting in plasma concentrations approximately twofold higher than in the untreated BLM group and not significantly different from healthy controls. Concomitantly, animals receiving ALA from day 10 exhibited accelerated body weight recovery, reaching pre-induction values by the end of the protocol. These parallel improvements in RAS peptide balance and clinical status further support the therapeutic potential of ALA in established pulmonary fibrosis. Our investigation was motivated by the established role of Ang II in promoting fibrotic effects. It was somewhat surprising that the plasma concentration of Ang II was not elevated in the BLM group, while the observed decrease in concentrations of Ang-(1–7) and ALA was anticipated. It is also important to note that tissue RAS and plasma RAS do not always exhibit the same trends in activity. ALA treatment helps restore balance to the peptides of the RAS, rather than reinstating the homeostatic balance of ALA alone, reinforcing the need for further investigation into the interplay between these peptides in the context of fibrotic conditions. These comprehensive findings not only challenge the current understanding of established fibrosis management but also provide new perspectives. Clinically, the ability of ALA to reduce TGF-β levels and improve pulmonary resistance positions it as a promising candidate for therapeutic interventions in advanced fibrosis. This approach could not only alleviate symptoms but also potentially slow disease progression. Individual variability in treatment response highlights the need for personalized strategies, possibly combining ALA with other antifibrotic therapies. Our research underscores the importance of exploring therapeutic approaches that effectively address fibrosis after its onset, offering a novel perspective in the field of pulmonary disease management. CONCLUSION This study pioneers the evaluation of ALA’s therapeutic potential in treating established pulmonary fibrosis. We identified a significant reduction in TGF-β levels in fibrotic animals treated with ALA, underscoring its capacity to modulate key fibrotic mechanisms. Although the structural impacts of pulmonary fibrosis on ventilatory mechanics were not fully reversed, ALA demonstrated a notable protective effect by reducing respiratory resistance, thereby improving animal well-being by alleviating respiratory discomfort. Our findings suggest that optimizing treatment parameters, such as dose adjustments or extended treatment durations, could further enhance thoracic cavity compliance and lung function. These insights provide a foundation for future research, paving the way for deeper exploration of ALA’s clinical applications. As preliminary results, they require validation in larger cohorts, longer-term models, and mechanistic studies (e.g., MrgD knockout). Ultimately, this could lead to innovative, tailored therapies for patients with advanced pulmonary fibrosis, offering hope for improved disease management. Declarations Funding: This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Author Contribution IAM, ATS and JF: Data curation, Formal analysis, Investigation, visualization and Writing – original draft.AB and GRG: Valiadation and Writing – original draft.AFKV: Resources and Writing – original draftKR: Funding acquisition, Validadtion, Project administration and Writing – review & editing Acknowledgement We extend our heartfelt thanks to Sarah Eller for her technical expertise in conducting the plasma peptide dosages, which were crucial for the accuracy of our findings. Data Availability The data that support the findings of this study are available within the article and its Supplementary Materials. Additional data, if applicable, are available from the corresponding author upon reasonable request. References Ashcroft, T., Simpson, J. M., & Timbrelli, V. (1988). Simple method of estimating severity of pulmonary fibrosis on a numerical scale. In J Clin Pathol (Vol. 41). Bachofen, H., & Scherrer, M. (1967). Lung Tissue Resistance in Diffuse Interstitial Pulmonary. In Journal oClinical Investigation (Vol. 46, Number 1). Blanco, A., Fernandes, R., Guimarães, G. R., & Rigatto, K. (2025). Alamandine reduces oxidative stress and preserves the epithelium in BLM-induced pulmonary fibrosis. European Journal of Pharmacology , 1004 , 177995. https://doi.org/10.1016/j.ejphar.2025.177995 Dowman, L., McDonald, C. F., Hill, C., Lee, A., Barker, K., Boote, C., Glaspole, I., Goh, N., Southcott, A., Burge, A., Ndongo, R., Martin, A., & Holland, A. E. (2013). The benefits of exercise training in interstitial lung disease: Protocol for a multicentre randomised controlled trial. BMC Pulmonary Medicine , 13 (1). https://doi.org/10.1186/1471-2466-13-8 Eaton, T., Young, P., Milne, D., & Wells, A. U. (2005). Six-minute walk, maximal exercise tests: Reproducibility in fibrotic interstitial pneumonia. American Journal of Respiratory and Critical Care Medicine , 171 (10), 1150–1157. https://doi.org/10.1164/rccm.200405-578OC Fernandes, R. S., Dias, H. B., de Souza Jaques, W. A., Becker, T., & Rigatto, K. (2021). Assessment of Alamandine in Pulmonary Fibrosis and Respiratory Mechanics in Rodents. JRAAS - Journal of the Renin-Angiotensin-Aldosterone System , 2021 . https://doi.org/10.1155/2021/9975315 Funke-Chambour, M., & Geiser, T. (2015). Idiopathic pulmonary fibrosis: The turning point is now! Swiss Medical Weekly , 145 . https://doi.org/10.4414/smw.2015.14139 Hübner, R. H., Gitter, W., El Mokhtari, N. E., Mathiak, M., Both, M., Bolte, H., Freitag-Wolf, S., & Bewig, B. (2008). Standardized quantification of pulmonary fibrosis in histological samples. BioTechniques , 44 (4), 507–517. https://doi.org/10.2144/000112729 Lafyatis, R. (2014). Transforming growth factor β—at the centre of systemic sclerosis. Nature Reviews Rheumatology , 10 (12), 706–719. https://doi.org/10.1038/nrrheum.2014.137 Liu, T., De Los Santos, F. G., & Phan, S. H. (2017). The Bleomycin Model of Pulmonary Fibrosis (pp. 27–42). https://doi.org/10.1007/978-1-4939-7113-8_2 Moeller, A., Ask, K., Warburton, D., Jack, G. #, & Kolb, M. (n.d.). The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Otoupalova, E., Smith, S., Cheng, G., & Thannickal, V. J. (2020). Oxidative Stress in Pulmonary Fibrosis. In Comprehensive Physiology (2nd ed., Vol. 10, pp. 509–547). Wiley. https://doi.org/10.1002/cphy.c190017 Raghu, G., Collard, H. R., Egan, J. J., Martinez, F. J., Behr, J., Brown, K. K., Colby, T. V., Cordier, J. F., Flaherty, K. R., Lasky, J. A., Lynch, D. A., Ryu, J. H., Swigris, J. J., Wells, A. U., Ancochea, J., Bouros, D., Carvalho, C., Costabel, U., Ebina, M., … Schünemann, H. J. (2011). An Official ATS/ERS/JRS/ALAT Statement: Idiopathic pulmonary fibrosis: Evidence-based guidelines for diagnosis and management. American Journal of Respiratory and Critical Care Medicine , 183 (6), 788–824. https://doi.org/10.1164/rccm.2009-040GL Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F., & Sporn, M. B. (1985). Type beta transforming growth factor: a bifunctional regulator of cellular growth. Proceedings of the National Academy of Sciences , 82 (1), 119–123. https://doi.org/10.1073/pnas.82.1.119 Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotra, L. A., Falangat, V., Kehrl, J. H., & Faucit, A. S. (1986). Transforming growth factor type ,8: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. In Proc. Nati. Acad. Sci. USA (Vol. 83). Santos, R. A. S., Dias, M. T. S., Bessa, A. de S. M., Barros, C. F., Itaborahy, M. F., da Silva, F. A., Gonçalves, S. C. de A., Rodrigues-Ribeiro, L., Ferraz, K. S., Davel, A. P., Nóbrega, N., Silva, B. D. da, Scalzo, S., Soares, P. A., Dutra, J. B. R., Lula, I., Feng, I. Z. L. F., Vieira-Machado, U. F., de Godoy, A. C. V., … Campagnole-Santos, M. J. (2026). Identification and Characterization of Alamandine-(1-5), a New Component of the Renin-Angiotensin System. Circulation Research , 138 (1). https://doi.org/10.1161/CIRCRESAHA.125.326174 Silva, A. V. (2021). Associations between clinical signs and pathological findings in toxicity testing. ALTEX , 38 (2), 198–214. https://doi.org/10.14573/altex.2003311 Sipriani, T. S., Dos Santos, R. A. S., & Rigatto, K. (2019). The Renin-Angiotensin System: Alamandine is reduced in patients with Idiopathic Pulmonary Fibrosis. Journal of Cardiology and Cardiovascular Medicine , 4 (3), 210–215. https://doi.org/10.29328/journal.jccm.1001070 Uhal, B. D., Li, X., Piasecki, C. C., & Molina-Molina, M. (2012). Angiotensin signalling in pulmonary fibrosis. In International Journal of Biochemistry and Cell Biology (Vol. 44, Number 3, pp. 465–468). Elsevier Ltd. https://doi.org/10.1016/j.biocel.2011.11.019 Wynn, T. A. (2011). Integrating mechanisms of pulmonary fibrosis. Journal of Experimental Medicine , 208 (7), 1339–1350. https://doi.org/10.1084/jem.20110551 Yang, C., Wu, X., Shen, Y., Liu, C., Kong, X., & Li, P. (2020). Alamandine attenuates angiotensin II-induced vascular fibrosis via inhibiting p38 MAPK pathway. European Journal of Pharmacology , 883 , 173384. https://doi.org/10.1016/j.ejphar.2020.173384 Zhao, K., Xu, T., Mao, Y., Wu, X., Hua, D., Sheng, Y., & Li, P. (2022). Alamandine alleviated heart failure and fibrosis in myocardial infarction mice. Biology Direct , 17 (1). https://doi.org/10.1186/s13062-022-00338-6 Table Table – Oxidative Stress Levels in Lung Tissue and Plasma Concentrations of Renin-Angiotensin System Peptides Oxidative Stress Levels in Lung Tissue CO (N = 5) ALA (N = 5) BLM (N = 5) BA (N = 5) GSH (nmol/mg protein) 10 ± 1.9 8.73 ± 0.71 6.81 ± 1.8 c 9.95 ± 2.5 Carbonil (nmol of DNPH/mg protein) 21 ± 18 9.3 ± 7.9 25 ± 23 7.95 ± 3.7 SH (ug/mg protein) 248 ± 49 71 ± 22 a,b 106 ± 56 a,b 245 ± 76 DCF (A.U./mg protein) 25 ± 20 63 ± 36 41 ± 7.3 24 ± 14 Plasma Concentrations of Renin-Angiotensin System Peptides (N=7; ng/mL) Angiotensin I 0,089 ± 0,020 0,068 ± 0,029 0,035 ± 0,022 ** 0,065 ± 0,024 Angiotensin II 0,046 ± 0,018 0,039 ± 0,011 0,029 ± 0,015 0,037 ± 0,009 Angiotensin A 0,051 ± 0,009 0,052 ± 0,017 0,034 ± 0,020 0,050 ± 0,021 Angiotensin 1-7 0,039 ± 0,012 0,031 ± 0,009 0,013 ± 0,009 #** 0,029 ± 0,012 Alamandine 0,100 ± 0,027 0,087 ± 0,033 0,038 ± 0,026 **@ 0,083 ± 0,034 CO: control group; ALA: alamandine-treated group; BLM: bleomycin-only group; BA: bleomycin plus ALA treatment group. GSH: Reduced Glutathione; Carbonyl: Protein Carbonyl Content; DNPH: 2,4-Dinitrophenylhydrazine; SH: Thiol Group and DCF: Dichlorofluorescein. Data are presented as means ± standard deviations. a p < 0.007 vs CO; b p < 0.005 vs BA; c p < 0.03 vs CO; ** p < 0.004 vs CO; # p < 0.028 vs ALA; @ p < 0.046 vs ALA and BS grup. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9066088","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607231808,"identity":"7d68a020-634c-46dc-986f-e075ff786097","order_by":0,"name":"Isabel Amaral Martins","email":"","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"Amaral","lastName":"Martins","suffix":""},{"id":607231811,"identity":"91245999-6f12-476c-9751-411f6d4f1aeb","order_by":1,"name":"Andresa Thomé Silveira","email":"","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":false,"prefix":"","firstName":"Andresa","middleName":"Thomé","lastName":"Silveira","suffix":""},{"id":607231812,"identity":"add89e7f-5125-47bf-87e1-45792f372faf","order_by":2,"name":"Juliane Flor","email":"","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":false,"prefix":"","firstName":"Juliane","middleName":"","lastName":"Flor","suffix":""},{"id":607231815,"identity":"ddf645ea-3957-44d5-b206-7ca1639f9842","order_by":3,"name":"Aline Blanco","email":"","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":false,"prefix":"","firstName":"Aline","middleName":"","lastName":"Blanco","suffix":""},{"id":607231817,"identity":"58cf36a4-b411-48b9-8983-4dc90e3f8750","order_by":4,"name":"Giuliano Rizzotto Guimarães","email":"","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":false,"prefix":"","firstName":"Giuliano","middleName":"Rizzotto","lastName":"Guimarães","suffix":""},{"id":607231820,"identity":"43e4c0b7-6209-4b0e-8b37-6d1801331e8e","order_by":5,"name":"Adriana Fernanda K. Vizuete","email":"","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"Fernanda K.","lastName":"Vizuete","suffix":""},{"id":607231828,"identity":"3270508e-726a-49dc-a9d7-bcc77e77c551","order_by":6,"name":"Katya Rigatto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACCQYGxgcMNlAeDwNDAjFamA0Y0kjUwiZBmhbJGbnPqnkS7OzN2Q8wPnjbxpBn3kBAi7REutltnoTkxJ09CcyGc9sYimUOENAix3OM7TbvjwMJBgcS2KR52xgSZxByGEhLMU/CAXuD8w/YfxOlRZq9jY0ZqIVxw40ENmaitEi2tzFLzgH5ZcbDZsk55ySKJQhpkTjMxvjhDSjE+JMPfnhTZpNHUAscGDAwNjCA44kELaNgFIyCUTAKcAAABh03Sq2tNa4AAAAASUVORK5CYII=","orcid":"","institution":"Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)","correspondingAuthor":true,"prefix":"","firstName":"Katya","middleName":"","lastName":"Rigatto","suffix":""}],"badges":[],"createdAt":"2026-03-08 18:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9066088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9066088/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104809775,"identity":"c009e75b-708c-4cf5-ab06-2e2db4c600a5","added_by":"auto","created_at":"2026-03-17 12:52:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54124,"visible":true,"origin":"","legend":"\u003cp\u003eTimeline of Treatment Interventions from Day 0 to Day 19. CO= control group; ALA= alamandine treatment; BLM= bleomycin treatment; and BA= bleomycin treatment on Day 0 followed by ALA from Days 10 to 19.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9066088/v1/1c6d700baca5704fc030a803.png"},{"id":104809238,"identity":"1ebbb0bd-e79b-4289-8cde-bc657f06eb2f","added_by":"auto","created_at":"2026-03-17 12:48:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61044,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of body weight gain. Panel A: Treatment Interventions from Day 0 to Day 9, p\u0026lt;0.01 versus BLM and BA. Panel B: Treatment Interventions from Day 10 to Day 19, p\u0026lt;0.001. CO= control group; ALA= alamandine treatment; BLM= bleomycin treatment; and BA= bleomycin treatment on Day 0 followed by ALA from Days 10 to 19.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9066088/v1/73d37fecb186d1635440ae75.png"},{"id":104810644,"identity":"caf76a8a-80b2-479c-9686-a1485ca1df91","added_by":"auto","created_at":"2026-03-17 12:55:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101926,"visible":true,"origin":"","legend":"\u003cp\u003ePanel A: Ashcroft score (p \u0026lt; 0.0001); Panel B: TGF beta (pg/mg; p \u0026lt; 0.005); Panel C: Resistance (cm H₂O/mL; p \u0026lt; 0.02); Panel D: Compliance (cm H₂O/mL; p \u0026lt; 0.03). The groups are defined as CO = control group; ALA = alamandine treatment; BLM = bleomycin treatment; BA = bleomycin treatment on Day 0 followed by ALA from Days 10 to 19.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9066088/v1/e8692f097b3b8df9ba04df89.png"},{"id":105032978,"identity":"296bee6c-d757-46a4-8aea-a4e564ca1887","added_by":"auto","created_at":"2026-03-20 07:08:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1036329,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9066088/v1/e76ad602-33aa-4831-b91f-d04ae12e38f6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAlamandine Attenuates Signs and Reduces Fibrotic Markers in a Rat Model of Established Pulmonary Fibrosis\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e● Novel therapeutic approach targets established pulmonary fibrosis.\u003c/p\u003e\u003cp\u003e● Significant improvement in respiratory mechanics with ALA treatment.\u003c/p\u003e\u003cp\u003e● ALA reduces TGF-β levels, underscoring its antifibrotic potential.\u003c/p\u003e\u003cp\u003e● Enhanced body weight gain indicates improved health status with ALA.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003ePulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by relentless progression, with a median survival of approximately 3 to 5 years (Funke-Chambour \u0026amp; Geiser, 2015; Raghu et al., 2011). Despite extensive research efforts, the etiology and pathophysiological mechanisms of PF remain incompletely understood (Raghu et al., 2011). \u0026nbsp;This lack of clarity contributes to the disease's high morbidity and mortality rates, compounded by limited therapeutic options(Dowman et al., 2013). Symptoms such as dyspnea, fatigue, and muscle weakness (Eaton et al., 2005) severely reduce functional capacity and significantly impact quality of life.\u003c/p\u003e\n\u003cp\u003eWhile traditional therapeutic approaches have primarily focused on prevention, addressing advanced stages of fibrosis is crucial. This gap in research emphasizes the urgent need for innovative therapeutic strategies. Effective therapies that intervene after fibrosis progression are essential for improving patient prognosis. \u0026nbsp;In the bleomycin model, fibrosis is typically established by days 7-14, with peak inflammation resolving and fibrotic remodeling beginning (Hübner et al., 2008; Moeller et al., n.d.). Our chosen therapeutic window (days 10-19) targets this established phase to evaluate reversal potential, though longer models may be needed for chronic effects.\u003c/p\u003e\n\u003cp\u003eFibrosis is characterized by fibroblast proliferation, extracellular matrix accumulation (Uhal et al., 2012), and oxidative stress (Otoupalova et al., 2020). Understanding these mechanisms and identifying potential therapeutic targets is vital for the development of effective treatments.\u003c/p\u003e\n\u003cp\u003eAlamandine (ALA), a peptide within the renin-angiotensin system, has demonstrated various beneficial effects, particularly in the cardiovascular realm. Its mechanism of action is believed to involve binding to MrgD receptors, which modulate inflammatory responses and oxidative stress. By potentially activating these receptors, ALA may inhibit the production of pro-fibrotic cytokines such as transforming growth factor-beta (TGF-β) in vascular models (Yang et al., 2020), thereby contributing to fibrosis reduction. This dual action of ALA could modulate both inflammation and oxidative stress, presenting a unique therapeutic potential, especially in established fibrosis conditions where tissue remodeling has already compromised lung function.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIndeed, our studies have shown that preventive ALA administration significantly attenuates fibrosis (Fernandes et al., 2021) and oxidative stress (Blanco et al., 2025)\u0026nbsp;in a bleomycin-induced model. Notably, plasma ALA concentrations in fibrotic patients are four times lower than in healthy controls, while levels of angiotensin II (Ang II), Ang I, and Ang-(1-7) do not differ significantly (Sipriani et al., 2019).\u003c/p\u003e\n\u003cp\u003eTransforming growth factor-beta plays a pivotal role in disease progression by inducing fibroblast transformation into myofibroblasts, contributing to excessive extracellular matrix deposition (Roberts et al., 1985, 1986). Modulating TGF-β levels is crucial for antifibrotic therapy, as improper regulation can exacerbate tissue injury (Yang et al., 2020). Targeting TGF-β could potentially attenuate fibrosis progression (Lafyatis, 2014). Our previous research indicates that preventive treatment with ALA reduces mesenchymal cells and myofibroblasts, restores total glutathione levels, and enhances antioxidant capacity, thereby supporting antifibrotic therapy through the control of oxidative stress (Blanco et al., 2025).\u003c/p\u003e\n\u003cp\u003eFurthermore, increased airway resistance compromises lung function in fibrotic patients, directly affecting their quality of life (Bachofen \u0026amp; Scherrer, 1967). Improving airway resistance is a key therapeutic goal that may alleviate symptoms and enhance exercise tolerance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBuilding on our findings from the preventive ALA treatment (Blanco et al., 2025; Fernandes et al., 2021) this study aims to explore the reversal effects of ALA in an experimental pulmonary fibrosis model. Specifically, we will evaluate whether ALA can: (1) modulate TGF-β signaling, (2) reduce airway resistance, (3) decrease oxidative stress, and (4) improve well-being in fibrotic animals.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eThis study utilized five-week-old male Wistar rats from the Animal Facility at the Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). The health of the rats was monitored throughout the experiment by assessing their general behavior and body weight. All procedures adhered to the Guide for the Care and Use of Laboratory Animals (USA -16) and received approval (number 749/21) from the Ethics Committee on Research at UFCSPA.\u003c/p\u003e\n\u003cp\u003eSample size (n = 5–6 per group) was empirically determined based on previous studies using the same bleomycin-induced pulmonary fibrosis model in our laboratory. Because the primary endpoints (Ashcroft score, thoracic chamber resistance and TGF-β levels in the present study) already showed clear statistical significance (p \u0026lt; 0.01–0.001), the group size provided sufficient power to detect differences while minimizing animal use in accordance with the 3Rs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBleomycin and ALA Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a 7-day acclimatization period, animals were ranked by body weight and distributed into four groups in a balanced manner (block distribution) to ensure each group had similar mean initial body weight (n=6 each): control group (CO), ALA-treated group (ALA), bleomycin-only group (BLM), and bleomycin plus ALA treatment group (BA). On day zero, the rats were anesthetized intraperitoneally with ketamine (80 mg/kg) and xylazine (10 mg/kg) and underwent fibrosis induction via intratracheal intubation using a 20G catheter. Through this catheter, a bleomycin solution (2.5 mg/kg, Bonar, Ache) was administered to the BLM and BA groups, while the CO and ALA groups received only a saline solution (0.9% NaCl).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe total volume of instillation was 0.2 mL per 200 grams of body weight. In the BA group, animals received bleomycin instillation on day zero and remained without further intervention until ALA treatment (50 mg/kg/day for both ALA and BA groups) starting on day 10, continuing daily until day 19 (Figure 1). This single dose was chosen based on previous preventive studies(Fernandes et al., 2021), but future work should explore dose-response relationships.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmediately following instillation, while still under anesthesia, the animals received a subcutaneous injection of dipyrone (500 mg/mL) diluted in 1 mL of saline to minimize post-procedural pain. From days 10 to 19, the ALA and BA groups received ALA, while the CO and BLM groups continued to receive saline solution administered subcutaneously (Figure 1). On day 20 all rats were euthanized for blood and tissue collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBody Weight Measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Body weight was measured (g) at standardized time points throughout the experiment: at the baseline (day 0), and subsequently on days 10 and 20. This systematic monitoring allowed for quantification of the animals' weight progression following the initiation of ALA treatment. Body weight measurements were used to calculate the percentage of weight gain, enabling normalization and precise comparative analysis between experimental groups. The animals were maintained on standard laboratory chow from the animal facility, with no additional nutritional supplements introduced during the experimental period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRespiratory mechanics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn the 20\u003csup\u003eth\u003c/sup\u003e day, the animals were re-anesthetized with ketamine and xylazine administered intraperitoneally, underwent tracheostomy for the insertion of a 2 mm rigid cannula, which was connected to a mechanical ventilator (FlexiVent, Scireq, Montréal, Canada) to measure respiratory mechanics (pulmonary and the thoracic chamber). Resistance (R), Elastance (E), and Compliance (C) of the lungs and thoracic cavity were evaluated through three sequential series to obtain average values. At the end, the rats were euthanized via an overdose of anesthesia. The organs were immediately harvested and stored at -80°C for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAshcroft scale\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter euthanasia, the left lung was inflated with a 10% formalin-buffered solution and immersed in the same solution for 48 hours. For histological analysis, the lungs were dehydrated, cleared, and embedded in paraffin. Sections of 5 micrometers were prepared using a Histocore Biocut microtome (Leica Microsystems, Wetzlar, Germany) and placed on labeled slides, then stained with Hematoxylin and Eosin. Azan Trichrome staining (TA; isocarmine acid dye) and the Weigert-van Gieson technique were utilized to identify collagen and elastic fibers, respectively. For each lung, 20 fields were examined at 400x magnification. Pulmonary fibrosis was graded according to the Ashcroft Scale(Ashcroft et al., 1988).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistological fibrosis was quantified using the Ashcroft score by two trained investigators who were blinded to the experimental groups. Each slide was scored independently, and the final score for each animal was calculated as the mean of the two evaluations. In cases of discrepant scores (difference \u0026gt; 2 points), a third evaluation was performed by an experienced pulmonary pathologist (also blinded), and the median of the three scores was used. Representative images at low (20x) and high (40x) magnifications are shown in Figure 2 to illustrate morphological changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGF-β1 Quantification in Lung Tissue by ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLung tissue samples were collected from all experimental groups and processed for TGF-β1 quantification. Tissues were carefully homogenized using a tissue homogenizer in cold phosphate-buffered saline (PBS) to preserve protein integrity. Homogenates were then centrifuged at 12,000 x g for 15 minutes at 4°C to remove cellular debris. Supernatants were aliquoted and immediately stored at −80°C to prevent protein degradation.\u003c/p\u003e\n\u003cp\u003eTotal protein concentration was quantified using bovine serum albumin as a standard. TGF-β1 levels were measured using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kit from Invitrogen Life Sciences (Catalog Number 88-50680; USA), following the manufacturer's recommended protocol. Briefly, samples were thawed on ice and prepared according to the kit instructions. The assay was performed using a microplate reader (SpectraMax M2, EzBiochrom, USA), which allowed for precise quantification of TGF-β1 concentrations. The absorbance was measured at 450 nm, with a reference wavelength of 570 nm for background correction. TGF-β1 levels were expressed as pg/mg of total protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of plasma concentrations of vasoactive substances by liquid chromatography-tandem mass spectrometry (LC-MS/MS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples containing 1 mL of carotid blood were collected in EDTA tubes with the P8340 protease inhibitor cocktail from (Sigma-Aldrich, Merck). Following centrifugation at 3,500 rpm for 10 minutes at refrigerated temperature, the plasma was aliquoted into sterile tubes for further analysis of renin-angiotensin system (RAS) peptides as follows: 950 μL of acetone was added to 50 μL of plasma, and the samples were vortexed for 60 seconds. Subsequently, the samples were centrifuged at 9,000 × g for 6 minutes, and the supernatant was collected, dried under nitrogen, and resuspended in 50 μL of acetonitrile immediately before analysis.\u003c/p\u003e\n\u003cp\u003eThe analytical system consisted of a Nexera-i LC-2040C coupled with an LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). Electrospray ionization parameters in positive mode were set as follows: capillary voltage at 4,500 V; desolvation line temperature at 250 °C; heating block temperature at 400 °C; drying gas flow at 10 L/min; and nebulizing gas flow at 3 L/min. Multiple reaction monitoring (MRM) was conducted using the following fragmentation patterns. \u003cstrong\u003eAng I\u003c/strong\u003e: m/z 649.10 → 110.10; 649.10 → 269.10; 649.10 → 426.25;\u0026nbsp;\u003cstrong\u003eAng II\u003c/strong\u003e: m/z 523.80 → 263.20; 523.80 → 70.20; 523.80 → 110.15;\u0026nbsp;\u003cstrong\u003eAng A\u003c/strong\u003e: m/z 501.80 → 70.10; 501.80 → 263.10; 501.80 → 110.05;\u0026nbsp;\u003cstrong\u003eAng-(1-7)\u003c/strong\u003e: m/z 450.30 → 110.10; 450.30 → 70.10; 450.30 → 392.65;\u0026nbsp;\u003cstrong\u003eAlamandine\u003c/strong\u003e: m/z 428.45 → 110.10; 428.45 → 211.15; 428.45 → 111.10.\u003c/p\u003e\n\u003cp\u003eChromatographic separation was performed using an Acquity UPLC® C18 column (2.1 x 50 mm, 1.7 μm particle size) (Waters Corporation, Ireland) in gradient elution mode at a flow rate of 0.3 mL/min. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B), both containing 0.1 % formic acid, programmed as follows: 0–0.5 min, 2% B; 0.5–3.0 min, 2–100% B; 3.0–3.5 min, 100% B; 3.5–3.8 min, 100–2% B; 3.8–8 min, 2% B. The column oven was maintained at 50 °C. Data were processed using LabSolutions software (Shimadzu, Kyoto, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using SPSS version 27.0 (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk normality test was conducted to assess the normality of data distribution. The variables were found to be parametric and are reported as mean ± standard deviation. For the analysis of body weight gain, the Generalized Estimating Equations (GEE) model was used, followed by the Least Significant Difference (LSD) test, which facilitated simultaneous intra- and inter-group comparisons. For normally distributed data, a linear model was employed within the GEE framework, while for non-normally distributed data, the Tweedie model with a logarithmic link function was applied. Additionally, one-way ANOVA with Tukey's post hoc test was conducted for other comparisons, and statistical significance was set at p \u0026lt; 0.05 for all analyses.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eAlamandine Attenuates Weight Loss in Pulmonary Fibrosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAt the beginning of the experiment (day 0), all groups exhibited comparable initial body weights (g): CO at 213\u0026thinsp;\u0026plusmn;\u0026thinsp;41, ALA at 213\u0026thinsp;\u0026plusmn;\u0026thinsp;59, BLM at 248\u0026thinsp;\u0026plusmn;\u0026thinsp;41, and BA at 207\u0026thinsp;\u0026plusmn;\u0026thinsp;49. During the first nine days, the BLM and BA groups demonstrated significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) reduced body weight gain (%) compared to the CO and ALA groups. Specifically, BLM showed a weight gain of 14\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9 and BA of 8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;13, in contrast to CO (26\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5) and ALA (24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7). This significant difference suggests that bleomycin-induced fibrosis likely caused respiratory discomfort, which consequently suppressed appetite in the BLM and BA groups during the early stages of the experimental protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel A).\u003c/p\u003e \u003cp\u003eFrom day 10 to 19, ALA treatment effectively mitigated body weight loss (%) observed in the initial phase, with the ALA (26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2) and BA (26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7) groups achieving body weight gain comparable to the CO group (22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2). Moreover, the BLM group (15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7) demonstrated significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) reduced body weight gain compared to the ALA and BA groups. As weight gain is directly associated with improved health status(Silva, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), this outcome indicates that ALA not only halted the progression of established fibrosis but also improved the overall condition of these animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel B).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImpact of Bleomycin on Pulmonary Mechanics: Resistance and Compliance\u003c/h2\u003e \u003cp\u003eLung mechanics analysis revealed a significant increase in respiratory resistance (cmH₂O/mL) in the BLM group (0.159\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047) compared to the CO group (0.104\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Pulmonary compliance showed a trend toward reduction in the BLM group (0.444\u0026thinsp;\u0026plusmn;\u0026thinsp;0.146) compared to the CO (0.630\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and ALA groups (0.102\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021), but the BA group showed no statistically significant differences compared to other groups, indicating a modest, non-significant improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panels C and D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAshcroft score\u003c/h2\u003e \u003cp\u003ePulmonary fibrosis was assessed using the Ashcroft scoring system, a standardized histopathological method for quantifying lung fibrosis severity. Our results demonstrated that bleomycin consistently induced significant pulmonary fibrosis. Notably, the BA group (treated with ALA) exhibited a statistically significant lower Ashcroft score (1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40) compared to the bleomycin-induced group (2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This substantial reduction strongly suggests that ALA treatment was highly effective in mitigating lung fibrosis progression and potentially reversing established fibrotic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eElevated TGF-β Levels in Bleomycin-Induced Pulmonary Fibrosis\u003c/h2\u003e \u003cp\u003eTGF-β levels were significantly elevated in the BLM group (3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 pg/mg of protein; n\u0026thinsp;=\u0026thinsp;5) compared to the CO (1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17; n\u0026thinsp;=\u0026thinsp;4), ALA (1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23; n\u0026thinsp;=\u0026thinsp;5), and BA groups (1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42; n\u0026thinsp;=\u0026thinsp;5; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0015). This marked increase in TGF-β in the BLM group suggests enhanced fibrotic processes characteristic of bleomycin-induced lung injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlasma Concentrations of Renin-Angiotensin System Peptides\u003c/h2\u003e \u003cp\u003eThere was a significant difference among treatments for Ang I (p\u0026thinsp;=\u0026thinsp;0.003), Ang-(1\u0026ndash;7) (p\u0026thinsp;=\u0026thinsp;0.002), and ALA (p\u0026thinsp;=\u0026thinsp;0.004), indicating an effect of ALA in the fibrosis model. Conversely, Ang II (p\u0026thinsp;=\u0026thinsp;0.19) and Ang A (p\u0026thinsp;=\u0026thinsp;0.193) did not show significant differences (Table).\u003c/p\u003e \u003cp\u003eAng I concentration was significantly reduced in the BLM group compared to the CO group, indicating altered peptide regulation. Ang II and Ang A concentrations did not differ significantly among the groups, suggesting stable levels despite the treatment and lung injury. Ang-(1\u0026ndash;7) and ALA levels were significantly lower in the BLM group compared to the CO group, supporting the impact of the bleomycin model on peptide levels. However, the BA group showed recovery toward control levels, particularly for Ang-(1\u0026ndash;7), indicating a potential protective effect of ALA on these peptides. Results of Tukey's multiple comparison test are presented in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eALA treatment significantly reduces TGF-β pathway activation, a critical component in the development of fibrosis (Lafyatis, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Roberts et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), and decreases thoracic chamber resistance, a consequence of fibrosis. These effects notably improved feeding behavior, a strong indicator of well-being, which contributed to restored weight gain in the group with established fibrosis that was treated with ALA.\u003c/p\u003e \u003cp\u003eConsistent with well-established literature (Liu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wynn, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), our bleomycin-induced fibrosis model showed significant increases in TGF-β levels, higher Ashcroft scores, and elevated respiratory resistance in the BLM group, confirming effective induction of pulmonary fibrosis. In contrast to prior reports that evaluated ALA exclusively in preventive settings, this study provides the first evidence of therapeutic benefit in a model of established pulmonary fibrosis. This breakthrough is particularly significant because individuals are often unaware of their condition until fibrosis is already present, thus highlighting the relevance and applicability of our findings.\u003c/p\u003e \u003cp\u003eSince its discovery, TGF-β has been established as a key fibrotic agent (Roberts et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). In the cardiovascular system, ALA has shown promising results in slowing fibrosis progression, evidenced by reductions in arterial pressure and key fibrotic markers (Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Research indicates that ALA effectively prevents the increase in collagen I, TGF-β, and connective tissue growth factor in vascular smooth muscle cells under Ang II exposure (Yang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, a comprehensive review has highlighted the potent antifibrotic effects of inhibiting TGF-β signaling across various animal models and organs (Lafyatis, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), further underscoring its role in fibrosis. This may occur via MrgD receptor activation, which downregulates TGF-β signaling and reduces fibroblast activation (Yang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our study supports these findings, showing that rats with established fibrosis treated with ALA exhibited significant reductions in fibrotic scores and TGF-β levels, demonstrating ALA's potential to suppress fibrotic pathways.\u003c/p\u003e \u003cp\u003eBy reducing thoracic chamber resistance and inhibiting the TGF-β pathway, ALA alleviated functional impairment in established pulmonary fibrosis. In contrast to previous studies that evaluated ALA exclusively in preventive protocols (Blanco et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Fernandes et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the present work demonstrates its therapeutic efficacy in an established disease model, with significant reductions in Ashcroft fibrosis score and improvement in physiological parameters such as body weight recovery and food intake. These findings support ALA as a candidate for repurposing in the treatment of pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eOf note, animals in the BA group exhibited markedly faster body weight recovery after day 10 post-bleomycin, reaching pre-induction values by day 20, whereas the untreated bleomycin group remained 12\u0026ndash;18% below baseline. This accelerated weight gain paralleled the significant reduction in TGF-β levels and is consistent with the concept that body weight trajectory is a sensitive overall status (Silva, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Such easily monitored parameters may therefore serve as valuable complementary endpoints in preclinical evaluation of anti-fibrotic drug candidates.\u003c/p\u003e \u003cp\u003eIn our study, animals treated with both BLM and ALA experienced significantly less respiratory distress compared to those treated with BLM alone. Notably, ALA treatment resulted in a remarkable 26% weight gain in the BA group between days 10 and 19, comparable to that of the ALA and CO groups. This increase represents a 10% greater weight gain than the BLM group, which showed only a 15.9% increase during the same period.\u003c/p\u003e \u003cp\u003eAdditionally, our findings demonstrated that ALA treatment not only reduced resistance within the pulmonary chamber in the BA group but also restored it to levels comparable to those of the CO and ALA groups. This is consistent with the literature, which reports that lung tissue resistance in individuals with pulmonary fibrosis is, on average, approximately four times greater than that in age-matched healthy individuals (Bachofen \u0026amp; Scherrer, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1967\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, while no significant differences in airway resistance were observed among the CO, ALA, and BA groups, ALA treatment showed a modest trend toward improvement in thoracic chamber compliance; this modest effect could be physiologically meaningful but requires confirmation in larger studies. Since the thoracic chamber, unlike the lung, is not directly affected by bleomycin, any observed improvements in the combined lung and thoracic system suggest an even greater impact on lung tissue alone.\u003c/p\u003e \u003cp\u003eWhile our findings demonstrate the potential efficacy of ALA in models of established pulmonary fibrosis, certain limitations must be acknowledged. First, the treatment duration was relatively short, and the dose may not have been sufficient to observe the full effects of ALA therapy. Future studies should explore longer treatment regimens, multiple doses for dose-response curves, and inclusion of standard treatments like pirfenidone to compare efficacy. Although our animal model provides valuable insights, translating these results to humans requires caution. Clinical trials are necessary to confirm the safety and efficacy of ALA in patients with pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eSurprisingly, plasma ALA concentrations did not increase significantly in treated animals despite clear antifibrotic efficacy in lung tissue. This observation, also reported for other short-half-life therapeutic peptides, suggests rapid tissue uptake and/or local metabolism rather than systemic accumulation \u0026mdash; a potentially advantageous pharmacokinetic profile that limits off-target effects while preserving activity at the site of injury. In addition, plasma ALA levels may have remained similar across groups because, according to pathophysiological demands, circulating ALA can be rapidly converted into the bioactive pentapeptide ALA-(1\u0026ndash;5), potentially masking detectable increases in its precursor even after exogenous administration (Santos et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, bleomycin administration alone markedly reduced circulating levels of Ang I, Ang-(1\u0026ndash;7), and ALA. Late ALA treatment prevented these reductions, resulting in plasma concentrations approximately twofold higher than in the untreated BLM group and not significantly different from healthy controls. Concomitantly, animals receiving ALA from day 10 exhibited accelerated body weight recovery, reaching pre-induction values by the end of the protocol. These parallel improvements in RAS peptide balance and clinical status further support the therapeutic potential of ALA in established pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eOur investigation was motivated by the established role of Ang II in promoting fibrotic effects. It was somewhat surprising that the plasma concentration of Ang II was not elevated in the BLM group, while the observed decrease in concentrations of Ang-(1\u0026ndash;7) and ALA was anticipated. It is also important to note that tissue RAS and plasma RAS do not always exhibit the same trends in activity. ALA treatment helps restore balance to the peptides of the RAS, rather than reinstating the homeostatic balance of ALA alone, reinforcing the need for further investigation into the interplay between these peptides in the context of fibrotic conditions.\u003c/p\u003e \u003cp\u003eThese comprehensive findings not only challenge the current understanding of established fibrosis management but also provide new perspectives. Clinically, the ability of ALA to reduce TGF-β levels and improve pulmonary resistance positions it as a promising candidate for therapeutic interventions in advanced fibrosis. This approach could not only alleviate symptoms but also potentially slow disease progression. Individual variability in treatment response highlights the need for personalized strategies, possibly combining ALA with other antifibrotic therapies. Our research underscores the importance of exploring therapeutic approaches that effectively address fibrosis after its onset, offering a novel perspective in the field of pulmonary disease management.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study pioneers the evaluation of ALA\u0026rsquo;s therapeutic potential in treating established pulmonary fibrosis. We identified a significant reduction in TGF-β levels in fibrotic animals treated with ALA, underscoring its capacity to modulate key fibrotic mechanisms. Although the structural impacts of pulmonary fibrosis on ventilatory mechanics were not fully reversed, ALA demonstrated a notable protective effect by reducing respiratory resistance, thereby improving animal well-being by alleviating respiratory discomfort.\u003c/p\u003e \u003cp\u003eOur findings suggest that optimizing treatment parameters, such as dose adjustments or extended treatment durations, could further enhance thoracic cavity compliance and lung function. These insights provide a foundation for future research, paving the way for deeper exploration of ALA\u0026rsquo;s clinical applications. As preliminary results, they require validation in larger cohorts, longer-term models, and mechanistic studies (e.g., MrgD knockout). Ultimately, this could lead to innovative, tailored therapies for patients with advanced pulmonary fibrosis, offering hope for improved disease management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eIAM, ATS and JF: Data curation, Formal analysis, Investigation, visualization and Writing \u0026ndash; original draft.AB and GRG: Valiadation and Writing \u0026ndash; original draft.AFKV: Resources and Writing \u0026ndash; original draftKR: Funding acquisition, Validadtion, Project administration and Writing \u0026ndash; review \u0026amp; editing\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe extend our heartfelt thanks to Sarah Eller for her technical expertise in conducting the plasma peptide dosages, which were crucial for the accuracy of our findings.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available within the article and its Supplementary Materials. Additional data, if applicable, are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAshcroft, T., Simpson, J. M., \u0026amp; Timbrelli, V. (1988). Simple method of estimating severity of pulmonary fibrosis on a numerical scale. In \u003cem\u003eJ Clin Pathol\u003c/em\u003e (Vol. 41).\u003c/li\u003e\n \u003cli\u003eBachofen, H., \u0026amp; Scherrer, M. (1967). Lung Tissue Resistance in Diffuse Interstitial Pulmonary. In \u003cem\u003eJournal oClinical Investigation\u003c/em\u003e (Vol. 46, Number 1).\u003c/li\u003e\n \u003cli\u003eBlanco, A., Fernandes, R., Guimar\u0026atilde;es, G. R., \u0026amp; Rigatto, K. (2025). Alamandine reduces oxidative stress and preserves the epithelium in BLM-induced pulmonary fibrosis. \u003cem\u003eEuropean Journal of Pharmacology\u003c/em\u003e, \u003cem\u003e1004\u003c/em\u003e, 177995. https://doi.org/10.1016/j.ejphar.2025.177995\u003c/li\u003e\n \u003cli\u003eDowman, L., McDonald, C. F., Hill, C., Lee, A., Barker, K., Boote, C., Glaspole, I., Goh, N., Southcott, A., Burge, A., Ndongo, R., Martin, A., \u0026amp; Holland, A. E. (2013). The benefits of exercise training in interstitial lung disease: Protocol for a multicentre randomised controlled trial. \u003cem\u003eBMC Pulmonary Medicine\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1). https://doi.org/10.1186/1471-2466-13-8\u003c/li\u003e\n \u003cli\u003eEaton, T., Young, P., Milne, D., \u0026amp; Wells, A. U. (2005). Six-minute walk, maximal exercise tests: Reproducibility in fibrotic interstitial pneumonia. \u003cem\u003eAmerican Journal of Respiratory and Critical Care Medicine\u003c/em\u003e, \u003cem\u003e171\u003c/em\u003e(10), 1150\u0026ndash;1157. https://doi.org/10.1164/rccm.200405-578OC\u003c/li\u003e\n \u003cli\u003eFernandes, R. S., Dias, H. B., de Souza Jaques, W. A., Becker, T., \u0026amp; Rigatto, K. (2021). Assessment of Alamandine in Pulmonary Fibrosis and Respiratory Mechanics in Rodents. \u003cem\u003eJRAAS - Journal of the Renin-Angiotensin-Aldosterone System\u003c/em\u003e, \u003cem\u003e2021\u003c/em\u003e. https://doi.org/10.1155/2021/9975315\u003c/li\u003e\n \u003cli\u003eFunke-Chambour, M., \u0026amp; Geiser, T. (2015). Idiopathic pulmonary fibrosis: The turning point is now! \u003cem\u003eSwiss Medical Weekly\u003c/em\u003e, \u003cem\u003e145\u003c/em\u003e. https://doi.org/10.4414/smw.2015.14139\u003c/li\u003e\n \u003cli\u003eH\u0026uuml;bner, R. H., Gitter, W., El Mokhtari, N. E., Mathiak, M., Both, M., Bolte, H., Freitag-Wolf, S., \u0026amp; Bewig, B. (2008). Standardized quantification of pulmonary fibrosis in histological samples. \u003cem\u003eBioTechniques\u003c/em\u003e, \u003cem\u003e44\u003c/em\u003e(4), 507\u0026ndash;517. https://doi.org/10.2144/000112729\u003c/li\u003e\n \u003cli\u003eLafyatis, R. (2014). Transforming growth factor \u0026beta;\u0026mdash;at the centre of systemic sclerosis.\u0026nbsp;\u003cem\u003eNature Reviews Rheumatology\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(12), 706\u0026ndash;719. https://doi.org/10.1038/nrrheum.2014.137\u003c/li\u003e\n \u003cli\u003eLiu, T., De Los Santos, F. G., \u0026amp; Phan, S. H. (2017).\u0026nbsp;\u003cem\u003eThe Bleomycin Model of Pulmonary Fibrosis\u003c/em\u003e (pp. 27\u0026ndash;42). https://doi.org/10.1007/978-1-4939-7113-8_2\u003c/li\u003e\n \u003cli\u003eMoeller, A., Ask, K., Warburton, D., Jack, G. #, \u0026amp; Kolb, M. (n.d.). \u003cem\u003eThe bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003eOtoupalova, E., Smith, S., Cheng, G., \u0026amp; Thannickal, V. J. (2020). Oxidative Stress in Pulmonary Fibrosis. In \u003cem\u003eComprehensive Physiology\u003c/em\u003e (2nd ed., Vol. 10, pp. 509\u0026ndash;547). Wiley. https://doi.org/10.1002/cphy.c190017\u003c/li\u003e\n \u003cli\u003eRaghu, G., Collard, H. R., Egan, J. J., Martinez, F. J., Behr, J., Brown, K. K., Colby, T. V., Cordier, J. F., Flaherty, K. R., Lasky, J. A., Lynch, D. A., Ryu, J. H., Swigris, J. J., Wells, A. U., Ancochea, J., Bouros, D., Carvalho, C., Costabel, U., Ebina, M., \u0026hellip; Sch\u0026uuml;nemann, H. J. (2011). An Official ATS/ERS/JRS/ALAT Statement: Idiopathic pulmonary fibrosis: Evidence-based guidelines for diagnosis and management. \u003cem\u003eAmerican Journal of Respiratory and Critical Care Medicine\u003c/em\u003e, \u003cem\u003e183\u003c/em\u003e(6), 788\u0026ndash;824. https://doi.org/10.1164/rccm.2009-040GL\u003c/li\u003e\n \u003cli\u003eRoberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stern, D. F., \u0026amp; Sporn, M. B. (1985). Type beta transforming growth factor: a bifunctional regulator of cellular growth. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, \u003cem\u003e82\u003c/em\u003e(1), 119\u0026ndash;123. https://doi.org/10.1073/pnas.82.1.119\u003c/li\u003e\n \u003cli\u003eRoberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotra, L. A., Falangat, V., Kehrl, J. H., \u0026amp; Faucit, A. S. (1986). Transforming growth factor type ,8: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. In \u003cem\u003eProc. Nati. Acad. Sci. USA\u003c/em\u003e (Vol. 83).\u003c/li\u003e\n \u003cli\u003eSantos, R. A. S., Dias, M. T. S., Bessa, A. de S. M., Barros, C. F., Itaborahy, M. F., da Silva, F. A., Gon\u0026ccedil;alves, S. C. de A., Rodrigues-Ribeiro, L., Ferraz, K. S., Davel, A. P., N\u0026oacute;brega, N., Silva, B. D. da, Scalzo, S., Soares, P. A., Dutra, J. B. R., Lula, I., Feng, I. Z. L. F., Vieira-Machado, U. F., de Godoy, A. C. V., \u0026hellip; Campagnole-Santos, M. J. (2026). Identification and Characterization of Alamandine-(1-5), a New Component of the Renin-Angiotensin System. \u003cem\u003eCirculation Research\u003c/em\u003e, \u003cem\u003e138\u003c/em\u003e(1). https://doi.org/10.1161/CIRCRESAHA.125.326174\u003c/li\u003e\n \u003cli\u003eSilva, A. V. (2021). Associations between clinical signs and pathological findings in toxicity testing.\u0026nbsp;\u003cem\u003eALTEX\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(2), 198\u0026ndash;214. https://doi.org/10.14573/altex.2003311\u003c/li\u003e\n \u003cli\u003eSipriani, T. S., Dos Santos, R. A. S., \u0026amp; Rigatto, K. (2019). The Renin-Angiotensin System: Alamandine is reduced in patients with Idiopathic Pulmonary Fibrosis. \u003cem\u003eJournal of Cardiology and Cardiovascular Medicine\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(3), 210\u0026ndash;215. https://doi.org/10.29328/journal.jccm.1001070\u003c/li\u003e\n \u003cli\u003eUhal, B. D., Li, X., Piasecki, C. C., \u0026amp; Molina-Molina, M. (2012). Angiotensin signalling in pulmonary fibrosis. In \u003cem\u003eInternational Journal of Biochemistry and Cell Biology\u003c/em\u003e (Vol. 44, Number 3, pp. 465\u0026ndash;468). Elsevier Ltd. https://doi.org/10.1016/j.biocel.2011.11.019\u003c/li\u003e\n \u003cli\u003eWynn, T. A. (2011). Integrating mechanisms of pulmonary fibrosis. \u003cem\u003eJournal of Experimental Medicine\u003c/em\u003e, \u003cem\u003e208\u003c/em\u003e(7), 1339\u0026ndash;1350. https://doi.org/10.1084/jem.20110551\u003c/li\u003e\n \u003cli\u003eYang, C., Wu, X., Shen, Y., Liu, C., Kong, X., \u0026amp; Li, P. (2020). Alamandine attenuates angiotensin II-induced vascular fibrosis via inhibiting p38 MAPK pathway. \u003cem\u003eEuropean Journal of Pharmacology\u003c/em\u003e, \u003cem\u003e883\u003c/em\u003e, 173384. https://doi.org/10.1016/j.ejphar.2020.173384\u003c/li\u003e\n \u003cli\u003eZhao, K., Xu, T., Mao, Y., Wu, X., Hua, D., Sheng, Y., \u0026amp; Li, P. (2022). Alamandine alleviated heart failure and fibrosis in myocardial infarction mice. \u003cem\u003eBiology Direct\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(1). https://doi.org/10.1186/s13062-022-00338-6\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable \u0026ndash; Oxidative Stress Levels in Lung Tissue and Plasma Concentrations of Renin-Angiotensin System Peptides\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"671\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 671px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOxidative Stress Levels in Lung Tissue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCO\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N = 5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eALA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N = 5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBLM\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N = 5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(N = 5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSH (nmol/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e10 \u0026plusmn; 1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e8.73 \u0026plusmn; 0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e6.81 \u0026plusmn; 1.8\u003cstrong\u003e\u003csup\u003e\u0026nbsp;c\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e9.95 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarbonil (nmol of DNPH/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e21 \u0026plusmn; 18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e9.3 \u0026plusmn; 7.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e25 \u0026plusmn; 23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e7.95 \u0026plusmn; 3.7\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSH (ug/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e248 \u0026plusmn; 49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e71 \u0026plusmn; 22\u003cstrong\u003e\u003csup\u003ea,b\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e106 \u0026plusmn; 56\u003cstrong\u003e\u003csup\u003ea,b\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e245 \u0026plusmn; 76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDCF (A.U./mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e25 \u0026plusmn; 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003e63 \u0026plusmn; 36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 118px;\"\u003e\n \u003cp\u003e41 \u0026plusmn; 7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e24 \u0026plusmn; 14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 671px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasma Concentrations of Renin-Angiotensin System Peptides (N=7; ng/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAngiotensin I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,089 \u0026plusmn; 0,020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,068 \u0026plusmn; 0,029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e0,035 \u0026plusmn; 0,022\u003cstrong\u003e\u003csup\u003e**\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0,065 \u0026plusmn; 0,024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAngiotensin II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,046 \u0026plusmn; 0,018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,039 \u0026plusmn; 0,011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e0,029 \u0026plusmn; 0,015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0,037 \u0026plusmn; 0,009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAngiotensin A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,051 \u0026plusmn; 0,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,052 \u0026plusmn; 0,017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e0,034 \u0026plusmn; 0,020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0,050 \u0026plusmn; 0,021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAngiotensin 1-7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,039 \u0026plusmn; 0,012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,031 \u0026plusmn; 0,009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e0,013 \u0026plusmn; 0,009\u003cstrong\u003e\u003csup\u003e#**\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0,029 \u0026plusmn; 0,012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAlamandine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,100 \u0026plusmn; 0,027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e0,087 \u0026plusmn; 0,033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 118px;\"\u003e\n \u003cp\u003e0,038 \u0026plusmn; 0,026\u003cstrong\u003e\u003csup\u003e**@\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e0,083 \u0026plusmn; 0,034\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCO: control group; ALA: alamandine-treated group; BLM: bleomycin-only group; BA: bleomycin plus ALA treatment group. GSH: Reduced Glutathione; Carbonyl: Protein Carbonyl Content; DNPH: 2,4-Dinitrophenylhydrazine; SH: Thiol Group and DCF: Dichlorofluorescein. Data are presented as means \u0026plusmn; standard deviations. \u003csup\u003ea\u003c/sup\u003ep \u003cu\u003e\u0026lt;\u0026nbsp;\u003c/u\u003e0.007 \u003cem\u003evs\u003c/em\u003e CO; \u003cstrong\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003ep \u003cu\u003e\u0026lt;\u003c/u\u003e 0.005 \u003cem\u003evs\u003c/em\u003e BA; \u003cstrong\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003ep \u003cu\u003e\u0026lt;\u003c/u\u003e 0.03 \u003cem\u003evs\u003c/em\u003e CO; \u003csup\u003e**\u003c/sup\u003ep \u003cu\u003e\u0026lt;\u003c/u\u003e 0.004 \u003cem\u003evs\u003c/em\u003e CO; \u003cstrong\u003e\u003csup\u003e#\u003c/sup\u003e\u003c/strong\u003ep \u003cu\u003e\u0026lt;\u003c/u\u003e 0.028 \u003cem\u003evs\u003c/em\u003e ALA; \u003cstrong\u003e\u003csup\u003e@\u003c/sup\u003e\u003c/strong\u003ep \u003cu\u003e\u0026lt;\u003c/u\u003e 0.046 vs ALA and BS grup.\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":"international-journal-of-peptide-research-and-therapeutics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijpr","sideBox":"Learn more about [International Journal of Peptide Research and Therapeutics](http://link.springer.com/journal/10989)","snPcode":"10989","submissionUrl":"https://submission.nature.com/new-submission/10989/3","title":"International Journal of Peptide Research and Therapeutics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pulmonary Fibrosis, Alamandine, TGF-β, Respiratory Mechanics, Therapeutic Intervention","lastPublishedDoi":"10.21203/rs.3.rs-9066088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9066088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Pulmonary fibrosis (PF) is a challenging interstitial lung disease with limited therapeutic options. This study explores the therapeutic effects of alamandine (ALA), a renin-angiotensin system peptide, in reversing established fibrotic progression in an experimental model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Male Wistar rats were divided into four groups (n=6): control (CO), ALA-treated (ALA), bleomycin-induced fibrosis (BLM), and bleomycin plus ALA treatment (BA). Fibrosis was induced by intratracheal bleomycin (2.5 mg/kg) on day 0. Subcutaneous ALA treatment (50 µg/kg/day) started on day 10 and continued until day 19. Respiratory mechanics, body weight, Ashcroft score, and lung transforming growth factor-beta (TGF-β) content were evaluated. Plasma RAS peptides were quantified by LC-MS/MS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBleomycin significantly increased respiratory resistance (0.159±0.047 vs 0.104±0.026 cmH₂O/mL/s in CO, p\u0026lt;0.01) and Ashcroft score, and reduced body weight gain. ALA treatment from day 10–20 markedly improved body weight gain (p\u0026lt;0.001), reduced Ashcroft score (1.21±0.40 vs 2.23±0.35 in BLM, p\u0026lt;0.0001), and decreased lung TGF-β1 content (1.10±0.42 vs 3.70±1.7 pg/mg protein in BLM, p\u0026lt;0.005). In addition to significantly reducing thoracic chamber resistance (p \u0026lt; 0.02), ALA treatment reversed TGF-β elevation (p \u0026lt; 0.005) from 3.70 ± 1.7 in the BLM group to 1.10 ± 0.42 in the BA group and was associated with qualitatively improved respiratory effort, suggesting attenuation of bleomycin-induced fibrogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Despite rapid tissue uptake, late ALA treatment attenuates histological fibrosis and TGF-β1 accumulation and improves clinical well-being in rats with established pulmonary fibrosis. These findings suggest potential therapeutic benefit that warrants further dose- and time-ranging studies.\u003c/p\u003e","manuscriptTitle":"Alamandine Attenuates Signs and Reduces Fibrotic Markers in a Rat Model of Established Pulmonary Fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 08:29:25","doi":"10.21203/rs.3.rs-9066088/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-09T07:39:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T16:22:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T13:54:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-21T09:20:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26553995620939726296701592347772974336","date":"2026-03-20T08:42:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27665959918351019593352947309637320079","date":"2026-03-19T07:51:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220439622567818185827840777958492375354","date":"2026-03-18T13:45:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-15T23:41:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-10T07:20:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T07:20:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Peptide Research and Therapeutics","date":"2026-03-08T18:43:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-peptide-research-and-therapeutics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ijpr","sideBox":"Learn more about [International Journal of Peptide Research and Therapeutics](http://link.springer.com/journal/10989)","snPcode":"10989","submissionUrl":"https://submission.nature.com/new-submission/10989/3","title":"International Journal of Peptide Research and Therapeutics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8d839a70-dbde-476f-ab63-e02172720dd5","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T07:57:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 08:29:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9066088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9066088","identity":"rs-9066088","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}