Alamandine Mitigates Inflammation and Oxidative Stress in OVA-Induced Asthma: A Novel Therapeutic Approach

Alamandine Mitigates Inflammation and Oxidative Stress in OVA-Induced Asthma: A Novel Therapeutic Approach | 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 Mitigates Inflammation and Oxidative Stress in OVA-Induced Asthma: A Novel Therapeutic Approach Ali Abbasi, Kazem Javanmardi, Hamideh Shahbazi, Ava soltani hekmat This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7720698/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background: Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness, mucus overproduction, and oxidative stress. Alamandine (Ala), a peptide of the renin–angiotensin system, has shown anti-inflammatory and antioxidant properties in several experimental models. This study aimed to investigate the potential protective effects of Ala in an ovalbumin (OVA)-induced model of allergic asthma in mice. Methods: Thirty-five male mice were divided into control, OVA, OVA+Ala, OVA+dexamethasone, and Ala-only groups. Asthma was induced by sensitization and challenge with OVA. Ala (50 μg/kg) and dexamethasone (2 mg/kg) were administered intraperitoneally during the challenge phase. Lung tissues and serum samples were analyzed for inflammatory cytokines, oxidative stress markers, and histopathological alterations. Results: Treatment with Ala significantly reduced airway inflammation, eosinophil infiltration, and mucus secretion compared with the OVA group. Ala also downregulated the expression of pro-inflammatory cytokines (IL-6 and TNF-α) and suppressed NF-κB activation. Furthermore, oxidative stress markers improved following Ala administration, indicating its antioxidant potential. The effects of Ala were comparable, in part, to those of dexamethasone. Conclusion: These findings suggest that Ala may exert protective effects against allergic airway inflammation and oxidative stress in an OVA-induced asthma model. The observed improvements in cytokine levels and tissue pathology indicate that Ala could help modulate inflammatory signaling pathways involved in asthma. However, further experimental and clinical studies are needed to confirm these results and to clarify the underlying mechanisms of Ala’s action in asthma management. Alamandine asthma oxidative stress inflammation cytokines NF-κB dexamethasone Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Asthma, a chronic inflammatory disease affecting over 300 million individuals globally (Subbarao et al. 2009 ), is influenced by various factors and is recognized by recurring wheezing, difficulty breathing, chest ‎tightness and coughing particularly at night or, in the early morning‎ (Rodríguez-Torres et al. 2021 ). Airway hyperresponsiveness (AHR) is a characteristic of asthma‎ (Brannan 2010 ). The inhalation of substances like house dust, pollen, animal dander, and smoking trigger the development of asthma, leading to an immune imbalance with an increase in T helper type 2 (Th2) cells in the airway (Perret et al. 2013 ). Th2 cytokines, such as interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic airway inflammation's development and maintenance (Peebles and Aronica 2019 ). These cytokines recruit and activate eosinophils, which cause persistent inflammation, airway hyperresponsiveness, and excessive mucus production in allergic asthma ((Schatz and Rosenwasser 2014 ; Lee et al. 2017 ). Consequently, targeting the suppression of Th2 cytokines has been identified as a crucial approach for managing allergic asthma ((Leόn 2017; Gandhi et al. 2018). Furthermore, the prevalence of this respiratory condition is influenced by various other factors. One significant factor is the nuclear factor-kappa B (NF-κB), a transcription factor that plays a central role in regulating inflammatory responses (Zhang et al. 2017 ). NF-κB can be activated by different stimuli, leading to its translocation into the nucleus, where it binds to DNA and controls the transcription of genes involved in asthmatic responses. This includes genes related to Th2 cytokines and mucin 5AC (MUC5AC), which is associated with excessive mucus production in the airways (Cai et al. 2018 ; Tian et al. 2019 ). Another factor that plays a role in in the pathogenesis of asthma is oxidative stress. Inflammation induces a lung oxidative stress reaction, resulting in the production of reactive oxygen species (ROS) (Andreadis et al. 2003 ). Reactive oxygen species have various effects on the pathogenesis of asthma, including the stimulation of pulmonary function impairment, mast cell degranulation, airway remodeling, and mucus secretion by the epithelium. These processes can exacerbate local lung inflammation (Zhang et al. 2018 ). The imbalance between oxidants and antioxidants is significant in the recurrence of airway inflammation in asthma. Both endogenous and exogenous oxidants stimulate inflammatory responses by amplifying the pro-inflammatory signaling pathways (Nadeem et al. 2014 ). Cytokines, which are proteins produced by types of cells have a crucial role in coordinating, maintaining ‎and boosting inflammatory responses, in asthma ‎ (Mahajan and Mehta 2006 ). These substances help regulate inflammation in the airways leading to increased activity of the muscles ‎surrounding the airways and contributing to bronchial constriction (Mahajan and Mehta 2006 ). Pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β contribute to increased airway inflammation and severity of asthma (Barnes 2008 ). IL-1β specifically plays a role in the development of inflammation in asthma. It does so by stimulating ‎the production of cytokines activating cell adhesion molecules and triggering eosinophil activation. ‎Research has shown that patients with asthma have elevated levels of IL-1β and TNF-α ‎in their ‎bronchoalveolar lavage fluid ‎ (BALF) ‎(Mahajan and Mehta 2006 ). Currently the main treatments for asthma are inhalation and bronchodilators. However, these ‎medications come with side effects when used long term and can impose a substantial financial ‎burden on patients‎ (Klimek et al. 2019 ). Additionally, ‎5–10% ‎of severe asthma patients do not respond well to these therapies and prolonged ‎use can lead to serious adverse effects ‎ (Mosnaim, md et al. 2022). Therefore, it is crucial to identify a treatment option, for asthma.‎ Oxidative damage plays a pivotal role in the pathogenesis of bronchial asthma, making it a potential therapeutic target for the treatment of asthmatic patients. A clinical study has demonstrated the usefulness of antioxidants in managing mild to moderate asthma (van Toan and Hanh 2013 ). In recent years, numerous studies have focused on discovering novel compounds with enhanced ‎antioxidant activity. One such compound may be almandine) Ala (, which possesses anti-inflammatory, ‎cardioprotective, and antioxidant properties(Hekmat et al. 2021 ). Ala is a newly identified member of the angiotensin family and has shown significant cardioprotective effects in rats treated with isoproterenol. This peptide shares similarities with Ang-(1–7) and binds to the Mas-related G-coupled receptor known as member D (MrgD). Ala effectively mitigated monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. It achieved this by reducing oxidative stress and inflammation, regulating inducible nitric oxide synthase, and ameliorating histopathological changes. In ventricles subjected to ischemia and reperfusion (I/R), Ala upregulates the expression of several antioxidant proteins, including SOD and catalase. Additionally, it downregulates the expression of caspase-9, Bax, and caspase-3 in I/R ventricles (Lautner et al. 2013 a). Furthermore, Ala reduces the levels of IL-1β and TNF-α in neonatal cardiomyocytes following an increase induced by LPS treatment (De Souza-Neto et al. 2019). Moreover, Ala significantly mitigates DOX-induced cardiotoxicity in rats by modulating antioxidant status, apoptosis, and inflammatory cytokines (Hekmat et al. 2021 ). Therefore, this study aims to investigate the therapeutic potential of Ala in asthma by examining its protective properties against ovalbumin (OVA)-induced allergic asthma in mice, with a focus on oxidative stress and inflammatory factors. Material and Methods The animals used in this study were provided by the Animal Laboratory of Fasa University of Medical Sciences. Thirty-five male mice, each weighing approximately 20 grams and confirmed to be free from murine-specific pathogens, were housed in a standard laboratory environment (temperature: 22 ± 2°C, humidity: 50–60%, with a 12:12-hour light/dark cycle, lights on at 7:00 AM). The mice were grouped in sets of five and had unrestricted access to standard laboratory chow and water. All experimental procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Fasa University of Medical Sciences (ethical code: FUMS.AEC.1402.001)." Experimental protocols The animals were assigned to five groups (n = 7) based on random allocation to meet the research ‎requirements ‎(Lautner et al. 2013 b). 1. Control group: In the control group, mini-osmotic pumps (model 1002; ALZET Osmotic Pumps, CA, USA) infused saline at a rate of 0.25 µl/h, replicating the administration method utilized for the experimental groups. Furthermore, mice in this group received an intraperitoneal (ip) injection of saline to ensure uniformity with the injection protocol applied in the other groups. ‎2. OVA group: ‎OVA-sensitized mice injected intraperitoneally with 20 µg of OVA (BP106-Genesco) plus 2 mg of Al (OH)3 on day 0, 7 and 14 and and OVA-challenged on day 22–28 with inhalation of ‎ ‎1% OVA ‎ solution using a nebulizer for a duration of 30 minutes.‎ 3. Ala Group: Mice in this category received Ala (Phoenix Pharmaceuticals Inc., CA, USA) for 14 days from day 15 to day 28 through mini-osmotic pumps at a dosage of 50 µg Ala/kg/day. 4. OVA + Ala group: Mice in this category underwent a treatment protocol, combining sensitization with OVA through intraperitoneal injections of 20 µg OVA plus 2 mg of Al (OH)3 on days 0, 7, and 14. Subsequently, The OVA-challenge was administered through inhalation of a 1% OVA solution using a nebulizer for a duration of 30 minutes from days 22 to 28. Concurrently, Ala was administered s via mini-osmotic pumps at a dosage of 50 µg Ala/kg/day for the entire 14-day period, spanning from day 15 to day 28. 5. OVA +‎ Dex group: This experimental group was similar to the Ala + OVA Group, with one distinction: instead of Ala, Dexa was administered at a dosage of 2 mg/kg (Zhang et al. 2018 ). The treatment protocol is illustrated in Fig. 1 . Collection of BALF To collect BALF, the mice were euthanized by administering an overdose of pentobarbital at a dosage of 50 mg/kg, 24 hours after the final OVA challenge. Tracheotomy was performed, and ice-cold phosphate-buffered saline (PBS) (0.5 mL) was instilled into the lungs. The fluid was collected by three successive aspirations via tracheal cannulation, resulting in a total volume of 1.5 mL (Zhang et al. 2018 ). Subsequently, BALF samples were centrifuged at 1500 rpm for 10 minutes at 4°C. The supernatants were then stored at -80°C for further analysis. BALF and Eosinophil Counts The sediment acquired from BALF underwent resuspension in 200 µL of phosphate-buffered saline. Following this, a smear was meticulously prepared from the cellular constituents, and ensuing staining was performed utilizing the Wright–Giemsa method. Subsequent to staining, a differential cell analysis, guided by staining and morphological criteria, was systematically carried out using a light microscope Enzyme-Linked Immunosorbent Assay (ELISA) Detection of Cytokines in BALF ELISA was performed using specific ELISA kits for mice IL-4, IL-5, IL-13, TNF-α, IL-1β, IL-6 and NF-κB (Zell Bio Company, Germany) following the manufacturer's instructions. Briefly, 100 µL of standard blank or sample was added to appropriate wells and incubated for 1 hour at 37°C. After aspiration, 100 µL of prepared detection reagent was added to each well and incubated for 1 hour at 37°C. Following aspiration and washing, 90 µL of substrate solution was added to the wells and incubated for 15 minutes at 37°C. Finally, 50 µL of stop solution was added, and the optical density (OD) was immediately measured at 450 nm using a BioTek ELISA reader. The cytokine concentrations were determined by comparing the OD of the samples to the standard curve. Measurement of Oxidative Stress in BALF Oxidative stress markers, including MDA (malondialdehyde), SOD (superoxide dismutase), and GPx (glutathione peroxidase), were assessed in BALF using commercially available kits according to the manufacturer's instructions. The MDA levels were determined by the TBA method, which involves the condensation of lipid hydroperoxide decomposition products with TBA, forming red compounds with an absorption peak at 532 nm. SOD activity was quantified by measuring the conversion of superoxide anion into hydrogen peroxide and oxygen with absorbance at 420 nm. The GPx assay kit involved the catalytic oxidation of glutathione by cumene hydroperoxide accomplished by GPx, and the decrease in absorption was measured at 340 nm using a spectrophotometer in the presence of NADPH and glutathione reductase. Measurement of IgE Levels in Serum The levels of IgE in the serum were measured by ELISA following the manufacturer's instructions. Lung tissue histopathology The left lungs were washed with PBS, fixed in 4% paraformaldehyde, and dehydrated using an alcohol gradient. Subsequently, the lungs were rendered transparent with xylene. After embedding in paraffin, 5 µm slices were obtained and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) solution. The stained sections were examined using an optical microscope. H&E staining was utilized to evaluate pathological changes in lung tissue, and a score ranging from 0 to 5 was assigned to assess the extent of lung tissue inflammation. PAS staining was employed to evaluate the degree of airway goblet cell proliferation ([CSL STYLE ERROR: reference with no printed form.]). Statistical analyses Data analysis was performed using GraphPad Prism software. The results are presented as mean ± standard deviation (SD). Group comparisons were conducted through a one-way Analysis of Variance (ANOVA) followed by Tukey's post-hoc test. A significance level of P ≤ 0.05 was considered indicative of statistical significance in all analyses. Results Effect of Ala on eosinophils in BALF The administration of OVA significantly elevated the eosinophil count to 78.1 ± 6.69 cells/mm³, in stark contrast to the control group's count of 5.66 ± 1.1 cells/mm³ (p < 0.001). Treatment with Ala led to a notable reduction in eosinophil count, bringing it down to 41.5 ± 3.03 cells/mm³ in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a similar efficacy as Ala, resulting in an eosinophil count of 43.5 ± 6.50 cells/mm³ in the Asthma + Dexa group (Fig. 2 ). 3 / 3 Effect of Ala on proinflammatory cytokines and NF-κB in BALF: Following OVA administration, IL-6 levels significantly increased to 46.0 ± 3.71 pg/mL, representing a notable surge compared to the control group's 12.1 ± 1.15 pg/mL (p < 0.001). Ala treatment led to a considerable reduction in IL-6 levels, bringing them down to 23.3 ± 2.92 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a similar impact to Ala, showing IL-6 levels of 26.4 ± 5.75 pg/mL in the OVA + Dexa group. In addition, IL-1β levels also experienced a significant increase to 172 ± 12.9 pg/mL following OVA administration, contrasting with the control group's 78.1 ± 16.2 pg/mL (p < 0.001). Ala treatment resulted in a reduction of IL-1β levels to 108 ± 7.65 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone exhibited a comparable effect to Ala, with IL-1β levels of 107 ± 10.6 pg/mL in the OVA + Dexa group. Furthermore, TNF-α levels significantly increased to 132 ± 34.0 pg/mL after OVA administration, in contrast to the control group's 59.6 ± 30.0 pg/mL (p < 0.001). Ala treatment led to a marked decrease in TNF-α levels, reducing them to 13.4 ± 10.1 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone exhibited a similar effect to Ala, with TNF-α levels of 9.62 ± 4.65 pg/mL in the OVA + Dexa group. Moreover, NF-κB levels significantly rose to 3.02 ± 0.419 pg/mL after OVA administration, compared to the control group's 1.27 ± 0.286 pg/mL (p < 0.001). Ala treatment led to a reduction in NF-κB levels to 1.27 ± 0.0881 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a similar effect to Ala, with NF-κB levels of 1.27 ± 0.127 pg/mL in the OVA + Dexa group (Fig. 3 ). Effect of Ala on Th2 cytokines in BALF:‎ The administration of OVA led to a significant elevation of IL-4 levels, reaching 115 ± 14.6 pg/mL, in comparison to the control group's 53.8 ± 2.83 pg/mL (p < 0.001). Ala treatment effectively reduced IL-4 levels to 54.8 ± 15.4 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone exhibited a similar effect to Ala, demonstrating IL-4 levels of 57.2 ± 7.67 pg/mL in the OVA + Dexa group. In the context of OVA administration, IL-5 levels experienced a substantial increase, reaching 356 ± 12.4 pg/mL, compared to the control group's 172 ± 4.53 pg/mL (p < 0.001). The introduction of Ala effectively reduced IL-5 levels to 261 ± 25.6 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a comparable effect to Ala, with IL-5 levels of 234 ± 22.0 pg/mL in the OVA + Dexa group. The administration of OVA also resulted in a significant elevation of IL-13 levels, reaching 181 ± 12 pg/mL, in comparison to the control group's 57 ± 6.82 pg/mL (p < 0.001). Ala treatment effectively reduced IL-13 levels to 153 ± 14.6 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone exhibited a similar effect to Ala, with IL-13 levels of 133 ± 13.9 pg/mL in the OVA + Dexa group (Fig. 4 ). Effect of Ala on the IgE level in serum OVA administration led to a significant increase in IgE levels, reaching 266 ± 23.2 pg/mL, in contrast to the control group's 158 ± 16.7 pg/mL (p < 0.001). Ala treatment effectively reduced IgE levels to 214 ± 37.7 pg/mL in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a similar effect to Ala, with IgE levels measuring 214 ± 13.1 pg/mL in the OVA + Dexa group (Fig. 5 ). Effect of Ala on oxidative stress in BALF:‎ OVA administration resulted in a significant increase in MDA levels, reaching 13.6 ± 3.89 µM—a substantial surge compared to the control group's 0.707 ± 0.231 µM (p < 0.001). Ala intervention effectively decreased MDA levels to 5.61 ± 2.00 µM in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a comparable yet distinct effect to Ala, showing MDA levels of 4.95 ± 1.16 µM in the OVA + Dexa group. On the contrary, OVA administration resulted in a significant reduction in SOD activity to 7.00 ± 1.04 IU/mL—a marked decrease compared to the control group's 81.7 ± 10.8 IU/mL (p < 0.001). Ala treatment elicited an elevation in SOD activity to 36.2 ± 1.75 IU/mL in the OVA + Ala group (p < 0.001). Dexamethasone exhibited a similar effect to Ala, with SOD activity measuring 47.2 ± 2.73 IU/mL in the OVA + Dexa group. In another aspect, OVA administration significantly decreased GPX activity to 0.046 ± 0.0061 U/mL—a notable decline compared to the control group's 0.24 ± 0.025 U/mL (p < 0.001). Ala treatment induced an increase in GPX activity to 0.17 ± 0.032 U/mL in the OVA + Ala group (p < 0.001). Dexamethasone demonstrated a similar yet discernible effect to Ala, with GPX activity measuring 0.19 ± 0.0097 U/mL in the OVA + Dexa group (Fig. 6 ). Effects of Ala on histopathologic changes in the lung: Figure 7 depicts a cross-section of mouse lung tissue stained with Hematoxylin and Eosin. The results obtained regarding the level of lung inflammation revealed that the OVA group exhibited the highest level of inflammation. Inflammatory indicators, such as lymphocyte secretion, presence of neutrophils, and tissue changes including epithelial cell accumulation, were significantly higher in this group compared to the other studied groups. Moreover, in this group, there was an observed increase in the thickness of the alveolar wall's epithelial layer, which comprises two types of pneumocytes cells (type 1 and 2) responsible for covering and secreting surfactant. This increase led to a reduction in the exchange of respiratory gases and an elevation in pulmonary secretions, ultimately resulting in pulmonary edema. Conversely, in the OVA + Ala and OVA + Dexa groups, these changes showed a significant decrease compared to the OVA group. The OVA + Ala group exhibited reduced lung inflammation, mucus secretion, intrapulmonary hemorrhage, and thickening of alveolar walls, along with a decreased level of inflammatory cell infiltration compared to the untreated OVA group. Similar improvements were observed in the OVA + Dexa group. The examination of images from the control group revealed normal bronchioles with ciliated cells and a regular appearance of the lung tissue. The alveoli were found to have a single layer of pneumocytic cells. Among these cells, type 1 alveolar cells, constituting approximately 95% of the alveolar surface and forming a barrier between the air and alveolar walls, were observed as thin pavement-like cells. Additionally, type 2 alveolar cells, responsible for surfactant secretion, appeared normal in this group. Overall, the tissue appeared completely normal, without any signs of thickening or evidence of cellular metaplasia. Discussion Asthma is a chronic inflammatory condition characterized by the infiltration of eosinophils and other inflammatory cells, leading to bronchial hyper-responsiveness and airway obstruction (Calderon et al. 2013 ). The increasing prevalence of asthma has driven extensive research into the discovery of new anti-asthmatic agents. Previous investigations have indicated that Ala possesses antioxidant and anti-inflammatory properties (Soltani Hekmat et al. 2021 ). Hence, the primary aim of this study was to evaluate the impact of Ala administration on inflammation in the context of asthma. Our results demonstrate that treatment with Ala effectively reduced the accumulation of eosinophils and inflammatory cytokines in the BALF, leading to mitigated pulmonary remodeling. During the development of allergic asthma, there is typically a dominant Th2 immune response. Th2 cells secrete inflammatory cytokines, including IL-4, IL-5, IL-9, and IL-13, with IL-4 and IL-13 playing crucial regulatory roles in asthma. These cytokines significantly contribute to airway inflammation by promoting the production of IgE and mucus by B cells, inducing remodeling of epithelial cells, activating M2 macrophages, and binding to IgE FcεRI receptors on mast cells and basophils, resulting in the release of histamine and leukotrienes. IL-5 plays a pivotal role in eosinophil differentiation, maturation, chemotaxis, and activation, often working in conjunction with IL-4 to increase eosinophil levels in individuals with allergic asthma(León and Ballesteros-Tato 2021 ; Yang et al. 2022 ). Our findings revealed a significant reduction in serum IgE levels in OVA-sensitized and challenged mice following treatment with Ala. Previous studies have highlighted the crucial role of the IL-4 cytokine in initiating isotype class switching, which is necessary for B cells to produce IgE (Vazquez et al. 2015 ). Considering that Ala was shown to decrease interleukin levels in this study, and previous research has also demonstrated its ability to reduce cytokines (Hekmat et al. 2021 ; Soltani Hekmat et al. 2021 ), it is plausible that the decrease in interleukins mediated by Ala contributed to the reduction in IgE levels. Moreover, we observed an increase in cytokine levels in the BALF of mice treated with OVA compared to the control group. Yan et al. have previously reported that Th2 cells in the lungs of asthmatic patients secrete significant amounts of IL-4, IL-5, and IL-13 (Yan et al. 2011 ). However, treatment with Ala effectively reduced the elevated cytokine levels, similar to the effect of Dexa. Dexa, a glucocorticoid drug, exerts potent anti-inflammatory effects against bronchial asthma by inhibiting the production of IL-4, IL-5, and IL-13, which are important cytokines in asthma (Westergaard et al. 2015 ). Numerous studies have demonstrated that Th2-mediated cytokines, including IL-4, IL-5, and IL-13, play crucial roles in regulating and exacerbating the inflammatory response in asthma (Thakur et al. 2019 ; Menzella et al. 2020 ). Additionally, another study reported that asthmatic mice lacking the IL-13 receptor exhibited significantly less airway remodeling compared to mice with wild-type asthma (Chen et al. 2013 ). Hence, it can be suggested that one of the mechanisms by which Ala improved asthma in this study was through the reduction of these cytokines. The ability of Ala to target and decrease these pro-inflammatory cytokines highlights its potential as a promising therapeutic agent for asthma treatment. Studies have reported a correlation between the number of eosinophils in the peripheral blood and bronchial lavage of asthmatic patients and the severity of the disease (Louis et al. 2000 ). In our study, we observed a significant increase in the percentage of eosinophils in the BALF of the OVA treatment group. However, this increase was prevented in the groups treated with Dexa and Ala. It has been suggested that the increase in eosinophils is associated with elevated production of IL-4, IgE, and TNF-α (Parlar and Arslan 2020). Another study reported that vitamin E treatment indirectly reduces IgE levels by suppressing the production of IL-4, which is involved in IgE production, and IL-5, which plays a role in eosinophil migration (Jiang et al. 2021 ). Therefore, it can be inferred that Ala treatment influences the percentage of eosinophils by reducing the levels of IgE, TNF-α, IL-4, and IL-5. Multiple studies have highlighted the activation of the NF-κB pathway in asthmatic tissues, and gene-deficient mouse models of allergic asthma have underscored the importance of NF-κB in the pathogenesis of the disease (Haeberle et al. 2002 ). In asthma, the activation of NF-κB leads to increased expression of genes responsible for producing inflammatory cytokines and chemokines, which, in turn, promotes the recruitment, activation, and survival of inflammatory cells (Sousa et al. 2010 ; El-Hashim et al. 2012 ). There have been studies that strongly support the involvement of NF κB, a transcription factor in the ‎development of asthma. It's particularly interesting to note that mice, without NF κB only experience ‎lung inflammation highlighting the crucial role of this factor in the disease’s progression (El-Hashim et al. 2012 ). Additionally inhibiting NF κB in a mouse model of asthma has shown results in reducing airway ‎inflammation and improving health ‎ (Sousa et al. 2010 ; Pinheiro and Simões E Silva 2012). In our investigation we observed that treatment with Ala resulted in decreased levels of NF κB in the ‎BALF. This suggests that Ala may contribute to resolving lung inflammation by regulating NF κB levels. ‎This mechanism further strengthens the use of Ala as a treatment, for asthma since it can help control ‎responses and lessen disease severity by targeting NF κB activation.‎ Numerous studies have highlighted the active participation of oxidative stress in the pathological development of asthma(Jiang et al. 2021 ). Oxidative stress is known to trigger damage, which contributes to the progress of asthma ‎(Nesi et al. 2017 ). The formation of MDA is widely recognized as an indicator of oxidative stress (Türk et al. 2022). ‎). One mechanism that leads to MDA formation involves peroxidation, which is caused by oxygen ‎radicals. Lipid peroxidation results in decreased membrane fluidity and impaired membrane function‎ (Kuzu et al. 2019 ). In our study we observed a rise in MDA levels in the group treated with OVA compared to the control ‎group. It has been reported that exposure to OVA disrupts balance and diminishes the defense ‎mechanism resulting in the production of highly reactive hydroxyl radicals, increased lipid peroxidation ‎and cellular damage (Tiwari et al. 2014 ). There is also a correlation between oxygen species (ROS) production and asthma severity since ROS ‎production is heightened in asthmatic patients BALF ‎ (Sahiner et al. 2018 ). Our study revealed that treatment with Ala reduced MDA levels in the BALF to levels, to those observed ‎in the control group.‎ Oxidative stress arises from an imbalance between the production of free radicals and their removal by antioxidants (Giulia et al. 2017). SOD is a crucial endogenous enzymatic antioxidant, and studies have shown reduced SOD activity in the BALF and airway epithelial cells of asthmatic patients (Comhair and Erzurum 2010 ). GPx is another important antioxidant enzyme in the lungs, and its activity has been found to be reduced in asthma (Rahman et al. 2006 ). Decreased GPx levels can inhibit the production of Th1-dependent cytokines while promoting Th2-related responses (Peterson et al. 1998 ). In our study, we observed a significant decrease in GPx activity in the OVA group compared to the control group. However, GPx activity in the Ala and Dexa treatment groups was similar to that in the control group. This suggests that Ala may be beneficial in the treatment of asthma by restoring the balance of oxidative stress. The mechanisms by which Ala regulates SOD and GPx activities likely involve its antioxidant properties (Li P, Chen X-R, Xu F, et al Hekmat AS,),. Ala has been shown to reduce oxidative stress, which in turn could lead to the preservation and enhancement of antioxidant enzyme activities. By reducing the oxidative burden, Ala may prevent the inactivation of SOD and GPx, thereby maintaining their levels and activities. Furthermore, by decreasing the levels of pro-inflammatory cytokines such as TNF-α and IL-6, Ala may reduce the oxidative stress induced by inflammation. Pro-inflammatory cytokines can generate ROS and deplete antioxidant defenses, so their reduction through Ala treatment could help restore the balance of oxidative stress and improve the activities of SOD and GPx. Considering that the production and release of pro-inflammatory cytokines play a pivotal role in inflammation and airway remodeling in asthma (Hough et al. 2020 ), we measured the levels of TNF-α, IL-1β, and IL-6, tree important pro-inflammatory cytokines, in the BALF of asthmatic mice (Bejeshk et al. 2019). We found that the levels of these cytokines were increased in the BALF of asthmatic mice, while treatment with Ala led to a decrease in their levels. Ala has been shown to reduce the levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in other studies (Hekmat et al. 2021 ). IL-6 plays an important role in various aspects of asthma pathology, such as airway remodeling, mucus overproduction, and inflammatory cell recruitment (Neveu et al. 2010). Administration of Ala resulted in a significant reduction in IL-6 levels in BALF, suggesting its potential to regulate IL-6 production or release, subsequently reducing airway inflammation. This reduction in IL-6 may help improve asthma symptoms and decrease airway remodeling. TNF-α, a cytokine involved in systemic inflammation, has been implicated in the pathogenesis of asthma, particularly in airway remodeling and bronchial hyperresponsiveness (Doeing and Solway 2013 ). TNF-α is implicated in the initiation of airway inflammation, airway hyperreactivity, and the promotion of profibrotic processes in allergic asthma. It is also involved in the recruitment of neutrophils and eosinophils in asthma (Rana et al. 2016 ). TNF-α promotes the expression of adhesion molecules on endothelial cells, facilitating the recruitment of inflammatory cells to the airways, and enhances the proliferation and survival of airway smooth muscle cells, contributing to airway remodeling and obstruction. Blocking the activity of TNF-α has been considered a potential therapeutic option in asthma (Babu et al. 2004). The decreased level of TNF-α observed in our study with Ala treatment suggests a broader anti-inflammatory effect of Ala on asthma. Our histopathological data further support these findings, showing that Ala prevents lung inflammation, bronchial wall thickening, and alveolar edema. The reduction in bronchial wall thickening and alveolar edema might be attributed to the direct effect of Ala on the smooth muscles of the bronchial wall. Smooth muscle cells are the main structural cells of the bronchi, and their remodeling is a crucial factor in airway obstruction. Ala’s effects could be mediated through a decrease in the expression of cell adhesion molecules and receptors for cytokines such as TNF-α on these smooth muscle cells. Additionally, the observed decrease in inflammatory cell infiltration and oxidative stress might also play a significant role in the protective effects of Ala. Limitations and Future Directions Although this study provides valuable insights into the potential therapeutic effects of alamandine (Ala) in allergic airway inflammation, several limitations should be acknowledged. The study was conducted in a murine model, which may not fully replicate the complex pathophysiology of human asthma. Only a single dose and route of administration were used, and the duration of treatment was relatively short. Future studies should explore dose-response relationships, different routes of administration, and long-term safety profiles. Moreover, while both Ala and dexamethasone were evaluated, possible pharmacological interactions between Ala and standard asthma therapies were not assessed. Additional investigations using larger animal cohorts and mechanistic studies focusing on pathways such as NF-κB, PI3K/Akt, and MAPK will help to better elucidate the molecular basis of Ala’s protective actions and support its translational potential in asthma management. In conclusion, our study demonstrates that alamandine (Ala) alleviates airway inflammation and oxidative stress in an OVA-induced asthma model. These effects appear to be associated with the downregulation of pro-inflammatory cytokines such as IL-6 and TNF-α, as well as the modulation of NF-κB activity. The observed improvements in inflammatory and histopathological parameters suggest that Ala may play a protective role in allergic airway inflammation. However, these findings are limited to an experimental animal model and should be interpreted with caution. Further investigations using different doses, longer treatment durations, and clinical studies are required to validate these results and better understand the underlying molecular mechanisms of Ala’s action. Collectively, our data provide preliminary evidence that Ala has potential as a complementary approach in asthma therapy, pending further confirmation through mechanistic and translational research. Figure 8 provides a summary of the impact of Ala on asthma induced by OVA, illustrating the multifaceted approach through which Ala mitigates airway inflammation and remodeling. In conclusion, our study unveils a novel action of the peptide Ala in reducing allergic lung inflammation. The data obtained from our experiments demonstrate that treatment with Ala in mice with allergic lung inflammation fulfills all the criteria to be considered a novel therapeutic intervention. Moreover, when combined with previous findings showcasing Ala's significant anti-inflammatory effects, these results underscore the potential for developing new pharmacological strategies based on Ala to control, prevent, and treat chronic inflammation-related diseases, including asthma. The promising therapeutic properties of Ala in mitigating airway inflammation, reducing cytokine levels, regulating eosinophil recruitment, and influencing NF-κB expression make it an attractive candidate for further investigation and potential clinical applications. Future studies exploring the precise mechanisms of Ala's actions and its safety and efficacy in human subjects will be essential to fully realize its potential as a novel therapeutic agent for managing asthma and other inflammatory diseases. Declarations Author contribution: The experimental design and conception were carried out by A.S.H. and K.J. A.A., H.SH and A.S.H. ‎‎were responsible for conducting the research. Data analysis was performed by A.S.H. and K.J., ‎and they also wrote the manuscript. All authors have thoroughly reviewed and given approval for ‎the final version of the manuscript.‎ All data were generated in-house, and no paper mill was used. Funding The authors disclose that they have received financial support for the research, authorship, and publication of this article. The study was funded by Fasa University of Medical Sciences, with grant number 401121. Data availability The datasets generated during and/or analyzed during the current study are available from the ‎corresponding author on reasonable request.‎ Ethical Approval This study obtained approval from the Ethics Committee of The Fasa University of Medical Sciences, and all procedures were meticulously conducted in strict adherence to the approved guidelines (IR. FUMS.AEC1402.001). Conflict of Interest The authors declare that they have no conflict of interest. Funding This research was financially supported by the Research Center of Fasa University of Medical Sciences, Fasa, Iran. The funding body had no role in the design of the study, data collection, analysis, interpretation of data, or in writing the manuscript. References P. Subbarao, P. J. Mandhane, and M. R. Sears, “Asthma: Epidemiology, etiology and risk factors,” Canadian Medical Association Journal, vol. 181, no. 9, pp. E181–E190, 2009. 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2","display":"","copyAsset":false,"role":"figure","size":60101,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of alamandine on OVA-induced eosinophil count in BALF. Twenty-four hours ‎after the last challenge, eosinophil collected from BALF were counted. The data is presented as ‎mean ± SD, n=7. Statistical comparisons were carried out through a one-way analysis of variance ‎‎(ANOVA), followed by Tukey's post hoc tests. The levels of significance are denoted as ***P \u0026lt; ‎‎0.001.‎\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/0bc40b71406f9cc72948b1fb.png"},{"id":97673173,"identity":"55083801-7f0b-4d30-b9e5-a28308411f6f","added_by":"auto","created_at":"2025-12-08 09:39:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101919,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of Alamandine on Cytokine Levels in the BALF of OVA-Induced Asthma Mice. The administration of 50 µg/kg/day of alamandine effectively reduced cytokine levels in OVA-induced asthma mice. Data is presented as mean ± SD, with a sample size of n=7. Statistical analyses involved a one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. Significance levels are denoted as ‎*P\u0026lt;0.05, ‎**P\u0026lt;0.01 and ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/224b04b33cf7d881e1d920a3.jpg"},{"id":97553555,"identity":"f5d28c2b-7761-4adf-9905-1d5b5b466114","added_by":"auto","created_at":"2025-12-05 17:59:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49135,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of Alamandine on TH2 Cytokine Levels in OVA-Induced Asthmatic Mice. ‎Treatment with 50 µg/kg/day of alamandine significantly decreased the levels of IL-4, IL-5, and ‎IL-13 in OVA-sensitized mice. Group comparisons were conducted using a one-way Analysis of ‎Variance (ANOVA) followed by Tukey's post-hoc test. Significance is indicated by **P\u0026lt;0.01 and ‎‎***P\u0026lt;0.001.‎\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/eacc966f44959411aa6f311c.jpg"},{"id":97553556,"identity":"713fc817-3077-4256-8c2e-552cf173588e","added_by":"auto","created_at":"2025-12-05 17:59:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":83266,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of alamandine on serum IgE levels. Treatment with 50 µg/kg/day of alamandine ‎significantly reduced IgE levels in OVA-sensitized mice. Data is presented as mean ± SD, n=7. ‎Statistical comparisons were carried out using a one-way analysis of variance (ANOVA) followed ‎by Tukey's post-hoc tests. Significance levels are denoted as *P\u0026lt;0.01 and ***P\u0026lt;0.001‎\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/6abf9baee861e4118372fb01.png"},{"id":97672545,"identity":"17ec52a3-c8ad-4e91-9c6c-cb1bc2054ddf","added_by":"auto","created_at":"2025-12-08 09:38:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46642,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of alamandine on malondialdehyde (MDA), superoxide dismutase (SOD), and ‎glutathione peroxidase (GPX) Levels in BALF of Rats. Significance is denoted as ‎*P\u0026lt;0.01‎, ‎‎**P\u0026lt;0.01 and ***P\u0026lt;0.01.‎\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/b768f741aadcd49006eb915b.jpg"},{"id":97553570,"identity":"b0c05f30-1e01-4033-a5f4-953b5dd5b06d","added_by":"auto","created_at":"2025-12-05 17:59:50","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":280172,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of alamandine on histopathological changes in the lung tissues of OVA-Induced ‎asthmatic mice In Vivo. The results revealed a substantial increase in lung inflammation, bronchial ‎wall thickness, and alveolar edema in OVA-Induced asthmatic mice, and these effects were ‎mitigated by alamandine. Significance is indicated as ‎*P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.01.‎Top of Form\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/ed9e8acd06177a477a60418a.jpg"},{"id":97553572,"identity":"78a79f14-d9b1-40ca-8407-19a7c76eee9d","added_by":"auto","created_at":"2025-12-05 17:59:51","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":50445,"visible":true,"origin":"","legend":"\u003cp\u003eThe figure illustrates the effects of Ala on OVA-induced asthma.‎\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/8aba9e037901c277a18d2c4f.jpg"},{"id":97678647,"identity":"eaff234f-58b7-42ce-b961-5a6ab6e59fd3","added_by":"auto","created_at":"2025-12-08 09:55:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1426041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7720698/v1/dd9f8c54-b6db-4dd1-ad1c-f33ab7d36c06.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eAlamandine Mitigates Inflammation and Oxidative Stress in OVA-Induced Asthma: A Novel Therapeutic Approach\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAsthma, a chronic inflammatory disease affecting over 300\u0026nbsp;million individuals globally (Subbarao et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), is influenced by various factors and is recognized by recurring wheezing, difficulty breathing, chest \u0026lrm;tightness and coughing particularly at night or, in the early morning\u0026lrm; (Rodr\u0026iacute;guez-Torres et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Airway hyperresponsiveness (AHR) is a characteristic of asthma\u0026lrm; (Brannan \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The inhalation of substances like house dust, pollen, animal dander, and smoking trigger the development of asthma, leading to an immune imbalance with an increase in T helper type 2 (Th2) cells in the airway (Perret et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Th2 cytokines, such as interleukin (IL)-4, IL-5, and IL-13, play a critical role in allergic airway inflammation's development and maintenance (Peebles and Aronica \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These cytokines recruit and activate eosinophils, which cause persistent inflammation, airway hyperresponsiveness, and excessive mucus production in allergic asthma ((Schatz and Rosenwasser \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Consequently, targeting the suppression of Th2 cytokines has been identified as a crucial approach for managing allergic asthma ((Leόn 2017; Gandhi et al. 2018).\u003c/p\u003e\u003cp\u003eFurthermore, the prevalence of this respiratory condition is influenced by various other factors. One significant factor is the nuclear factor-kappa B (NF-κB), a transcription factor that plays a central role in regulating inflammatory responses (Zhang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). NF-κB can be activated by different stimuli, leading to its translocation into the nucleus, where it binds to DNA and controls the transcription of genes involved in asthmatic responses. This includes genes related to Th2 cytokines and mucin 5AC (MUC5AC), which is associated with excessive mucus production in the airways (Cai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnother factor that plays a role in in the pathogenesis of asthma is oxidative stress. Inflammation induces a lung oxidative stress reaction, resulting in the production of reactive oxygen species (ROS) (Andreadis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Reactive oxygen species have various effects on the pathogenesis of asthma, including the stimulation of pulmonary function impairment, mast cell degranulation, airway remodeling, and mucus secretion by the epithelium. These processes can exacerbate local lung inflammation (Zhang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The imbalance between oxidants and antioxidants is significant in the recurrence of airway inflammation in asthma. Both endogenous and exogenous oxidants stimulate inflammatory responses by amplifying the pro-inflammatory signaling pathways (Nadeem et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCytokines, which are proteins produced by types of cells have a crucial role in coordinating, maintaining \u0026lrm;and boosting inflammatory responses, in asthma \u0026lrm; (Mahajan and Mehta \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These substances help regulate inflammation in the airways leading to increased activity of the muscles \u0026lrm;surrounding the airways and contributing to bronchial constriction (Mahajan and Mehta \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β contribute to increased airway inflammation and severity of asthma (Barnes \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIL-1β specifically plays a role in the development of inflammation in asthma. It does so by stimulating \u0026lrm;the production of cytokines activating cell adhesion molecules and triggering eosinophil activation. \u0026lrm;Research has shown that patients with asthma have elevated levels of IL-1β and TNF-α \u0026lrm;in their \u0026lrm;bronchoalveolar lavage fluid \u0026lrm; (BALF) \u0026lrm;(Mahajan and Mehta \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Currently the main treatments for asthma are inhalation and bronchodilators. However, these \u0026lrm;medications come with side effects when used long term and can impose a substantial financial \u0026lrm;burden on patients\u0026lrm; (Klimek et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, \u0026lrm;5\u0026ndash;10% \u0026lrm;of severe asthma patients do not respond well to these therapies and prolonged \u0026lrm;use can lead to serious adverse effects \u0026lrm; (Mosnaim, md et al. 2022). Therefore, it is crucial to identify a treatment option, for asthma.\u0026lrm;\u003c/p\u003e\u003cp\u003eOxidative damage plays a pivotal role in the pathogenesis of bronchial asthma, making it a potential therapeutic target for the treatment of asthmatic patients. A clinical study has demonstrated the usefulness of antioxidants in managing mild to moderate asthma (van Toan and Hanh \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, numerous studies have focused on discovering novel compounds with enhanced \u0026lrm;antioxidant activity. One such compound may be almandine) Ala (, which possesses anti-inflammatory, \u0026lrm;cardioprotective, and antioxidant properties(Hekmat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ala is a newly identified member of the angiotensin family and has shown significant cardioprotective effects in rats treated with isoproterenol. This peptide shares similarities with Ang-(1\u0026ndash;7) and binds to the Mas-related G-coupled receptor known as member D (MrgD). Ala effectively mitigated monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. It achieved this by reducing oxidative stress and inflammation, regulating inducible nitric oxide synthase, and ameliorating histopathological changes. In ventricles subjected to ischemia and reperfusion (I/R), Ala upregulates the expression of several antioxidant proteins, including SOD and catalase. Additionally, it downregulates the expression of caspase-9, Bax, and caspase-3 in I/R ventricles (Lautner et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003ea). Furthermore, Ala reduces the levels of IL-1β and TNF-α in neonatal cardiomyocytes following an increase induced by LPS treatment (De Souza-Neto et al. 2019). Moreover, Ala significantly mitigates DOX-induced cardiotoxicity in rats by modulating antioxidant status, apoptosis, and inflammatory cytokines (Hekmat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, this study aims to investigate the therapeutic potential of Ala in asthma by examining its protective properties against ovalbumin (OVA)-induced allergic asthma in mice, with a focus on oxidative stress and inflammatory factors.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eThe animals used in this study were provided by the Animal Laboratory of Fasa University of Medical Sciences. Thirty-five male mice, each weighing approximately 20 grams and confirmed to be free from murine-specific pathogens, were housed in a standard laboratory environment (temperature: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, humidity: 50\u0026ndash;60%, with a 12:12-hour light/dark cycle, lights on at 7:00 AM). The mice were grouped in sets of five and had unrestricted access to standard laboratory chow and water. All experimental procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Fasa University of Medical Sciences (ethical code: FUMS.AEC.1402.001).\"\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental protocols\u003c/h2\u003e\u003cp\u003eThe animals were assigned to five groups (n\u0026thinsp;=\u0026thinsp;7) based on random allocation to meet the research \u0026lrm;requirements \u0026lrm;(Lautner et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e1. Control group: In the control group, mini-osmotic pumps (model 1002; ALZET Osmotic Pumps, CA, USA) infused saline at a rate of 0.25 \u0026micro;l/h, replicating the administration method utilized for the experimental groups. Furthermore, mice in this group received an intraperitoneal (ip) injection of saline to ensure uniformity with the injection protocol applied in the other groups.\u003c/p\u003e\u003cp\u003e\u0026lrm;2. OVA group: \u0026lrm;OVA-sensitized mice injected intraperitoneally with 20 \u0026micro;g of OVA (BP106-Genesco) plus 2 mg of Al (OH)3 on day 0, 7 and 14 and and OVA-challenged on day 22\u0026ndash;28 with inhalation of \u0026lrm; \u0026lrm;1% OVA \u0026lrm; solution using a nebulizer for a duration of 30 minutes.\u0026lrm;\u003c/p\u003e\u003cp\u003e3. Ala Group: Mice in this category received Ala (Phoenix Pharmaceuticals Inc., CA, USA) for 14 days from day 15 to day 28 through mini-osmotic pumps at a dosage of 50 \u0026micro;g Ala/kg/day.\u003c/p\u003e\u003cp\u003e4. OVA\u0026thinsp;+\u0026thinsp;Ala group: Mice in this category underwent a treatment protocol, combining sensitization with OVA through intraperitoneal injections of 20 \u0026micro;g OVA plus 2 mg of Al (OH)3 on days 0, 7, and 14. Subsequently, The OVA-challenge was administered through inhalation of a 1% OVA solution using a nebulizer for a duration of 30 minutes from days 22 to 28. Concurrently, Ala was administered s via mini-osmotic pumps at a dosage of 50 \u0026micro;g Ala/kg/day for the entire 14-day period, spanning from day 15 to day 28.\u003c/p\u003e\u003cp\u003e5. OVA +\u0026lrm; Dex group: This experimental group was similar to the Ala\u0026thinsp;+\u0026thinsp;OVA Group, with one distinction: instead of Ala, Dexa was administered at a dosage of 2 mg/kg (Zhang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The treatment protocol is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCollection of BALF\u003c/h3\u003e\n\u003cp\u003eTo collect BALF, the mice were euthanized by administering an overdose of pentobarbital at a dosage of 50 mg/kg, 24 hours after the final OVA challenge. Tracheotomy was performed, and ice-cold phosphate-buffered saline (PBS) (0.5 mL) was instilled into the lungs. The fluid was collected by three successive aspirations via tracheal cannulation, resulting in a total volume of 1.5 mL (Zhang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Subsequently, BALF samples were centrifuged at 1500 rpm for 10 minutes at 4\u0026deg;C. The supernatants were then stored at -80\u0026deg;C for further analysis.\u003c/p\u003e\n\u003ch3\u003eBALF and Eosinophil Counts\u003c/h3\u003e\n\u003cp\u003eThe sediment acquired from BALF underwent resuspension in 200 \u0026micro;L of phosphate-buffered saline. Following this, a smear was meticulously prepared from the cellular constituents, and ensuing staining was performed utilizing the Wright\u0026ndash;Giemsa method. Subsequent to staining, a differential cell analysis, guided by staining and morphological criteria, was systematically carried out using a light microscope\u003c/p\u003e\n\u003ch3\u003eEnzyme-Linked Immunosorbent Assay (ELISA) Detection of Cytokines in BALF\u003c/h3\u003e\n\u003cp\u003eELISA was performed using specific ELISA kits for mice IL-4, IL-5, IL-13, TNF-α, IL-1β, IL-6 and NF-κB (Zell Bio Company, Germany) following the manufacturer's instructions. Briefly, 100 \u0026micro;L of standard blank or sample was added to appropriate wells and incubated for 1 hour at 37\u0026deg;C. After aspiration, 100 \u0026micro;L of prepared detection reagent was added to each well and incubated for 1 hour at 37\u0026deg;C. Following aspiration and washing, 90 \u0026micro;L of substrate solution was added to the wells and incubated for 15 minutes at 37\u0026deg;C. Finally, 50 \u0026micro;L of stop solution was added, and the optical density (OD) was immediately measured at 450 nm using a BioTek ELISA reader. The cytokine concentrations were determined by comparing the OD of the samples to the standard curve.\u003c/p\u003e\n\u003ch3\u003eMeasurement of Oxidative Stress in BALF\u003c/h3\u003e\n\u003cp\u003eOxidative stress markers, including MDA (malondialdehyde), SOD (superoxide dismutase), and GPx (glutathione peroxidase), were assessed in BALF using commercially available kits according to the manufacturer's instructions. The MDA levels were determined by the TBA method, which involves the condensation of lipid hydroperoxide decomposition products with TBA, forming red compounds with an absorption peak at 532 nm. SOD activity was quantified by measuring the conversion of superoxide anion into hydrogen peroxide and oxygen with absorbance at 420 nm. The GPx assay kit involved the catalytic oxidation of glutathione by cumene hydroperoxide accomplished by GPx, and the decrease in absorption was measured at 340 nm using a spectrophotometer in the presence of NADPH and glutathione reductase.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of IgE Levels in Serum\u003c/h2\u003e\u003cp\u003eThe levels of IgE in the serum were measured by ELISA following the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLung tissue histopathology\u003c/h3\u003e\n\u003cp\u003eThe left lungs were washed with PBS, fixed in 4% paraformaldehyde, and dehydrated using an alcohol gradient. Subsequently, the lungs were rendered transparent with xylene. After embedding in paraffin, 5 \u0026micro;m slices were obtained and stained with hematoxylin and eosin (H\u0026amp;E) and periodic acid-Schiff (PAS) solution. The stained sections were examined using an optical microscope. H\u0026amp;E staining was utilized to evaluate pathological changes in lung tissue, and a score ranging from 0 to 5 was assigned to assess the extent of lung tissue inflammation. PAS staining was employed to evaluate the degree of airway goblet cell proliferation ([CSL STYLE ERROR: reference with no printed form.]).\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eData analysis was performed using GraphPad Prism software. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Group comparisons were conducted through a one-way Analysis of Variance (ANOVA) followed by Tukey's post-hoc test. A significance level of P\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered indicative of statistical significance in all analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003eEffect of Ala on eosinophils in BALF\u003c/h2\u003e\u003cp\u003eThe administration of OVA significantly elevated the eosinophil count to 78.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.69 cells/mm\u0026sup3;, in stark contrast to the control group's count of 5.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 cells/mm\u0026sup3; (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with Ala led to a notable reduction in eosinophil count, bringing it down to 41.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03 cells/mm\u0026sup3; in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a similar efficacy as Ala, resulting in an eosinophil count of 43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50 cells/mm\u0026sup3; in the Asthma\u0026thinsp;+\u0026thinsp;Dexa group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e3 / 3\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Ala on proinflammatory cytokines and NF-κB in BALF:\u003c/h2\u003e\u003cp\u003eFollowing OVA administration, IL-6 levels significantly increased to 46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71 pg/mL, representing a notable surge compared to the control group's 12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment led to a considerable reduction in IL-6 levels, bringing them down to 23.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a similar impact to Ala, showing IL-6 levels of 26.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eIn addition, IL-1β levels also experienced a significant increase to 172\u0026thinsp;\u0026plusmn;\u0026thinsp;12.9 pg/mL following OVA administration, contrasting with the control group's 78.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.2 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment resulted in a reduction of IL-1β levels to 108\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone exhibited a comparable effect to Ala, with IL-1β levels of 107\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eFurthermore, TNF-α levels significantly increased to 132\u0026thinsp;\u0026plusmn;\u0026thinsp;34.0 pg/mL after OVA administration, in contrast to the control group's 59.6\u0026thinsp;\u0026plusmn;\u0026thinsp;30.0 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment led to a marked decrease in TNF-α levels, reducing them to 13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone exhibited a similar effect to Ala, with TNF-α levels of 9.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.65 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eMoreover, NF-κB levels significantly rose to 3.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.419 pg/mL after OVA administration, compared to the control group's 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.286 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment led to a reduction in NF-κB levels to 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0881 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a similar effect to Ala, with NF-κB levels of 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.127 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Ala on Th2 cytokines in BALF:\u0026lrm;\u003c/h2\u003e\u003cp\u003eThe administration of OVA led to a significant elevation of IL-4 levels, reaching 115\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6 pg/mL, in comparison to the control group's 53.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment effectively reduced IL-4 levels to 54.8\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone exhibited a similar effect to Ala, demonstrating IL-4 levels of 57.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.67 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eIn the context of OVA administration, IL-5 levels experienced a substantial increase, reaching 356\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4 pg/mL, compared to the control group's 172\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The introduction of Ala effectively reduced IL-5 levels to 261\u0026thinsp;\u0026plusmn;\u0026thinsp;25.6 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a comparable effect to Ala, with IL-5 levels of 234\u0026thinsp;\u0026plusmn;\u0026thinsp;22.0 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eThe administration of OVA also resulted in a significant elevation of IL-13 levels, reaching 181\u0026thinsp;\u0026plusmn;\u0026thinsp;12 pg/mL, in comparison to the control group's 57\u0026thinsp;\u0026plusmn;\u0026thinsp;6.82 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment effectively reduced IL-13 levels to 153\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone exhibited a similar effect to Ala, with IL-13 levels of 133\u0026thinsp;\u0026plusmn;\u0026thinsp;13.9 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEffect of Ala on the IgE level in serum\u003c/h2\u003e\u003cp\u003eOVA administration led to a significant increase in IgE levels, reaching 266\u0026thinsp;\u0026plusmn;\u0026thinsp;23.2 pg/mL, in contrast to the control group's 158\u0026thinsp;\u0026plusmn;\u0026thinsp;16.7 pg/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment effectively reduced IgE levels to 214\u0026thinsp;\u0026plusmn;\u0026thinsp;37.7 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a similar effect to Ala, with IgE levels measuring 214\u0026thinsp;\u0026plusmn;\u0026thinsp;13.1 pg/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eEffect of Ala on oxidative stress in BALF:\u0026lrm;\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eOVA administration resulted in a significant increase in MDA levels, reaching 13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89 \u0026micro;M\u0026mdash;a substantial surge compared to the control group's 0.707\u0026thinsp;\u0026plusmn;\u0026thinsp;0.231 \u0026micro;M (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala intervention effectively decreased MDA levels to 5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 \u0026micro;M in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a comparable yet distinct effect to Ala, showing MDA levels of 4.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 \u0026micro;M in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eOn the contrary, OVA administration resulted in a significant reduction in SOD activity to 7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 IU/mL\u0026mdash;a marked decrease compared to the control group's 81.7\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8 IU/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment elicited an elevation in SOD activity to 36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75 IU/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone exhibited a similar effect to Ala, with SOD activity measuring 47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73 IU/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group.\u003c/p\u003e\u003cp\u003eIn another aspect, OVA administration significantly decreased GPX activity to 0.046\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0061 U/mL\u0026mdash;a notable decline compared to the control group's 0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 U/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Ala treatment induced an increase in GPX activity to 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032 U/mL in the OVA\u0026thinsp;+\u0026thinsp;Ala group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Dexamethasone demonstrated a similar yet discernible effect to Ala, with GPX activity measuring 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0097 U/mL in the OVA\u0026thinsp;+\u0026thinsp;Dexa group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEffects of Ala on histopathologic changes in the lung:\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e depicts a cross-section of mouse lung tissue stained with Hematoxylin and Eosin. The results obtained regarding the level of lung inflammation revealed that the OVA group exhibited the highest level of inflammation. Inflammatory indicators, such as lymphocyte secretion, presence of neutrophils, and tissue changes including epithelial cell accumulation, were significantly higher in this group compared to the other studied groups. Moreover, in this group, there was an observed increase in the thickness of the alveolar wall's epithelial layer, which comprises two types of pneumocytes cells (type 1 and 2) responsible for covering and secreting surfactant. This increase led to a reduction in the exchange of respiratory gases and an elevation in pulmonary secretions, ultimately resulting in pulmonary edema. Conversely, in the OVA\u0026thinsp;+\u0026thinsp;Ala and OVA\u0026thinsp;+\u0026thinsp;Dexa groups, these changes showed a significant decrease compared to the OVA group. The OVA\u0026thinsp;+\u0026thinsp;Ala group exhibited reduced lung inflammation, mucus secretion, intrapulmonary hemorrhage, and thickening of alveolar walls, along with a decreased level of inflammatory cell infiltration compared to the untreated OVA group. Similar improvements were observed in the OVA\u0026thinsp;+\u0026thinsp;Dexa group. The examination of images from the control group revealed normal bronchioles with ciliated cells and a regular appearance of the lung tissue. The alveoli were found to have a single layer of pneumocytic cells. Among these cells, type 1 alveolar cells, constituting approximately 95% of the alveolar surface and forming a barrier between the air and alveolar walls, were observed as thin pavement-like cells. Additionally, type 2 alveolar cells, responsible for surfactant secretion, appeared normal in this group. Overall, the tissue appeared completely normal, without any signs of thickening or evidence of cellular metaplasia.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAsthma is a chronic inflammatory condition characterized by the infiltration of eosinophils and other inflammatory cells, leading to bronchial hyper-responsiveness and airway obstruction (Calderon et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The increasing prevalence of asthma has driven extensive research into the discovery of new anti-asthmatic agents. Previous investigations have indicated that Ala possesses antioxidant and anti-inflammatory properties (Soltani Hekmat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hence, the primary aim of this study was to evaluate the impact of Ala administration on inflammation in the context of asthma. Our results demonstrate that treatment with Ala effectively reduced the accumulation of eosinophils and inflammatory cytokines in the BALF, leading to mitigated pulmonary remodeling. During the development of allergic asthma, there is typically a dominant Th2 immune response. Th2 cells secrete inflammatory cytokines, including IL-4, IL-5, IL-9, and IL-13, with IL-4 and IL-13 playing crucial regulatory roles in asthma. These cytokines significantly contribute to airway inflammation by promoting the production of IgE and mucus by B cells, inducing remodeling of epithelial cells, activating M2 macrophages, and binding to IgE FcεRI receptors on mast cells and basophils, resulting in the release of histamine and leukotrienes. IL-5 plays a pivotal role in eosinophil differentiation, maturation, chemotaxis, and activation, often working in conjunction with IL-4 to increase eosinophil levels in individuals with allergic asthma(Le\u0026oacute;n and Ballesteros-Tato \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur findings revealed a significant reduction in serum IgE levels in OVA-sensitized and challenged mice following treatment with Ala. Previous studies have highlighted the crucial role of the IL-4 cytokine in initiating isotype class switching, which is necessary for B cells to produce IgE (Vazquez et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Considering that Ala was shown to decrease interleukin levels in this study, and previous research has also demonstrated its ability to reduce cytokines (Hekmat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Soltani Hekmat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), it is plausible that the decrease in interleukins mediated by Ala contributed to the reduction in IgE levels.\u003c/p\u003e\u003cp\u003eMoreover, we observed an increase in cytokine levels in the BALF of mice treated with OVA compared to the control group. Yan et al. have previously reported that Th2 cells in the lungs of asthmatic patients secrete significant amounts of IL-4, IL-5, and IL-13 (Yan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, treatment with Ala effectively reduced the elevated cytokine levels, similar to the effect of Dexa. Dexa, a glucocorticoid drug, exerts potent anti-inflammatory effects against bronchial asthma by inhibiting the production of IL-4, IL-5, and IL-13, which are important cytokines in asthma (Westergaard et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Numerous studies have demonstrated that Th2-mediated cytokines, including IL-4, IL-5, and IL-13, play crucial roles in regulating and exacerbating the inflammatory response in asthma (Thakur et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Menzella et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, another study reported that asthmatic mice lacking the IL-13 receptor exhibited significantly less airway remodeling compared to mice with wild-type asthma (Chen et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Hence, it can be suggested that one of the mechanisms by which Ala improved asthma in this study was through the reduction of these cytokines. The ability of Ala to target and decrease these pro-inflammatory cytokines highlights its potential as a promising therapeutic agent for asthma treatment.\u003c/p\u003e\u003cp\u003eStudies have reported a correlation between the number of eosinophils in the peripheral blood and bronchial lavage of asthmatic patients and the severity of the disease (Louis et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In our study, we observed a significant increase in the percentage of eosinophils in the BALF of the OVA treatment group. However, this increase was prevented in the groups treated with Dexa and Ala. It has been suggested that the increase in eosinophils is associated with elevated production of IL-4, IgE, and TNF-α (Parlar and Arslan 2020). Another study reported that vitamin E treatment indirectly reduces IgE levels by suppressing the production of IL-4, which is involved in IgE production, and IL-5, which plays a role in eosinophil migration (Jiang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, it can be inferred that Ala treatment influences the percentage of eosinophils by reducing the levels of IgE, TNF-α, IL-4, and IL-5. Multiple studies have highlighted the activation of the NF-κB pathway in asthmatic tissues, and gene-deficient mouse models of allergic asthma have underscored the importance of NF-κB in the pathogenesis of the disease (Haeberle et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn asthma, the activation of NF-κB leads to increased expression of genes responsible for producing inflammatory cytokines and chemokines, which, in turn, promotes the recruitment, activation, and survival of inflammatory cells (Sousa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; El-Hashim et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). There have been studies that strongly support the involvement of NF κB, a transcription factor in the \u0026lrm;development of asthma. It's particularly interesting to note that mice, without NF κB only experience \u0026lrm;lung inflammation highlighting the crucial role of this factor in the disease\u0026rsquo;s progression (El-Hashim et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Additionally inhibiting NF κB in a mouse model of asthma has shown results in reducing airway \u0026lrm;inflammation and improving health \u0026lrm; (Sousa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pinheiro and Sim\u0026otilde;es E Silva 2012). In our investigation we observed that treatment with Ala resulted in decreased levels of NF κB in the \u0026lrm;BALF. This suggests that Ala may contribute to resolving lung inflammation by regulating NF κB levels. \u0026lrm;This mechanism further strengthens the use of Ala as a treatment, for asthma since it can help control \u0026lrm;responses and lessen disease severity by targeting NF κB activation.\u0026lrm;\u003c/p\u003e\u003cp\u003eNumerous studies have highlighted the active participation of oxidative stress in the pathological development of asthma(Jiang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Oxidative stress is known to trigger damage, which contributes to the progress of asthma \u0026lrm;(Nesi et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The formation of MDA is widely recognized as an indicator of oxidative stress (T\u0026uuml;rk et al. 2022). \u0026lrm;). One mechanism that leads to MDA formation involves peroxidation, which is caused by oxygen \u0026lrm;radicals. Lipid peroxidation results in decreased membrane fluidity and impaired membrane function\u0026lrm; (Kuzu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study we observed a rise in MDA levels in the group treated with OVA compared to the control \u0026lrm;group. It has been reported that exposure to OVA disrupts balance and diminishes the defense \u0026lrm;mechanism resulting in the production of highly reactive hydroxyl radicals, increased lipid peroxidation \u0026lrm;and cellular damage (Tiwari et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). There is also a correlation between oxygen species (ROS) production and asthma severity since ROS \u0026lrm;production is heightened in asthmatic patients BALF \u0026lrm; (Sahiner et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our study revealed that treatment with Ala reduced MDA levels in the BALF to levels, to those observed \u0026lrm;in the control group.\u0026lrm; Oxidative stress arises from an imbalance between the production of free radicals and their removal by antioxidants (Giulia et al. 2017).\u003c/p\u003e\u003cp\u003eSOD is a crucial endogenous enzymatic antioxidant, and studies have shown reduced SOD activity in the BALF and airway epithelial cells of asthmatic patients (Comhair and Erzurum \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). GPx is another important antioxidant enzyme in the lungs, and its activity has been found to be reduced in asthma (Rahman et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Decreased GPx levels can inhibit the production of Th1-dependent cytokines while promoting Th2-related responses (Peterson et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In our study, we observed a significant decrease in GPx activity in the OVA group compared to the control group. However, GPx activity in the Ala and Dexa treatment groups was similar to that in the control group. This suggests that Ala may be beneficial in the treatment of asthma by restoring the balance of oxidative stress. The mechanisms by which Ala regulates SOD and GPx activities likely involve its antioxidant properties (Li P, Chen X-R, Xu F, et al Hekmat AS,),. Ala has been shown to reduce oxidative stress, which in turn could lead to the preservation and enhancement of antioxidant enzyme activities. By reducing the oxidative burden, Ala may prevent the inactivation of SOD and GPx, thereby maintaining their levels and activities. Furthermore, by decreasing the levels of pro-inflammatory cytokines such as TNF-α and IL-6, Ala may reduce the oxidative stress induced by inflammation. Pro-inflammatory cytokines can generate ROS and deplete antioxidant defenses, so their reduction through Ala treatment could help restore the balance of oxidative stress and improve the activities of SOD and GPx.\u003c/p\u003e\u003cp\u003eConsidering that the production and release of pro-inflammatory cytokines play a pivotal role in inflammation and airway remodeling in asthma (Hough et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), we measured the levels of TNF-α, IL-1β, and IL-6, tree important pro-inflammatory cytokines, in the BALF of asthmatic mice (Bejeshk et al. 2019). We found that the levels of these cytokines were increased in the BALF of asthmatic mice, while treatment with Ala led to a decrease in their levels. Ala has been shown to reduce the levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in other studies (Hekmat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). IL-6 plays an important role in various aspects of asthma pathology, such as airway remodeling, mucus overproduction, and inflammatory cell recruitment (Neveu et al. 2010). Administration of Ala resulted in a significant reduction in IL-6 levels in BALF, suggesting its potential to regulate IL-6 production or release, subsequently reducing airway inflammation. This reduction in IL-6 may help improve asthma symptoms and decrease airway remodeling. TNF-α, a cytokine involved in systemic inflammation, has been implicated in the pathogenesis of asthma, particularly in airway remodeling and bronchial hyperresponsiveness (Doeing and Solway \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTNF-α is implicated in the initiation of airway inflammation, airway hyperreactivity, and the promotion of profibrotic processes in allergic asthma. It is also involved in the recruitment of neutrophils and eosinophils in asthma (Rana et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). TNF-α promotes the expression of adhesion molecules on endothelial cells, facilitating the recruitment of inflammatory cells to the airways, and enhances the proliferation and survival of airway smooth muscle cells, contributing to airway remodeling and obstruction. Blocking the activity of TNF-α has been considered a potential therapeutic option in asthma (Babu et al. 2004). The decreased level of TNF-α observed in our study with Ala treatment suggests a broader anti-inflammatory effect of Ala on asthma. Our histopathological data further support these findings, showing that Ala prevents lung inflammation, bronchial wall thickening, and alveolar edema. The reduction in bronchial wall thickening and alveolar edema might be attributed to the direct effect of Ala on the smooth muscles of the bronchial wall. Smooth muscle cells are the main structural cells of the bronchi, and their remodeling is a crucial factor in airway obstruction. Ala\u0026rsquo;s effects could be mediated through a decrease in the expression of cell adhesion molecules and receptors for cytokines such as TNF-α on these smooth muscle cells. Additionally, the observed decrease in inflammatory cell infiltration and oxidative stress might also play a significant role in the protective effects of Ala.\u003c/p\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eLimitations and Future Directions\u003c/h2\u003e\u003cp\u003eAlthough this study provides valuable insights into the potential therapeutic effects of alamandine (Ala) in allergic airway inflammation, several limitations should be acknowledged. The study was conducted in a murine model, which may not fully replicate the complex pathophysiology of human asthma. Only a single dose and route of administration were used, and the duration of treatment was relatively short. Future studies should explore dose-response relationships, different routes of administration, and long-term safety profiles. Moreover, while both Ala and dexamethasone were evaluated, possible pharmacological interactions between Ala and standard asthma therapies were not assessed. Additional investigations using larger animal cohorts and mechanistic studies focusing on pathways such as NF-κB, PI3K/Akt, and MAPK will help to better elucidate the molecular basis of Ala\u0026rsquo;s protective actions and support its translational potential in asthma management.\u003c/p\u003e\u003cp\u003eIn conclusion, our study demonstrates that alamandine (Ala) alleviates airway inflammation and oxidative stress in an OVA-induced asthma model. These effects appear to be associated with the downregulation of pro-inflammatory cytokines such as IL-6 and TNF-α, as well as the modulation of NF-κB activity. The observed improvements in inflammatory and histopathological parameters suggest that Ala may play a protective role in allergic airway inflammation. However, these findings are limited to an experimental animal model and should be interpreted with caution. Further investigations using different doses, longer treatment durations, and clinical studies are required to validate these results and better understand the underlying molecular mechanisms of Ala\u0026rsquo;s action. Collectively, our data provide preliminary evidence that Ala has potential as a complementary approach in asthma therapy, pending further confirmation through mechanistic and translational research.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e provides a summary of the impact of Ala on asthma induced by OVA, illustrating the multifaceted approach through which Ala mitigates airway inflammation and remodeling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn conclusion, our study unveils a novel action of the peptide Ala in reducing allergic lung inflammation. The data obtained from our experiments demonstrate that treatment with Ala in mice with allergic lung inflammation fulfills all the criteria to be considered a novel therapeutic intervention. Moreover, when combined with previous findings showcasing Ala's significant anti-inflammatory effects, these results underscore the potential for developing new pharmacological strategies based on Ala to control, prevent, and treat chronic inflammation-related diseases, including asthma.\u003c/p\u003e\u003cp\u003eThe promising therapeutic properties of Ala in mitigating airway inflammation, reducing cytokine levels, regulating eosinophil recruitment, and influencing NF-κB expression make it an attractive candidate for further investigation and potential clinical applications. Future studies exploring the precise mechanisms of Ala's actions and its safety and efficacy in human subjects will be essential to fully realize its potential as a novel therapeutic agent for managing asthma and other inflammatory diseases.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe experimental design and conception were carried out by A.S.H. and K.J. A.A., H.SH and A.S.H. \u0026lrm;\u0026lrm;were responsible for conducting the research. Data analysis was performed by A.S.H. and K.J., \u0026lrm;and they also wrote the manuscript. All authors have thoroughly reviewed and given approval for \u0026lrm;the final version of the manuscript.\u0026lrm; All data were generated in-house, and no paper mill was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors disclose that they have received financial support for the research, authorship, and publication of this article. The study was funded by Fasa University of Medical Sciences, with grant number 401121.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the \u0026lrm;corresponding author on reasonable request.\u0026lrm;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study obtained approval from the Ethics Committee of The Fasa University of Medical Sciences, and all procedures were meticulously conducted in strict adherence to the approved guidelines (IR. FUMS.AEC1402.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the Research Center of Fasa University of Medical Sciences, Fasa, Iran. The funding body had no role in the design of the study, data collection, analysis, interpretation of data, or in writing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP. Subbarao, P. J. Mandhane, and M. R. Sears, \u0026ldquo;Asthma: Epidemiology, etiology and risk factors,\u0026rdquo; Canadian Medical Association Journal, vol. 181, no. 9, pp. E181\u0026ndash;E190, 2009.\u003c/li\u003e\n\u003cli\u003eRodr\u0026iacute;guez-Torres, J.; L\u0026oacute;pez-L\u0026oacute;pez, L.; Cabrera-Martos, I.; Torres-S\u0026aacute;nchez, I.; Prados-Rom\u0026aacute;n, E.; Ort\u0026iacute;z-Rubio, A.; Valenza, M.C. Symptom severity is associated with signs of central sensitization in patients with asthma. Clin. Respir. J. 2021, 15, 1219\u0026ndash;1226.\u003c/li\u003e\n\u003cli\u003eBrannan JD: Bronchial hyperresponsiveness in the assessment of asthma control: Airway hyperresponsiveness in asthma: its measurement and clinical significance. Chest. 138 (Suppl):11S\u0026ndash;17S. 2010. View Article: Google Scholar: PubMed/NCBI\u003c/li\u003e\n\u003cli\u003ePerret, J. L., Dharmage, S. C., Matheson, M. C., Johns, D. P., Gurrin, L. C., Burgess, J. A., et al. (2013). 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An evidence-based analysis of house dust mite allergen immunotherapy: a call for more rigorous clinical studies. J. Allergy Clin. Immunol. 132, 1322\u0026ndash;1336. doi: 10.1016/j.jaci.2013.09.004\u003c/li\u003e\n\u003cli\u003eLe\u0026oacute;n B, Ballesteros-Tato A. Modulating Th2 Cell Immunity for the Treatment of Asthma. Front Immunol. 2021 Feb 10; 12:637948. doi: 10.3389/fimmu.2021.637948. PMID: 33643321; PMCID: PMC7902894.\u003c/li\u003e\n\u003cli\u003eYang Z, Li X, Fu R, Hu M, Wei Y, Hu X, Tan W, Tong X, Huang F. Therapeutic Effect of Renifolin F on Airway Allergy in an Ovalbumin-Induced Asthma Mouse Model In Vivo. Molecules. 2022 Jun 12;27(12):3789. doi: 10.3390/molecules27123789. PMID: 35744915; PMCID: PMC9227769.\u003c/li\u003e\n\u003cli\u003eVazquez MI, Catalan-Dibene J, Zlotnik A. B cells responses and cytokine production are regulated by their immune microenvironment. Cytokine. 2015 Aug;74(2):318-26. doi: 10.1016/j.cyto.2015.02.007. Epub 2015 Mar 2. PMID: 25742773; PMCID: PMC4475485.\u003c/li\u003e\n\u003cli\u003eSoltani Hekmat A, Chenari A, Alipanah H, Javanmardi K. Protective effect of alamandine on doxorubicin-induced nephrotoxicity in rats. BMC Pharmacol Toxicol. 2021 May 29;22(1):31. doi: 10.1186/s40360-021-00494-x. PMID: 34049594; PMCID: PMC8164237.\u003c/li\u003e\n\u003cli\u003eYan S, Ci X, Chen N, Chen C, Li X, Chu X, Li J, Deng X. Anti-inflammatory effects of ivermectin in mouse model of allergic asthma. Inflamm Res. 2011 Jun;60(6):589-96. doi: 10.1007/s00011-011-0307-8. Epub 2011 Jan 29. PMID: 21279416.\u003c/li\u003e\n\u003cli\u003eWestergaard CG, Porsbjerg C, Backer V. The effect of Varenicline on smoking cessation in a group of young asthma patients. Respir Med. 2015 Nov;109(11):1416-22. doi: 10.1016/j.rmed.2015.07.017. Epub 2015 Jul 30. PMID: 26427627.\u003c/li\u003e\n\u003cli\u003eThakur VR, Khuman V, Beladiya JV, Chaudagar KK, Mehta AA. An experimental model of asthma in rats using ovalbumin and lipopolysaccharide allergens. Heliyon. 2019 Nov 19;5(11):e02864. doi: 10.1016/j.heliyon. 2019.e02864. PMID: 31768443; PMCID: PMC6872797.\u003c/li\u003e\n\u003cli\u003eMenzella F, Ruggiero P, Galeone C, Scelfo C, Bagnasco D, Facciolongo N. Significant improvement in lung function and asthma control after benralizumab treatment for severe refractory eosinophilic asthma. Pulm Pharmacol Ther. 2020 Oct; 64:101966. doi: 10.1016/j.pupt.2020.101966. Epub 2020 Oct 8. PMID: 33039666.\u003c/li\u003e\n\u003cli\u003eChen YC, Dong GH, Lin KC, Lee YL. Gender difference of childhood overweight and obesity in predicting the risk of incident asthma: a systematic review and meta-analysis. Obes Rev. 2013 Mar;14(3):222-31. doi: 10.1111/j.1467-789X.2012.01055. x. Epub 2012 Nov 12. PMID: 23145849.\u003c/li\u003e\n\u003cli\u003eLouis R, Leyder E, Malaise M, Bartsch P, Louis E. Lack of association between adult asthma and the tumour necrosis factor alpha-308 polymorphism gene. Eur Respir J. 2000 Oct;16(4):604-8. doi: 10.1034/j.1399-3003.2000.16d06. x. PMID: 11106199.\u003c/li\u003e\n\u003cli\u003eParlar A, Arslan SO. CB2 Agonist (AM1241) Improving Effect on Ovalbumin-Induced Asthma in Rats. Iran J Pharm Res. 2020 Winter;19(1):3-17. doi: 10.22037/ijpr.2019.1101002. PMID: 32922464; PMCID: PMC7462485.\u003c/li\u003e\n\u003cli\u003eJiang J, Mehrabi Nasab E, Athari SM, Athari SS. Effects of vitamin E and selenium on allergic rhinitis and asthma pathophysiology. Respir Physiol Neurobiol. 2021 Apr;286:103614. doi: 10.1016/j.resp.2020.103614. Epub 2021 Jan 7. PMID: 33422684.\u003c/li\u003e\n\u003cli\u003eHaeberle, H.A., R. Takizawa, A. Casola, A.R. Brasier, H.J. Dieterich, N. Van Rooijen, et al. 2002. Respiratory syncytial virus-induced activation of nuclear factor-kappaB in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways. Journal of Infectious Diseases 186: 1199\u0026ndash;1206\u003c/li\u003e\n\u003cli\u003eSousa LP, Lopes F, Silva DM, Tavares LP, Vieira AT, Rezende BM, et al. PDE4 inhibition drives resolution of neutrophilic inflammation by inducing apoptosis in a PKA-PI3K/Akt-dependent and NF-kappaB-independent manner. J Leukoc Biol (2010) 87(5):895\u0026ndash;904. doi:10.1189/jlb.0809540\u003c/li\u003e\n\u003cli\u003eEl-Hashim AZ, Renno WM, Raghupathy R, Abduo HT, Akhtar S, Benter IF. Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-kappaB-dependent pathways. Br J Pharmacol (2012) 166(6):1964\u0026ndash;76. doi:10.1111/j.1476-5381.2012. 01905.x\u003c/li\u003e\n\u003cli\u003eJiang J, Mehrabi Nasab E, Athari SM, Athari SS (2021) Effects of vitamin E and selenium on allergic rhinitis and asthma pathophysiology. Respir Physiol Neurobiol 286:103614. https://doi.org/10.1016/j.resp.2020.103614\u003c/li\u003e\n\u003cli\u003eR.T. Nesi, E. Kennedy-Feitosa, M. Lanzetti, et al. Inflammatory and oxidative stress markers in experimental allergic asthma Inflammation, 40 (4) (2017), pp. 1166-1176\u003c/li\u003e\n\u003cli\u003eT\u0026uuml;rk E, G\u0026uuml;ven\u0026ccedil; M, Cellat M, et al (2020) Zingerone protects liver and kidney tissues by preventing oxidative stress, inflammation, and apoptosis in methotrexate-treated rats. Drug Chem Toxicol 1\u0026ndash;12. https://doi.org/10.1080/01480545.2020.1804397\u003c/li\u003e\n\u003cli\u003eKuzu M, Yildirim S, Kandemir FM, et al (2019) Protective effect of morin on doxorubicin-induced hepatorenal toxicity in rats. Chem Biol Interact 308:89\u0026ndash;100. https://doi.org/10.1016/j.cbi.2019.05.017\u003c/li\u003e\n\u003cli\u003eTiwari M, Dwivedi UN, Kakkar P (2014) Tinospora cordifolia extract modulates COX-2, iNOS, ICAM-1, proinflammatory cytokines and redox status in murine model of asthma. J Ethnopharmacol 153:326\u0026ndash;337. https://doi.org/10.1016/j.jep.2014.01.031\u003c/li\u003e\n\u003cli\u003eSahiner UM, Birben E, Erzurum S, Sackesen C, Kalayci \u0026Ouml;. Oxidative stress in asthma: Part of the puzzle. Pediatr Allergy Immunol. 2018 Dec;29(8):789-800. doi: 10.1111/pai.12965. Epub 2018 Oct 1. PMID: 30069955.\u003c/li\u003e\n\u003cli\u003eNesi G, Sestito S, Digiacomo M, Rapposelli S. Oxidative Stress, Mitochondrial Abnormalities and Proteins Deposition: Multitarget Approaches in Alzheimer\u0026apos;s Disease. Curr Top Med Chem. 2017;17(27):3062-3079. doi: 10.2174/1568026617666170607114232. PMID: 28595557.\u003c/li\u003e\n\u003cli\u003eComhair SA, Erzurum SC. Redox control of asthma: molecular mechanisms and therapeutic opportunities. Antioxid Redox Signal. 2010; 12:93---124.\u003c/li\u003e\n\u003cli\u003eRahman I, Biswas SK, Kode A (2006) Oxidant and antioxidant balance in the airways and airway diseases. 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PMID: 30528469.\u003c/li\u003e\n\u003cli\u003eS. Rana, M. Shahzad, and A. Shabbir, \u0026ldquo;Pistacia integerrima ameliorates airway inflammation by attenuation of TNF\u0026alpha;, IL-4, and IL-5 expression levels, and pulmonary edema by elevation of AQP1 and AQP5 expression levels in mouse model of ovalbumin-induced allergic asthma,\u0026rdquo; Phytomed, vol. 23, no. 8, pp. 838\u0026ndash;845, 2016\u003c/li\u003e\n\u003cli\u003eK. S. Babu, D. E. Davies, and S. T. Holgate, \u0026ldquo;Role of tumor necrosis factor alpha in asthma,\u0026rdquo; Immunology and Allergy Clinics, vol. 24, no. 4, pp. 583\u0026ndash;597, 2004.\u003c/li\u003e\n\u003cli\u003eNeveu WA, Allard JL, Raymond DM, Bourassa LM, Burns SM, Bunn JY, Irvin CG, Kaminsky DA, Rincon M. Elevation of IL-6 in the allergic asthmatic airway is independent of inflammation but associates with loss of central airway function. Respir Res. 2010 Mar 8;11(1):28. doi: 10.1186/1465-9921-11-28. PMID: 20205953; PMCID: PMC2842243.\u003c/li\u003e\n\u003cli\u003eDoeing DC, Solway J. Airway smooth muscle in the pathophysiology and treatment of asthma. J Appl Physiol (1985). 2013 Apr;114(7):834-43. doi: 10.1152/japplphysiol.00950.2012. Epub 2013 Jan 10. PMID: 23305987; PMCID: PMC3633438.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alamandine, asthma, oxidative stress, inflammation, cytokines, NF-κB, dexamethasone","lastPublishedDoi":"10.21203/rs.3.rs-7720698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7720698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003cbr\u003e\nAsthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness, mucus overproduction, and oxidative stress. Alamandine (Ala), a peptide of the renin–angiotensin system, has shown anti-inflammatory and antioxidant properties in several experimental models. This study aimed to investigate the potential protective effects of Ala in an ovalbumin (OVA)-induced model of allergic asthma in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003cbr\u003e\nThirty-five male mice were divided into control, OVA, OVA+Ala, OVA+dexamethasone, and Ala-only groups. Asthma was induced by sensitization and challenge with OVA. Ala (50 μg/kg) and dexamethasone (2 mg/kg) were administered intraperitoneally during the challenge phase. Lung tissues and serum samples were analyzed for inflammatory cytokines, oxidative stress markers, and histopathological alterations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003cbr\u003e\nTreatment with Ala significantly reduced airway inflammation, eosinophil infiltration, and mucus secretion compared with the OVA group. Ala also downregulated the expression of pro-inflammatory cytokines (IL-6 and TNF-α) and suppressed NF-κB activation. Furthermore, oxidative stress markers improved following Ala administration, indicating its antioxidant potential. The effects of Ala were comparable, in part, to those of dexamethasone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003cbr\u003e\nThese findings suggest that Ala may exert protective effects against allergic airway inflammation and oxidative stress in an OVA-induced asthma model. The observed improvements in cytokine levels and tissue pathology indicate that Ala could help modulate inflammatory signaling pathways involved in asthma. However, further experimental and clinical studies are needed to confirm these results and to clarify the underlying mechanisms of Ala’s action in asthma management.\u003c/p\u003e","manuscriptTitle":"Alamandine Mitigates Inflammation and Oxidative Stress in OVA-Induced Asthma: A Novel Therapeutic Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 17:59:45","doi":"10.21203/rs.3.rs-7720698/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-11-28T09:35:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91431825854516606528262280503088123820","date":"2025-11-28T08:53:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191606016159640937083796921839355626233","date":"2025-11-27T12:06:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-27T11:44:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-23T13:49:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-17T11:24:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-16T08:13:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2025-10-16T08:08:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2a180bef-96a1-4b1a-817f-e21347ebfb38","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-05T17:59:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 17:59:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7720698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7720698","identity":"rs-7720698","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}