Syringic Acid ameliorates Bleomycin induced Pulmonary Inflammation and Fibrosis in rats via maintenance of endogenous anti-oxidants and downregulation of pro-inflammatory markers

Syringic Acid ameliorates Bleomycin induced Pulmonary Inflammation and Fibrosis in rats via maintenance of endogenous anti-oxidants and downregulation of pro-inflammatory markers | 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 Syringic Acid ameliorates Bleomycin induced Pulmonary Inflammation and Fibrosis in rats via maintenance of endogenous anti-oxidants and downregulation of pro-inflammatory markers Lakshmi BVS, krupavaram bethala This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4350558/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Chronic administration of bleomycin (BLM), a chemotherapeutic drug, has been linked to idiopathic pulmonary fibrosis (IPF). It has been observed that syringic acid, a phenolic compound, has antiapoptotic, anti-inflammatory, and antioxidant properties. To assess syringic acid's therapeutic potential against lung fibrosis caused by BLM and determine a potential mechanism of action. Methods: Sprague-Dawley rats were inducted into IPF after receiving 7.5 IU/kg of BLM intratracheally. Syringic acid (50 mg/kg, i.p.) was administered to rats for 14 days, after which different parameters in the lung and bronchoalveolar lavage fluid (BALF) were measured. Results: Altered BALF differential cell counts, elevated lung index, hydroxyproline, NO, and MDA plasma levels, and reduction in GSH, GPx, SOD, and CAT in group receiving BLM were evidence of pulmonary toxicity. Administering 50 mg/kg of syringic acid significantly reduced (p < 0.001) the changes brought about by BLM. The expression of TNF-α was greatly reduced by syringic acid when it was stimulated by BLM. The BLM-treated group's histological analysis revealed significant lung damage with alveolar septal thickening, interstitial infiltration, collapsing alveolar gaps, and an elevated Ashcroft and Szapiel score. Syringic acid was used to lessen these effects. The syringic acid group (p<0.01) markedly reduced Szapiel score, collagen deposition, lung edema, and fibrotic alterations. It also inhibited the infiltration of myofibroblasts and inflammatory cells, primarily macrophages and lymphocytes. By inhibiting the TGF-β1/NF-κB pathways, syringic acid mitigates BLM-induced IPF. This, in turn, improves the control of oxidant and pro-inflammatory markers (TNF-α) to decrease collagen deposition during pulmonary fibrosis. Conclusion: Finally, it is concluded that Syringic acid can protect the lung against BLM-induced pulmonary oxidative stress, inflammation and fibrosis. Bleomycin Pulmonary fibrosis Syringic acid Anti-fibrotic Anti-inflammatory Antioxidant Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Idiopathic pulmonary fibrosis (IPF) is a distinct type of slowly developing, chronic lung disease with no known cause that is linked to oxidative stress, inflammation, and the buildup of fibroblasts and myofibroblasts. In the early stages of the disease, this can result in abnormal extracellular collagen deposition. Regretfully, the prognosis for IPF is bleak, and pirfenidone remains the only viable medication, even after much research. The 5-year death rate is 80% in the absence of lung transplantation. Novel treatment agents with enhanced efficacy are therefore required [2]. Many plant-derived bioactive chemicals, including vinblastine, camptothecin, and paclitaxel, have been identified and are being utilized extensively to treat different kinds of cancer in the previous few decades. Creating more effective treatment agents with increased action against lung cancer is the requirement. Nowadays, there is a lot of interest in functional foods and nutraceuticals for the prevention of several chronic illnesses, like cancer and cardiovascular disease [3]. The two most significant classes of bioactive substances and secondary metabolites found in plants are flavonoids and phenolic acids [4]. Syringic acid (SA), also known as 4-hydroxy-3, 5-dimethoxybenzoic acid, is a significant phenolic chemical that occurs naturally and is present in a wide variety of plants and foods. Swiss chard, dates, walnuts, olives, spices and pumpkin are the primary sources of SA [5,6]. Research has indicated that large amounts of SA can be found in plants such as Tagetese recta Linn flower [10], Hemidesmus indicus [9], barley, maize, millet, oat, rice, rye, sorghum, and wheat [7]. It demonstrates the wide range of biological processes that support the preservation of human health. SA has been shown to have a number of biological properties, including antioxidant, antiproliferative [11], anti-endotoxic [12], and anti-cancer [13]. The administration of SA could suppress hepatic fibrosis in chronic liver injury [14]. SA decreased proliferation in leukemia cells and induced apoptosis by raising the level caspase 3, 8, and 9 activities [15]. Because intratracheal BLM treatment in rats results in alveolar cell injury, an inflammatory response, fibroblast proliferation, and collagen content deposition, bleomycin (BLM)-induced pulmonary fibrosis is a commonly used animal model of human IPF [16]. Reactive oxygen species (ROS) and proteolytic enzymes are known to be released by activated inflammatory cells that accumulate in the lungs, causing parenchymal damage and escalating the severity of injury [17]. Research evaluating various antioxidant compounds as a preventative measure in BLM-induced lung fibrosis in rats, including N-acetylcysteine, erdosteine [18], caffeic acid phenethyl ester [17], melatonin [19], ginkgo biloba [20], cordyceps [21], and resveratrol [22], discovered that these compounds typically prevented or lessened lung fibrosis based on Ashcroft's criteria and lung hydroxyproline content. Consequently, using antioxidant techniques to prevent or treat BLM-induced lung fibrosis may make sense. We looked into SA's ability to prevent IPF using this approach. We assessed the histological and biochemical evidence of lung fibrosis brought on by exposure to BLM, and we investigated inflammatory markers and oxidative stress in a model of injured lungs in rats. Methods Chemicals and kits Bleomycin was obtained from Chemex Company (Argentina), bovine serum albumin, Ellman’s reagent (DTNB), thiobarbituric acid and Bradford reagent were purchased from Sigma–Aldrich. Ammonium molybdate, butylated hydroxytoluene, trichloroacetic acid, buffered formalin, HCl and perchloric acid were purchased from Merck Company. Commercial glutathione peroxidase (GPX) and superoxide dismutase (SOD) kits were purchased from RANSEL, Randox Com,UK. TNF-α commercial enzyme-linked immunosorbentassay (ELISA) kit was provided by Hangzhou, Eastbiopharm. BLM-induced lung fibrosis in experimental animals [23] We purchased thirty-two male Sprague Dawley rats, weighing between 150 and 250 grammes, from Sanzyme Ltd. in GaghanPahad, Hyderabad. The rats were kept in conventional settings, with 12 hour cycles of light and dark, 24±1º C temperature and 65±10% humidity, inside cages made of polypropylene. Water from the tap and standard pelletized feed were given. The Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2018-19/MPCOL/9) authorised the protocols for every pharmaceutical trial. The rats were divided into four groups equally and at random, and they received the following care: Group 1: The animals received normal saline (0.5mL/d, p.o.) for 14 days. Group 2: Syringic acid group; 50 mg/kg was continued intraperitoneally (i.p.) for 14 days, Group 3: BLM was applied intratracheally (7.5 units/kg, single dose) on day 1. Normal saline (0.5 mL/d, p.o.) was given for 14 consecutive days. Group 4: BLM was applied intratracheally (7.5 units/kg, single dose) on day 1. Syringic acid (50 mg/kg/d, i.p.) was given for 14 consecutive days. Under ether anaesthesia, a single intratracheal injection of BLM was administered. Saline was given orally to the Control group in place of BLM. Rats were weighed before and after the trial began. Rat blood samples were obtained for haematological parameters following the tests. After receiving BLM injections for 14 days, every rat was killed. Following the animals' overdose on anesthesia-induced death, the lung tissue samples were cleaned in cold saline and weighed in order to determine the lung/body mass index. By dividing the lung weight (g) by the body weight (g) and multiplying the result by 100, the lung index was calculated. Thereafter employed for histological and biochemical examination. The right section of the lung was placed in liquid nitrogen and stored at −70 °C until the assay for thiobarbituric acid-reactive substances (TBARS) a lipid peroxidation product, superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), and glutathione peroxidase (GPx) contents. The left part of the lung was placed in formaldehyde solution for routine histopathological examination by light microscopy. Determination of hydroxyproline in the lung tissue A colorimetric assay was used to determine the left lung's total collagen content [25, 26]. The left lung was dried at 80◦C until it achieved a steady weight. The dry lung was hydrolyzed overnight in a glass vial with 12-N HCl at 120C while being vacuum-operated. The sample volume was adjusted to 30 mL using distilled water, and the pH was adjusted to 7 using NaOH. After combining the sample solution (1.0 mL) with 1.0 mL of chloramine T solution (0.05 mol/L), the mixture was incubated for 20 minutes at room temperature. After adding a 20% dimethyl benzaldehyde solution to the mixture, it was incubated for 20 minutes at 60 °C. Every sample was analysed by measuring its absorbance at 557 nm. The results are expressed in micrograms of hydroxyproline per gram of wet lung weight using the standard hydroxyproline curve [24, 25]. Biochemical assays Two hundred milligrams of frozen lung tissue specimen was dissected into pieces on dry ice, homogenized in 1.15 % KCl buffer (1:9, w/v) using a manual glass homogenizer for approximately 5min and flushed with centrifugation for approximately 10s to remove large debris. The supernatant was used for analysis. Using a commercial kit, the activity of SOD was measured (RANSOD kit, Randox Com, UK). For SOD, absorption was measured at 412 nm and reported as (IU/mg protein). Goth's colorimetric approach was used to measure the catalase (CAT) activity. After homogenised lung tissue was incubated with H2O2, the reaction was stopped after 10 minutes by mixing in ammonium molybdate. While the ammonium molybdate reacts with the sample's residual H2O2 to form a yellow complex, the CAT enzyme breaks down the H2O2 in the samples to produce H2O and O2. Using a spectrophotometer, absorption was measured at 410 nm. The expression for CAT activity was (μmol H2O2/min/mg protein) [26]. A commercial kit (RANSEL kit, Randox Com, UK) based on the Paglia and Valentine method [27] was used to measure the GPX activity. At 340 nm, absorption was measured, and (IU/mg of protein) was the expression for enzyme activity. Using Ellman's approach, the GSH level in lung tissue was determined. Ellman's reagent was applied to 40μl of lung tissue homogenate after it had been combined with 2ml of buffer phosphate. The GSH content of the yellow complex was measured using a spectrophotometer set to 410 nm, and was reported as (nmol/mg protein) [28]. Thiobarbituric acid reactive substances (TBARS) production was used to measure the amount of lipid peroxidation [29]. To summarise, 0.8 ml of a solution containing 0.25N HCl, 0.375% (w/v) thiobarbituric acid, and 15% (w/v) trichloroacetic acid (TCA) was mixed with 0.2 ml of homogenised tissue. Centrifugation was used for five minutes at 5000 RPM to precipitate and remove the protein. After being transferred to test tubes containing 0.02% butylated hydroxyl toluene, the supernatants were bain-marie heated for 15 minutes at 100°C. After that, samples were chilled and centrifuged for five minutes at 2000 RPM to get rid of the precipitant. When measuring absorption at 532 nm, MDA concentrations were reported as (nmol/mgprotein). Determination of nitric oxide (NO) The Griess method was used to determine the nitrite levels, which serve as an indicator of NO generation. First, acetonitrile (1:2, v/v) was used to deproteinize tissue samples. Subsequently, 100 μL of supernatant and 100 μL of Griess reagent were applied to a microplate well. After that, samples were incubated in microplate wells for 30 minutes at 37 °C, and the absorbance of the samples was eventually found at 546 nm. Based on a typical linear curve established by 0–150 mol/ml sodium nitrite, the quantity of nitric oxide metabolites in the samples was ascertained. For tissue samples, the data were given in moles per gram (gr) and per litre (L) [30, 31]. Bronchoalveolar lavage Fluid differential cell count The process of obtaining bronchoalveolar lavage fluid involved injecting 3 ml of saline three times, for a total of 9 ml, and then gently aspirating the fluid out of the lung after inserting an intratracheal catheter into a trachea. The recovery ratio of lavage fluid with this catheter was roughly 80% and did not show any discernible variation across the groups. Using a hemocytometer, the total number of cells in the bronchoalveolar lavage fluid was determined. Smear slides were produced and stained with Giemsa solution to allow for differential counts of leukocytes in the broncholaveolar lavage fluid. Three hundred cells were counted differently for each sample. Measurement of tumor necrosis factor-α Tumor necrosis factor-α concentration was measured using an enzyme-linked immunosorbent assay kit (Xpressbio life scientific products). The determinations were done according to the test kit instructions. Histopathological studies Inflammatory and fibrotic evidences in histopathology studies The tissue from the left lung was blocked in paraffin and put in 10% buffered formalin for histopathological analysis. Hematoxylin and eosin (H&E) and Masson's trichrome (MT) were used to stain 4 μm sections in order to assess collagen deposition, alveolar thickness, and inflammatory cell infiltration. The Ashcroft score [33] measured fibrotic lesions from 0 to 8, while the Szapiel score [32] graded alveolitis and inflammation from 0 to 3. Grading of inflammatory and fibrotic lesions was done on three slides per rat, with ten fields each slide. Statistical analysis The Data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc.). The result was expressed as Mean ± SEM and analyzed using one-way ANOVA followed by Tukey–Kramer multiple comparison tests. Previously, the normal distribution of data was evaluated. Differences were considered statistically significant at (p < 0.05). Results Effect of syringic acid on body weight and lung index BLM application caused significant body weight loss after 14 days compared with the control group (p<0.05). In addition, zingerone (50 and 100 mg/kg) administration increased body weight compared with BLM group (p< 0.05) (Fig. 1a). BLM significantly increased lung index at 14th day compared with control group (p< 0.05). Zingerone (50 and 100 mg/kg After 14 days, BLM treatment resulted in a substantial reduction in body weight as compared to the control group (p<0.05).Moreover, delivery of 50 mg/kg of syringic acid resulted in an increase in body weight as compared to the BLM group (p< 0.01). On day 14, BLM exhibited a substantial rise in lung index as compared to the control group (p<0.001). When compared to the BLM group, syringic acid (50 mg/kg) dramatically reduced the lung index (Table 1, Fig 1). Hydroxyproline assay Using hydroxyproline measurements, we calculated the amount of collagen deposited in the lung tissues. Bleomycin markedly raised the amount of hydroxyproline in the rat lungs as compared to the control group (P < 0.001). When compared to the BLM-treated group, the hydroxyproline content was considerably lower in the animals treated with syringic acid (50 mg/kg). The group treated with syringic acid plus bleomycin had a substantial decrease in hydroxyproline content as compared to the group treated with BLM (P < 0.01). (Figure 3) Antioxidant defense system markers When compared to the control group, BLM reduced the activity of the enzymes SOD, CAT, and GPX at the same level (p < 0.001). Syringic acid pretreatment at a dose of 50 mg/kg boosted the activity of CAT, GPX, and SOD. These effects demonstrated a strong recovery and were comparable to the BLM group (p < 0.01) (Table 2). When compared to the control group, BLM decreased the levels of GSH in lung tissue (p < 0.001). GSH levels were elevated by 50 mg/kg (p < 0.001) of syringic acid. Syringic acid 50 mg/kg (p < 0.01) reduced lung tissue MDA and NO levels, while BLM raised MDA and NO levels (p < 0.001) (Fig.2). Total and differential cell count in Bronchoalveolar lavage fluid The impact of syringic acid on total cell counts and the differential in bronchoalveolar lavage fluid between the experimental and control groups of rats is displayed in Table 3. When compared to control rats, bleomycin therapy resulted in a substantial increase in the overall cell count in the bronchoalveolar lavage fluid (p < 0.001). The total cell count in rats given syringic acid treatment stayed at levels comparable to those of control rats. Rats treated to bleomycin had significantly higher neutrophil and eosinophil counts in their lungs, according to the differential cell count. The increase in blood cells in the bronchoalveolar lavage caused by bleomycin was considerably decreased after a 14-day syringic acid treatment. Although the bleomycin-induced group had a lower percentage of lymphocytes and alveolar macrophages, syringic acid treatment dramatically reversed these effects (p < 0.01). Pro-inflammatoy marker-Tumor necrosis factor-α concentration Fig. 4a, b shows the plasma levels of tumour necrosis factor-α. On day 14, the plasma levels of tumour necrosis factor-α protein in the rats in the bleomycin-administered group were still higher than those in the control group. At the conclusion of the trial, it was discovered that syringic acid treatment reduced the increase in tumour necrosis factor-α level caused by bleomycin. Histological changes On day 14, histopathological abnormalities in the lungs were found using Masson's trichome staining (Fig. 6) and hematoxylin and eosin staining (Fig. 5). Typical open alveoli, interalveolar gaps with typical terminal bronchi, normal bronchiolar epithelial appearance, thin interalveolar septa, absence of inflammatory cells, and fibrosis were all seen in normal lung tissues. Rats in the bleomycin-administered group displayed deformed tissue architecture, including moderate to severe bleeding, congestion, emphysema-related sloughing of the bronchial epithelium from the basement membrane, areas of increased alveolar thickening, increased fibrosis, and leukocyte accumulation in the alveolar walls. In contrast to the bleomycin-treated group, the lungs of the rats treated with syringic acid exhibited fewer leukocytes and less thickening of the alveoli. When localizing collagen as a distinct area in a histological preparation, Masson staining is seen to be a dependable technique. In comparison to the control group, the bleomycin-treated group showed larger fibrotic regions and an elevated grade of collagen deposition. In contrast to the bleomycin group, collagen formation was noticeably reduced in syringic acid. The Szapiel examination's pathology score was used to determine the lung sections' semi-quantitative assessment. On day 14, the bleomycin-induced group's Szapiel core was discovered to be considerably higher than that of the control group. On the fourteenth day, the Szapiel scores of the group treated with syringic acid shown a notable decline in comparison to the group treated with bleomycin. Discussion One popular experimental model used to examine pathophysiology and look into novel medications for the treatment of IPF is the BLM-induced PF model [34]. Many studies suggest that oxidative stress and inflammation in the respiratory system may be linked to the development of lung fibrosis [35]. The findings supported the involvement of oxidative stress (shown by decreased GSH) and inflammation (neutrophil infiltration) in the BLM-induced lung damage. The reported alterations were considerably reduced by syringic acid. Weight loss is a well-known side effect of BLM administration in the treatment of cancer and pulmonary fibrosis models [35]. In line with earlier research, BLM given intratracheally reduced animal weight and raised body lung index in our investigation [36]. According to previous research, BLM's effects on hunger and protein catabolism may be the cause of these effects [37]. Previous research indicates that during the early stages of pulmonary fibrosis, the inflammatory responses stimulate and facilitate damage to the cells that line capillaries and alveolar epithelium. Later on, fibroblasts promote the formation of extracellular matrix (ECM) and collagen secretion [38]. Fibrosis results from excessive collagen remodelling and deposition, which compromises the lung tissue's structural integrity. Since hydroxyproline makes up the majority of collagen fibres, the tissue lung's hydroxyproline content reveals the amount of collagen and the degree of pulmonary fibrosis [39]. In the current investigation, intratracheal injection of BLM considerably elevated lung hydroxyproline levels, in line with findings by Huang et al. [40] and Ramezani et al. [41]. Syringic acid treatment considerably decreased the increase in hydroxyproline levels in the lung tissue. Accordingly, a histological analysis of the lung tissues in the group that received BLM treatment revealed that the drug caused duct hyperplasia, collagen buildup, and inflammatory cell penetration. In pulmonary fibrosis animal models, BLM enhances the generation of ROS [42]. By employing Masson's trichome staining of lung sections for collagen deposition, this discovery was further supported. In the current study, bleomycin triggered the deposition, aggregation, and deposition of collagen in the peribronchial and perialveolar tissues, obliterating the alveolar gaps as small fibrils. However, the group that received syringic acid treatment had significantly less collagen deposition, which may have been caused by syringic acid's inhibitory action. [43] The ability of BLM to produce ROS is one of the generally acknowledged reasons for the lung damage and tissue remodelling it induces. It is known that BLM binds to DNA and Fe2+ to create a complex [44, 45]. Redox cycling occurs in the DNA/Fe2+/BLM complex, producing ROS such hydroxyl and superoxide radicals. We assessed the levels of oxidative stress markers in the current investigation. Apart from the inflammation and collagen deposition caused by BLM, we discovered that it also considerably increased the levels of lipid peroxide and significantly decreased the levels of antioxidant enzymes such as GPx, CAT, and SOD when compared to the control group. These findings corroborated earlier research assessing the function of oxidative stress in BLM-induced lung fibrosis [46]. Furthermore, we saw that syringic acid improved oxidative stress indicators as CAT, MDA, and SOD. Rat lung tissues in the syringic acid + BLM group had MDA content that was relatively similar to the control group's. In these mice, concurrent administration of syringic acid effectively mitigated the inflammatory consequences of BLM therapy, or syringic acid's antioxidant properties produced an at least partially preventive impact. Reduced oxidative stress has been linked to anti-inflammatory benefits of the majority of antioxidant medicines investigated for treatment of BLM-induced lung fibrosis models [47, 48]. Yinfang et al. reported that syringic acid had antioxidant qualities in this regard by lowering lipid peroxidation, which was indicated by a drop in the MDA level. Furthermore, by reducing oxidative stress, syringic acid is said to be a direct scavenger of free oxygen radicals in non-alcoholic fatty liver disease brought on by a high-fat diet. It has been established that NO plays a critical role in the aetiology of lung disorders, particularly pulmonary fibrosis. Peroxynitrite, a highly reactive nitrogen species, is created when NO interacts with superoxide free radicals, causing nitrosative stress and significant lung tissue damage. When compared to the BLM group, the group that received syringic acid saw a considerable drop in the level of NO. Bleomycin administered intratracheally causes interstitial inflammation in addition to the oxidative stress already discussed. This inflammation is accompanied by a notable increase in leukocyte recruitment. Leukocytes, including neutrophils, lymphocytes, and macrophages, are important for tissue remodelling and inflammation [49]. In the bronchoalveolar lavage fluid, the bleomycin-treated group exhibited a significant decrease in macrophages and a significant rise in total cells, neutrophils, and lymphocytes. This is consistent with earlier research by Sriram et al. (2009)[51] and Gong et al. (2005)[50]. Rats treated with syringic acid had similar counts of neutrophils, lymphocytes, macrophages, and total cells as the rats in the control group. Inhibited leukocytes recruitment, which directly impacted inflammation and tissue repair, might partly account for the preventive effect of Syringic acid on bleomycin-induced pulmonary fibrosis, which may be due to its ability to interfere with free radical-mediated reactions. Furthermore, among the complex webs of cellular and molecular interactions that control the fibrotic process, tumour necrosis factor-α, a strong pro-inflammatory cytokine, functions as a key player [52]. According to El-Medany et al. (2005) [53], there was a notable increase in tumour necrosis factor-α expression in the group that received bleomycin in this investigation. Bleomycin is known to induce inflammation-mediated tissue injury, which may be brought on by the generation of free radicals, which could activate nuclear factor kappa-B and enhance the synthesis of tumour necrosis factor-α [54, 55]. The activity of nuclear factor kappa-B is inhibited by syringic acid [56]. TNF-α expression was significantly decreased by syringic acid, possibly due to its inhibitory influence on nuclear factor kappa-B activity. NO, iNOS, TNF-α, COX-2, and PGE2 release were all inhibited by syringic acid. Additionally, SA lessened the significant phosphorylation of the PTEN/AKT/NF-kB pathway that was caused by IL-1β [57]. The lung interstitium experiences an excessive amount of collagen deposition as a result of an increase in fibroblast count. Inhibiting fibroblast proliferation and excessive collagen synthesis is one method of attenuating fibrosis [58]. Sections of the lungs stained with Masson's trichome showed that the syringic acid-treated group had less collagen deposition. According to this study, the pulmonary response to the bleomycin challenge involves both a decrease in lung antioxidant capacity and a quick onset of oxidative stress. By inhibiting inflammation and having the ability to scavenge reactive oxygen species, syringic acid's inhibitory impact decreased oxidative stress. Further research is necessary to elucidate the protective mechanism of syringic acid on this model and explore its impact on alternative animal models of lung fibrosis. Conclusion Conventional corticosteroid therapy typically has no effect on pulmonary fibrosis [59]. In lung tissue, bleomycin inoculation led to decreased antioxidant capacity, increased levels of inflammatory cytokines, fibrotic alterations, and collagen accumulation; in contrast, syringic acid demonstrated pneumoprotective properties by boosting antioxidant defense, lowering inflammatory cytokine levels, and preventing collagen accumulation. Accordingly, the current findings imply that syringic acid successfully guards against the lung damage brought on by bleomycin challenge. Abbreviations Fig. Figure i.p. intra peritoneal kg kilogram mg milligram ml millilitre p.o. Per oral w/w weight/ weight w/v weight/ volume NO nitric oxide GSH reduced glutathione MDA malondialdehyde CAT catalase SOD superoxide dismutase GPx glutathione peroxidase TNF-α tumor necrosis factor alpha NFkB nuclear factor kappa-light-chain-enhancer of activated B cells Declarations Authors’ contributions BVSL and BKV wrote the manuscript. BVSL and BKV designed and performed the study. All authors participated in data analysis and approved the final manuscript. Funding There was no specific fund for this study. Availability of data and materials Data analyzed and used for this manuscript are available within the manuscript. Ethics approval and consent to participate The Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2018-19/MPCOL/9) authorized the protocols for every pharmaceutical trial. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. Author details 1. 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Sadeghi, et al., Protective and therapeutic effects of ethanolic extract of Nasturtium officinale (watercress) and vitamin E against bleomycin-induced pulmonary fibrosis in rats, Research in Pharmaceutical Sciences. 2021; 16 (1): 94. D-x Yang, J. Qiu, H.-H. Zhou, Y. Yu, D-l Zhou, Y. Xu, et al., Dihydroartemisinin alleviates oxidative stress in bleomycin-induced pulmonary fibrosis, Life Sci. 2018; 205: 176–183. Zhou X., Zhang G., Li C., Hou J., Inhibitory effects of Hu-qi-yin on the bleomycin-induced pulmonary fibrosis in rats. Journal of Ethnopharmacology 2007; 111: 255-264. CasparyWJ,LanzoDA,NiziakC(1982)Effectofdeoxyribonucleicacid on the production of reduced oxygen by bleomycin and iron. Biochemistry 21:334–338 El-Khouly D, El-Bakly W, Awad AS, El-Mesallamy HO, El-Demerdash E. Thymoquinone blocks lung injury and fibrosis by attenuating bleomycin-induced oxidative stress and activation of nuclear factor Kappa-B in rats. Toxicology 2012; 302:106–113 Yinfang Li, Li Zhang, Xiaohua Wang, Wei Wu , Rui Qin. Effect of Syringic acid on antioxidant biomarkers and associated inflammatory markers in mice model of asthma, Drug Dev Res . 2019 Mar; 80(2):253-261. Yildirim Z, Kotuk M, Erdogan H, Iraz M, Yagmurca M, Kuku I, Fadillioglu E. Preventive effect of melatonin on bleomycininduced lung fibrosis in rats. J Pineal Res. 2006; 40:27–33. MengliC,CheungFWK,MingHungC,PakKwanH,Siu-PoI,YickHin L, Chun-Tao C, Wing Keung L. Protective roles of Cordyceps on lung fibrosis in cellular and rat models. J ethnopharmacol. 2012; 143(2):448–54. Xin Wei, X., Han, J., Chen, Z., Qi, B., Wang, G., Ma, Y., Zheng, H., Luo, Y., Wei, Y., Chen, L., Aphosphoinositide3-kinase-γinhibitor,AS605240preventsbleomycin-inducedpulmonaryfibrosisinrats.BiochemicalandBiophysicalResearchCommunications. 2010; 397:311–317. Gong, L., Li, X., Wang, H., Zhang, L., Chen, F., Cai, Y., Qi, X., Liu, L., Liu, Y., Xiong-fei Wu, X., Huang, C., Ren, J., EffectofFeitaionbleomycin-inducedpulmonaryfibrosisinrats.JournalofEthnopharmacology. 2005; 96:537–544. Sriram, N., Kalayarasan, S., Sudhandiran, G., Epigallocatechin-3-gallateaugmentsantioxidantactivitiesandinhibitsinflammationduringbleomycin-inducedexperimentalpulmonaryfibrosisthroughNrf2–Keap1signaling.PulmonaryPharmacologyandTherapeutics 2009; 22:221–236. Razzaque, M.S., Taguchi, T., Pulmonaryfibrosis:cellularandmolecularevents.PathologyInternational.2003; 53: 133–145. El-Rakhawy, F.I., El-Medany, G., 2005. Attenuationofbleomycininducedlungfibrosisinratsbymesna.EuropeanJournalofPharmacology. 2005; 509:61–70. Ortiz, L.A., Champion, H.C., Lasky, J.A., Gambelli, F., Gozal, E., Hoyle, G.W., Beasley, M.B., Hyman, A.L., Friedman, M., Kadowitz, J., 2002. EnalaprilprotectsmicefrompulmonaryhypertensionbyinhibitingTNF-mediatedactivationofNF-kBandAP-1.AmericanJournalofPhysiology–LungCellularandMolecularPhysiology282,1209–1221. Kalayarasan, S., Sriram, N., Sudhandiran, G., Diallylsulfideattenuatesbleomycin-inducedpulmonaryfibrosis:criticalroleofiNOS,NF-nB,TNF-αandIL-1β.LifeSciences. 2008; 82:1142–1153. Oluwatobi T Somade., Olubisi E Adeyi et al., Syringic and ascorbic acids prevent NDMA-induced pulmonary fibrogenesis, inflammation, apoptosis, and oxidative stress through the regulation of PI3K-Akt/PKB-mTOR-PTEN signaling pathway. Metabol Open 2022; 14;1-5. Yao Li., Yurun Zhu et al., Syringic acid inhibits IL-1β-induced inflammation in mice chondrocytes and ameliorates the progression of osteoarthritis via the PTEN/AKT/NF-κB pathway. Journal of functional foods, 2023; 107: 105683. Gong, L., Li, X., Wang, H., Zhang, L., Chen, F., Cai, Y., Qi, X., Liu, L., Liu, Y., Xiong-fei Wu, X., Huang, C., Ren, J., 2005. EffectofFeitaionbleomycin-inducedpulmonaryfibrosisinrats.JournalofEthnopharmacology. 2005; 96: 537–544. Green, F.H.Y., Overviewofpulmonaryfibrosis.Chest 2002; 122:334–339. Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4350558","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301363926,"identity":"6b55283b-9ccf-49ce-b8c2-1a73ac9209b5","order_by":0,"name":"Lakshmi BVS","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFCCxAZmCIOHgeEDkGJjJ6ylsRmmhXEGSAszQS0JjHAtzDwgmpAW/vbk9seFbXYM5uy9Bz/b/Nomz8fMwPjhYw5uLRJnHjY2z2xLZrDsOZcsndt327CNmYFZcuY2PNbcAPqFF6jM4EaOgXRuz21GIJuNmRePFnmIlnoGg/tvjH9b9ty2J6jFAKLlMJDBYybN8ON2IkEthkC/zOY5d5zH4EyOmWVvw+3kNmbGZrx+kTue/uAzT1m1nMHxM8Y3fvy5bTu/vfngh4/4vA8CjGwMPBBGG5hsIKAeBP5gMEbBKBgFo2AUIAAAGJlReTKaYNwAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Lakshmi","middleName":"","lastName":"BVS","suffix":""},{"id":301363927,"identity":"2998f3b2-8f02-46be-8c67-ed36ccf69f99","order_by":1,"name":"krupavaram bethala","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"krupavaram","middleName":"","lastName":"bethala","suffix":""}],"badges":[],"createdAt":"2024-04-30 16:42:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4350558/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4350558/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56387798,"identity":"8c33beb5-1c57-4af4-8402-1668ea1b25ac","added_by":"auto","created_at":"2024-05-13 14:02:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Syringic acid on the rat lung index of BLM-induced pulmonary fibrosis. \u003c/strong\u003eValues are presented as mean ± SEM (n=8). *P \u0026lt;0.001 Indicate significant difference compared with the control group; **P \u0026lt;0.01 vs BLM-treated rats. BLM: Bleomycin\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4350558/v1/8ce75cfe8bf771d92d3cc613.png"},{"id":56386902,"identity":"881722ce-ba72-4ea1-90b7-b9e43ca37be3","added_by":"auto","created_at":"2024-05-13 13:54:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Syringic acid on the tissue oxidative stress markers (NO metabolite level) in animals. \u003c/strong\u003eValues are presented as mean ± SEM (n=8). *P \u0026lt;0.001 Indicate significant difference compared with the control group; **P \u0026lt;0.01 vs BLM-treated rats. BLM: Bleomycin\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4350558/v1/da0f77be2245cb11bc80e65f.png"},{"id":56386905,"identity":"0cda90ed-171c-4d99-a95a-daee4433c646","added_by":"auto","created_at":"2024-05-13 13:54:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Syringic acid on hydroxyproline content in the rat lung index of BLM-induced pulmonary fibrosis. \u003c/strong\u003eValues are presented as mean ± SEM (n=8). *P \u0026lt;0.001 Indicate significant difference compared with the control group; **P \u0026lt;0.01 vs BLM-treated rats. BLM: Bleomycin\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4350558/v1/2439d005c9c757db2135f2c8.png"},{"id":56386903,"identity":"ed35bc7a-c122-4e3b-815c-cc610e0a97ec","added_by":"auto","created_at":"2024-05-13 13:54:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16487,"visible":true,"origin":"","legend":"\u003cp\u003ea, b. The tumor necrosis factor-α and grade of fibrosis in rats subjected to various treatments. Values are presented as mean ± SEM (n=8). *P \u0026lt;0.001 Indicate significant difference compared with the control group; **P \u0026lt;0.01 vs BLM-treated rats. BLM: Bleomycin\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4350558/v1/b2339a12d6828a1365b0256a.png"},{"id":92985068,"identity":"7ddd41cb-bf8c-471e-a522-edc24f1a2ec7","added_by":"auto","created_at":"2025-10-07 21:31:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":829348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4350558/v1/b2cce8c1-739a-4b0c-94f0-e2ccd1ca73c4.pdf"},{"id":56386901,"identity":"0d41c322-f613-4dd5-a92e-65c8ba414023","added_by":"auto","created_at":"2024-05-13 13:54:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17818,"visible":true,"origin":"","legend":"","description":"","filename":"tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4350558/v1/b01162458120581dbba44d0c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Syringic Acid ameliorates Bleomycin induced Pulmonary Inflammation and Fibrosis in rats via maintenance of endogenous anti-oxidants and downregulation of pro-inflammatory markers","fulltext":[{"header":"Background","content":"\u003cp\u003eIdiopathic pulmonary fibrosis (IPF) is a distinct type of slowly developing, chronic lung disease with no known cause that is linked to oxidative stress, inflammation, and the buildup of fibroblasts and myofibroblasts. In the early stages of the disease, this can result in abnormal extracellular collagen deposition. Regretfully, the prognosis for IPF is bleak, and pirfenidone remains the only viable medication, even after much research. The 5-year death rate is 80% in the absence of lung transplantation. Novel treatment agents with enhanced efficacy are therefore required [2].\u003c/p\u003e\n\u003cp\u003eMany plant-derived bioactive chemicals, including vinblastine, camptothecin, and paclitaxel, have been identified and are being utilized extensively to treat different kinds of cancer in the previous few decades. Creating more effective treatment agents with increased action against lung cancer is the requirement. Nowadays, there is a lot of interest in functional foods and nutraceuticals for the prevention of several chronic illnesses, like cancer and cardiovascular disease [3]. The two most significant classes of bioactive substances and secondary metabolites found in plants are flavonoids and phenolic acids [4].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSyringic acid (SA), also known as 4-hydroxy-3, 5-dimethoxybenzoic acid, is a significant phenolic chemical that occurs naturally and is present in a wide variety of plants and foods. Swiss chard, dates, walnuts, olives, spices and pumpkin are the primary sources of SA [5,6]. Research has indicated that large amounts of SA can be found in plants such as Tagetese recta Linn flower [10], Hemidesmus indicus [9], barley, maize, millet, oat, rice, rye, sorghum, and wheat [7]. It demonstrates the wide range of biological processes that support the preservation of human health. SA has been shown to have a number of biological properties, including antioxidant, antiproliferative [11], anti-endotoxic [12], and anti-cancer [13]. \u0026nbsp;The administration of SA could suppress hepatic fibrosis in chronic liver injury [14]. SA decreased proliferation in leukemia cells and induced apoptosis by raising the level caspase 3, 8, and 9 activities [15]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause intratracheal BLM treatment in rats results in alveolar cell injury, an inflammatory response, fibroblast proliferation, and collagen content deposition, bleomycin (BLM)-induced pulmonary fibrosis is a commonly used animal model of human IPF [16].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReactive oxygen species (ROS) and proteolytic enzymes are known to be released by activated inflammatory cells that accumulate in the lungs, causing parenchymal damage and escalating the severity of injury [17]. Research evaluating various antioxidant compounds as a preventative measure in BLM-induced lung fibrosis in rats, including N-acetylcysteine, erdosteine [18], caffeic acid phenethyl ester [17], melatonin [19], ginkgo biloba [20], cordyceps [21], and resveratrol [22], discovered that these compounds typically prevented or lessened lung fibrosis based on Ashcroft\u0026apos;s criteria and lung hydroxyproline content.\u003c/p\u003e\n\u003cp\u003eConsequently, using antioxidant techniques to prevent or treat BLM-induced lung fibrosis may make sense. We looked into SA\u0026apos;s ability to prevent IPF using this approach. We assessed the histological and biochemical evidence of lung fibrosis brought on by exposure to BLM, and we investigated inflammatory markers and oxidative stress in a model of injured lungs in rats.\u003c/p\u003e"},{"header":"Methods ","content":"\u003cp\u003e\u003cstrong\u003eChemicals and kits\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBleomycin was obtained from Chemex Company (Argentina), bovine serum albumin, Ellman\u0026rsquo;s reagent (DTNB), thiobarbituric acid and Bradford reagent were purchased from Sigma\u0026ndash;Aldrich. Ammonium molybdate, butylated hydroxytoluene, trichloroacetic acid, buffered formalin, HCl and perchloric acid were purchased from Merck Company. Commercial glutathione peroxidase (GPX) and superoxide dismutase (SOD) kits were purchased from RANSEL, Randox Com,UK. TNF-\u0026alpha; commercial enzyme-linked immunosorbentassay (ELISA) kit was provided by Hangzhou, Eastbiopharm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBLM-induced lung fibrosis in experimental animals [23]\u0026nbsp;\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe purchased thirty-two male Sprague Dawley rats, weighing between 150 and 250 grammes, from Sanzyme Ltd. in GaghanPahad, Hyderabad. The rats were kept in conventional settings, with 12 hour cycles of light and dark, 24\u0026plusmn;1\u0026ordm; C temperature and 65\u0026plusmn;10% humidity, inside cages made of polypropylene. Water from the tap and standard pelletized feed were given. The Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2018-19/MPCOL/9) authorised the protocols for every pharmaceutical trial. The rats were divided into four groups equally and at random, and they received the following care:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGroup 1: The animals received normal saline (0.5mL/d, p.o.) for 14 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGroup 2: Syringic acid group; 50 mg/kg was continued intraperitoneally (i.p.) for 14 days,\u003c/p\u003e\n\u003cp\u003eGroup 3: BLM was applied intratracheally (7.5 units/kg, single dose) on day 1. Normal saline (0.5 mL/d, p.o.) was given for 14 consecutive days.\u003c/p\u003e\n\u003cp\u003eGroup 4: BLM was applied intratracheally (7.5 units/kg, single dose) on day 1. Syringic acid (50 mg/kg/d, i.p.) was given for 14 consecutive days.\u003c/p\u003e\n\u003cp\u003eUnder ether anaesthesia, a single intratracheal injection of BLM was administered. Saline was given orally to the Control group in place of BLM. Rats were weighed before and after the trial began. Rat blood samples were obtained for haematological parameters following the tests. After receiving BLM injections for 14 days, every rat was killed. Following the animals\u0026apos; overdose on anesthesia-induced death, the lung tissue samples were cleaned in cold saline and weighed in order to determine the lung/body mass index. By dividing the lung weight (g) by the body weight (g) and multiplying the result by 100, the lung index was calculated. Thereafter employed for histological and biochemical examination.\u0026nbsp;The right section of the lung was placed in liquid nitrogen and stored at \u0026minus;70 \u0026deg;C until the assay for thiobarbituric acid-reactive substances (TBARS) a lipid peroxidation product, superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), and glutathione peroxidase (GPx) contents. The left part of the lung was placed in formaldehyde solution for routine histopathological examination by light microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of hydroxyproline in the lung tissue\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA colorimetric assay was used to determine the left lung\u0026apos;s total collagen content [25, 26]. The left lung was dried at 80◦C until it achieved a steady weight. The dry lung was hydrolyzed overnight in a glass vial with 12-N HCl at 120C while being vacuum-operated. The sample volume was adjusted to 30 mL using distilled water, and the pH was adjusted to 7 using NaOH. After combining the sample solution (1.0 mL) with 1.0 mL of chloramine T solution (0.05 mol/L), the mixture was incubated for 20 minutes at room temperature. After adding a 20% dimethyl benzaldehyde solution to the mixture, it was incubated for 20 minutes at 60 \u0026deg;C. Every sample was analysed by measuring its absorbance at 557 nm.\u0026nbsp;\u0026nbsp;The results are expressed in micrograms of hydroxyproline per gram of wet lung weight using the standard hydroxyproline curve [24, 25].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo hundred milligrams of frozen lung tissue specimen was dissected into pieces on dry ice, homogenized in 1.15 % KCl buffer (1:9, w/v) using a manual glass homogenizer for approximately 5min and flushed with centrifugation for approximately 10s to remove large debris. The supernatant was used for analysis.\u003c/p\u003e\n\u003cp\u003eUsing a commercial kit, the activity of SOD was measured (RANSOD kit, Randox Com, UK). For SOD, absorption was measured at 412 nm and reported as (IU/mg protein). Goth\u0026apos;s colorimetric approach was used to measure the catalase (CAT) activity. After homogenised lung tissue was incubated with H2O2, the reaction was stopped after 10 minutes by mixing in ammonium molybdate. While the ammonium molybdate reacts with the sample\u0026apos;s residual H2O2 to form a yellow complex, the CAT enzyme breaks down the H2O2 in the samples to produce H2O and O2. Using a spectrophotometer, absorption was measured at 410 nm. The expression for CAT activity was (\u0026mu;mol H2O2/min/mg protein) [26]. \u0026nbsp;A commercial kit (RANSEL kit, Randox Com, UK) based on the Paglia and Valentine method [27] was used to measure the GPX activity. At 340 nm, absorption was measured, and (IU/mg of protein) was the expression for enzyme activity. Using Ellman\u0026apos;s approach, the GSH level in lung tissue was determined. Ellman\u0026apos;s reagent was applied to 40\u0026mu;l of lung tissue homogenate after it had been combined with 2ml of buffer phosphate. The GSH content of the yellow complex was measured using a spectrophotometer set to 410 nm, and was reported as (nmol/mg protein) [28]. \u0026nbsp;Thiobarbituric acid reactive substances (TBARS) production was used to measure the amount of lipid peroxidation [29]. To summarise, 0.8 ml of a solution containing 0.25N HCl, 0.375% (w/v) thiobarbituric acid, and 15% (w/v) trichloroacetic acid (TCA) was mixed with 0.2 ml of homogenised tissue. Centrifugation was used for five minutes at 5000 RPM to precipitate and remove the protein. After being transferred to test tubes containing 0.02% butylated hydroxyl toluene, the supernatants were bain-marie heated for 15 minutes at 100\u0026deg;C. After that, samples were chilled and centrifuged for five minutes at 2000 RPM to get rid of the precipitant. When measuring absorption at 532 nm, MDA concentrations were reported as (nmol/mgprotein).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of nitric oxide (NO)\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Griess method was used to determine the nitrite levels, which serve as an indicator of NO generation. First, acetonitrile (1:2, v/v) was used to deproteinize tissue samples. Subsequently, 100 \u0026mu;L of supernatant and 100 \u0026mu;L of Griess reagent were applied to a microplate well. After that, samples were incubated in microplate wells for 30 minutes at 37 \u0026deg;C, and the absorbance of the samples was eventually found at 546 nm. Based on a typical linear curve established by 0\u0026ndash;150 mol/ml sodium nitrite, the quantity of nitric oxide metabolites in the samples was ascertained. For tissue samples, the data were given in moles per gram (gr) and per litre (L) [30, 31].\u003c/p\u003e\n\u003ch3\u003eBronchoalveolar lavage Fluid\u0026nbsp;differential cell count\u003c/h3\u003e\n\u003cp\u003eThe process of obtaining bronchoalveolar lavage fluid involved injecting 3 ml of saline three times, for a total of 9 ml, and then gently aspirating the fluid out of the lung after inserting an intratracheal catheter into a trachea. The recovery ratio of lavage fluid with this catheter was roughly 80% and did not show any discernible variation across the groups. Using a hemocytometer, the total number of cells in the bronchoalveolar lavage fluid was determined. Smear slides were produced and stained with Giemsa solution to allow for differential counts of leukocytes in the broncholaveolar lavage fluid. Three hundred cells were counted differently for each sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of tumor necrosis factor-\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor necrosis factor-\u0026alpha; concentration was measured using an enzyme-linked immunosorbent assay kit (Xpressbio life scientific products). The determinations were done according to the test kit instructions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological studies\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammatory and fibrotic evidences in histopathology studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue from the left lung was blocked in paraffin and put in 10% buffered formalin for histopathological analysis. Hematoxylin and eosin (H\u0026amp;E) and Masson\u0026apos;s trichrome (MT) were used to stain 4 \u0026mu;m sections in order to assess collagen deposition, alveolar thickness, and inflammatory cell infiltration. The Ashcroft score [33] measured fibrotic lesions from 0 to 8, while the Szapiel score [32] graded alveolitis and inflammation from 0 to 3. Grading of inflammatory and fibrotic lesions was done on three slides per rat, with ten fields each slide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc.). The result was expressed as Mean \u0026plusmn; SEM and analyzed using one-way ANOVA followed by Tukey\u0026ndash;Kramer multiple comparison tests. Previously, the normal distribution of data was evaluated. Differences were considered statistically significant at (p \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of syringic acid on body weight and lung index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBLM application caused significant body weight loss after 14 days compared with the control group (p\u0026lt;0.05). In addition, zingerone (50 and 100 mg/kg) administration increased body weight compared with BLM group (p\u0026lt; 0.05) (Fig. 1a).\u003c/p\u003e\n\u003cp\u003eBLM significantly increased lung index at 14th day compared with control group (p\u0026lt; 0.05). Zingerone (50 and 100 mg/kg\u003c/p\u003e\n\u003cp\u003eAfter 14 days, BLM treatment resulted in a substantial reduction in body weight as compared to the control group (p\u0026lt;0.05).Moreover, delivery of 50 mg/kg of syringic acid resulted in an increase in body weight as compared to the BLM group (p\u0026lt; 0.01). On day 14, BLM exhibited a substantial rise in lung index as compared to the control group (p\u0026lt;0.001). When compared to the BLM group, syringic acid (50 mg/kg) dramatically reduced the lung index (Table 1, Fig 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHydroxyproline assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing hydroxyproline measurements, we calculated the amount of collagen deposited in the lung tissues. Bleomycin markedly raised the amount of hydroxyproline in the rat lungs as compared to the control group (P \u0026lt; 0.001). When compared to the BLM-treated group, the hydroxyproline content was considerably lower in the animals treated with syringic acid (50 mg/kg). The group treated with syringic acid plus bleomycin had a substantial decrease in hydroxyproline content as compared to the group treated with BLM (P \u0026lt; 0.01). (Figure 3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntioxidant defense system markers\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen compared to the control group, BLM reduced the activity of the enzymes SOD, CAT, and GPX at the same level (p \u0026lt; 0.001). Syringic acid pretreatment at a dose of 50 mg/kg boosted the activity of CAT, GPX, and SOD. These effects demonstrated a strong recovery and were comparable to the BLM group (p \u0026lt; 0.01) (Table 2). When compared to the control group, BLM decreased the levels of GSH in lung tissue (p \u0026lt; 0.001). GSH levels were elevated by 50 mg/kg (p \u0026lt; 0.001) of syringic acid. Syringic acid 50 mg/kg (p \u0026lt; 0.01) reduced lung tissue MDA and NO levels, while BLM raised MDA and NO levels (p \u0026lt; 0.001) (Fig.2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal and differential cell count in Bronchoalveolar lavage fluid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of syringic acid on total cell counts and the differential in bronchoalveolar lavage fluid between the experimental and control groups of rats is displayed in Table 3. When compared to control rats, bleomycin therapy resulted in a substantial increase in the overall cell count in the bronchoalveolar lavage fluid (p \u0026lt; 0.001). The total cell count in rats given syringic acid treatment stayed at levels comparable to those of control rats. Rats treated to bleomycin had significantly higher neutrophil and eosinophil counts in their lungs, according to the differential cell count. The increase in blood cells in the bronchoalveolar lavage caused by bleomycin was considerably decreased after a 14-day syringic acid treatment. Although the bleomycin-induced group had a lower percentage of lymphocytes and alveolar macrophages, syringic acid treatment dramatically reversed these effects (p \u0026lt; 0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePro-inflammatoy marker-Tumor necrosis factor-\u0026alpha; concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 4a, b shows the plasma levels of tumour necrosis factor-\u0026alpha;. On day 14, the plasma levels of tumour necrosis factor-\u0026alpha; protein in the rats in the bleomycin-administered group were still higher than those in the control group. At the conclusion of the trial, it was discovered that syringic acid treatment reduced the increase in tumour necrosis factor-\u0026alpha; level caused by bleomycin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn day 14, histopathological abnormalities in the lungs were found using Masson\u0026apos;s trichome staining (Fig. 6) and hematoxylin and eosin staining (Fig. 5). Typical open alveoli, interalveolar gaps with typical terminal bronchi, normal bronchiolar epithelial appearance, thin interalveolar septa, absence of inflammatory cells, and fibrosis were all seen in normal lung tissues. Rats in the bleomycin-administered group displayed deformed tissue architecture, including moderate to severe bleeding, congestion, emphysema-related sloughing of the bronchial epithelium from the basement membrane, areas of increased alveolar thickening, increased fibrosis, and leukocyte accumulation in the alveolar walls. In contrast to the bleomycin-treated group, the lungs of the rats treated with syringic acid exhibited fewer leukocytes and less thickening of the alveoli.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen localizing collagen as a distinct area in a histological preparation, Masson staining is seen to be a dependable technique. In comparison to the control group, the bleomycin-treated group showed larger fibrotic regions and an elevated grade of collagen deposition. In contrast to the bleomycin group, collagen formation was noticeably reduced in syringic acid. The Szapiel examination\u0026apos;s pathology score was used to determine the lung sections\u0026apos; semi-quantitative assessment. On day 14, the bleomycin-induced group\u0026apos;s Szapiel core was discovered to be considerably higher than that of the control group. On the fourteenth day, the Szapiel scores of the group treated with syringic acid shown a notable decline in comparison to the group treated with bleomycin.\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp\u003eOne popular experimental model used to examine pathophysiology and look into novel medications for the treatment of IPF is the BLM-induced PF model [34]. Many studies suggest that oxidative stress and inflammation in the respiratory system may be linked to the development of lung fibrosis [35].\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe findings supported the involvement of oxidative stress (shown by decreased GSH) and inflammation (neutrophil infiltration) in the BLM-induced lung damage. The reported alterations were considerably reduced by syringic acid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWeight loss is a well-known side effect of BLM administration in the treatment of cancer and pulmonary fibrosis models [35]. In line with earlier research, BLM given intratracheally reduced animal weight and raised body lung index in our investigation [36]. According to previous research, BLM's effects on hunger and protein catabolism may be the cause of these effects [37]. Previous research indicates that during the early stages of pulmonary fibrosis, the inflammatory responses stimulate and facilitate damage to the cells that line capillaries and alveolar epithelium. Later on, fibroblasts promote the formation of extracellular matrix (ECM) and collagen secretion [38]. Fibrosis results from excessive collagen remodelling and deposition, which compromises the lung tissue's structural integrity.\u003c/p\u003e\n\u003cp\u003eSince hydroxyproline makes up the majority of collagen fibres, the tissue lung's hydroxyproline content reveals the amount of collagen and the degree of pulmonary fibrosis [39]. In the current investigation, intratracheal injection of BLM considerably elevated lung hydroxyproline levels, in line with findings by Huang et al. [40] and Ramezani et al. [41]. Syringic acid treatment considerably decreased the increase in hydroxyproline levels in the lung tissue.\u0026nbsp;\u003cbr\u003e\u0026nbsp;Accordingly, a histological analysis of the lung tissues in the group that received BLM treatment revealed that the drug caused duct hyperplasia, collagen buildup, and inflammatory cell penetration. In pulmonary fibrosis animal models, BLM enhances the generation of ROS [42].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy employing Masson's trichome staining of lung sections for collagen deposition, this discovery was further supported. In the current study, bleomycin triggered the deposition, aggregation, and deposition of collagen in the peribronchial and perialveolar tissues, obliterating the alveolar gaps as small fibrils. However, the group that received syringic acid treatment had significantly less collagen deposition, which may have been caused by syringic acid's inhibitory action. [43]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ability of BLM to produce ROS is one of the generally acknowledged reasons for the lung damage and tissue remodelling it induces. It is known that BLM binds to DNA and Fe2+ to create a complex [44, 45]. Redox cycling occurs in the DNA/Fe2+/BLM complex, producing ROS such hydroxyl and superoxide radicals. We assessed the levels of oxidative stress markers in the current investigation. Apart from the inflammation and collagen deposition caused by BLM, we discovered that it also considerably increased the levels of lipid peroxide and significantly decreased the levels of antioxidant enzymes such as GPx, CAT, and SOD when compared to the control group. These findings corroborated earlier research assessing the function of oxidative stress in BLM-induced lung fibrosis [46].\u003c/p\u003e\n\u003cp\u003eFurthermore, we saw that syringic acid improved oxidative stress indicators as CAT, MDA, and SOD. Rat lung tissues in the syringic acid + BLM group had MDA content that was relatively similar to the control group's. In these mice, concurrent administration of syringic acid effectively mitigated the inflammatory consequences of BLM therapy, or syringic acid's antioxidant properties produced an at least partially preventive impact. Reduced oxidative stress has been linked to anti-inflammatory benefits of the majority of antioxidant medicines investigated for treatment of BLM-induced lung fibrosis models [47, 48].\u003c/p\u003e\n\u003cp\u003eYinfang et al. reported that syringic acid had antioxidant qualities in this regard by lowering lipid peroxidation, which was indicated by a drop in the MDA level. Furthermore, by reducing oxidative stress, syringic acid is said to be a direct scavenger of free oxygen radicals in non-alcoholic fatty liver disease brought on by a high-fat diet. It has been established that NO plays a critical role in the aetiology of lung disorders, particularly pulmonary fibrosis. Peroxynitrite, a highly reactive nitrogen species, is created when NO interacts with superoxide free radicals, causing nitrosative stress and significant lung tissue damage. When compared to the BLM group, the group that received syringic acid saw a considerable drop in the level of NO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBleomycin administered intratracheally causes interstitial inflammation in addition to the oxidative stress already discussed. This inflammation is accompanied by a notable increase in leukocyte recruitment. Leukocytes, including neutrophils, lymphocytes, and macrophages, are important for tissue remodelling and inflammation [49]. In the bronchoalveolar lavage fluid, the bleomycin-treated group exhibited a significant decrease in macrophages and a significant rise in total cells, neutrophils, and lymphocytes. This is consistent with earlier research by Sriram et al. (2009)[51] and Gong et al. (2005)[50]. Rats treated with syringic acid had similar counts of neutrophils, lymphocytes, macrophages, and total cells as the rats in the control group.\u0026nbsp;Inhibited leukocytes recruitment, which directly impacted inflammation and tissue\u0026nbsp;repair,\u0026nbsp;might\u0026nbsp;partly\u0026nbsp;account\u0026nbsp;for\u0026nbsp;the\u0026nbsp;preventive\u0026nbsp;effect of Syringic acid on bleomycin-induced pulmonary fibrosis, which\u0026nbsp;may be due to its ability to interfere with free radical-mediated\u0026nbsp;reactions.\u003c/p\u003e\n\u003cp\u003eFurthermore, among the complex webs of cellular and molecular interactions that control the fibrotic process, tumour necrosis factor-α, a strong pro-inflammatory cytokine, functions as a key player [52]. According to El-Medany et al. (2005) [53], there was a notable increase in tumour necrosis factor-α expression in the group that received bleomycin in this investigation. Bleomycin is known to induce inflammation-mediated tissue injury, which may be brought on by the generation of free radicals, which could activate nuclear factor kappa-B and enhance the synthesis of tumour necrosis factor-α [54, 55]. The activity of nuclear factor kappa-B is inhibited by syringic acid [56].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNF-α expression was significantly decreased by syringic acid, possibly due to its inhibitory influence on nuclear factor kappa-B activity. NO, iNOS, TNF-α, COX-2, and PGE2 release were all inhibited by syringic acid. Additionally, SA lessened the significant phosphorylation of the PTEN/AKT/NF-kB pathway that was caused by IL-1β [57].\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe lung interstitium experiences an excessive amount of collagen deposition as a result of an increase in fibroblast count. Inhibiting fibroblast proliferation and excessive collagen synthesis is one method of attenuating fibrosis [58]. Sections of the lungs stained with Masson's trichome showed that the syringic acid-treated group had less collagen deposition.\u003c/p\u003e\n\u003cp\u003eAccording to this study, the pulmonary response to the bleomycin challenge involves both a decrease in lung antioxidant capacity and a quick onset of oxidative stress. By inhibiting inflammation and having the ability to scavenge reactive oxygen species, syringic acid's inhibitory impact decreased oxidative stress. Further research is necessary to elucidate the protective mechanism of syringic acid on this model and explore its impact on alternative animal models of lung fibrosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eConventional corticosteroid therapy typically has no effect on pulmonary fibrosis [59]. In lung tissue, bleomycin inoculation led to decreased antioxidant capacity, increased levels of inflammatory cytokines, fibrotic alterations, and collagen accumulation; in contrast, syringic acid demonstrated pneumoprotective properties by boosting antioxidant defense, lowering inflammatory cytokine levels, and preventing collagen accumulation. Accordingly, the current findings imply that syringic acid successfully guards against the lung damage brought on by bleomycin challenge.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFig. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Figure \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ei.p. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;intra peritoneal\u003c/p\u003e\n\u003cp\u003ekg \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; kilogram\u003c/p\u003e\n\u003cp\u003emg \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;milligram\u003c/p\u003e\n\u003cp\u003eml \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; millilitre\u003c/p\u003e\n\u003cp\u003ep.o. \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; Per oral\u003c/p\u003e\n\u003cp\u003ew/w \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;weight/ weight\u003c/p\u003e\n\u003cp\u003ew/v \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;weight/ volume\u003c/p\u003e\n\u003cp\u003eNO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;nitric oxide\u003c/p\u003e\n\u003cp\u003eGSH \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;reduced glutathione\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp; \u0026nbsp; \u0026nbsp;malondialdehyde\u003c/p\u003e\n\u003cp\u003eCAT \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;catalase\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;superoxide dismutase\u003c/p\u003e\n\u003cp\u003eGPx \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;glutathione peroxidase\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; \u0026nbsp; \u0026nbsp;\u0026nbsp;tumor necrosis factor alpha\u003c/p\u003e\n\u003cp\u003eNFkB \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;nuclear factor kappa-light-chain-enhancer of activated B cells\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthors\u0026rsquo; contributions\u003c/p\u003e\n\u003cp\u003eBVSL and BKV wrote the manuscript. BVSL and BKV designed and performed the study. All authors participated in data analysis and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThere was no specific fund for this study.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eData analyzed and used for this manuscript are available within the manuscript.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe Institutional Animal Ethics Committee (Reg no: MRCP/CPCSEA/IAEC/2018-19/MPCOL/9) authorized the protocols for every pharmaceutical trial.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAuthor details\u003c/p\u003e\n\u003cp\u003e1. Department of Pharmacology, School of Allied and Healthcare Sciences, Malla Reddy University, Hyderabad, Telangana, India. 2. Department of Pharmacology, School of Pharmacy, KPJ Healthcare University College, Nilai, Malaysia.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRaghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, Colby TV, Cordier JF, Flaherty KR, Lasky JA et al, An official ATS/ERS/JRS/ALAT statement idiopathic pulmonary fibrosis:evidence based guidelines for diagnosis and management. Am. J. Respir. Crit. Care Med. 2011; 183: 788\u0026ndash;824.\u003c/li\u003e\n \u003cli\u003eCottin V, The role of pirfenidone in the treatment of idiopathic pulmonaryfibrosis. Respir. Res. 2013; 14 (1): 5. doi:10.1186/1465-9921-14S1-S5\u003c/li\u003e\n \u003cli\u003eChalabi N, Bernard-Gallon DJ, Vasson MP, Bignon YJ. Personalized Med, Medicinal plants as potential sources of lead compounds with anti-platelet and anti-coagulant activities. 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Thymoquinone blocks lung injury and fibrosis by attenuating bleomycin-induced oxidative stress and activation of nuclear factor Kappa-B in rats. Toxicology 2012; 302:106\u0026ndash;113\u003c/li\u003e\n \u003cli\u003eYinfang Li, Li Zhang, Xiaohua Wang, Wei Wu\u003csup\u003e\u0026nbsp;\u003c/sup\u003e, Rui Qin. Effect of Syringic acid on antioxidant biomarkers and associated inflammatory markers in mice model of asthma, Drug Dev Res . 2019 Mar; 80(2):253-261.\u003c/li\u003e\n \u003cli\u003eYildirim Z, Kotuk M, Erdogan H, Iraz M, Yagmurca M, Kuku I, Fadillioglu E. Preventive effect of melatonin on bleomycininduced lung fibrosis in rats. J Pineal Res. 2006; 40:27\u0026ndash;33.\u003c/li\u003e\n \u003cli\u003eMengliC,CheungFWK,MingHungC,PakKwanH,Siu-PoI,YickHin L, Chun-Tao C, Wing Keung L. Protective roles of Cordyceps on lung fibrosis in cellular and rat models. J ethnopharmacol. 2012; 143(2):448\u0026ndash;54.\u003c/li\u003e\n \u003cli\u003eXin Wei, X., Han, J., Chen, Z., Qi, B., Wang, G., Ma, Y., Zheng, H., Luo, Y., Wei, Y., Chen, L., Aphosphoinositide3-kinase-\u0026gamma;inhibitor,AS605240preventsbleomycin-inducedpulmonaryfibrosisinrats.BiochemicalandBiophysicalResearchCommunications. 2010; 397:311\u0026ndash;317.\u003c/li\u003e\n \u003cli\u003eGong, L., Li, X., Wang, H., Zhang, L., Chen, F., Cai, Y., Qi, X., Liu, L., Liu, Y., Xiong-fei Wu, X., Huang, C., Ren, J., EffectofFeitaionbleomycin-inducedpulmonaryfibrosisinrats.JournalofEthnopharmacology. 2005; 96:537\u0026ndash;544.\u003c/li\u003e\n \u003cli\u003eSriram, N., Kalayarasan, S., Sudhandiran, G., Epigallocatechin-3-gallateaugmentsantioxidantactivitiesandinhibitsinflammationduringbleomycin-inducedexperimentalpulmonaryfibrosisthroughNrf2\u0026ndash;Keap1signaling.PulmonaryPharmacologyandTherapeutics 2009; 22:221\u0026ndash;236.\u003c/li\u003e\n \u003cli\u003eRazzaque, M.S., Taguchi, T., Pulmonaryfibrosis:cellularandmolecularevents.PathologyInternational.2003; 53: 133\u0026ndash;145.\u003c/li\u003e\n \u003cli\u003eEl-Rakhawy, F.I., El-Medany, G., 2005. Attenuationofbleomycininducedlungfibrosisinratsbymesna.EuropeanJournalofPharmacology. 2005; 509:61\u0026ndash;70.\u003c/li\u003e\n \u003cli\u003eOrtiz, L.A., Champion, H.C., Lasky, J.A., Gambelli, F., Gozal, E., Hoyle, G.W., Beasley, M.B., Hyman, A.L., Friedman, M., Kadowitz, J., 2002. EnalaprilprotectsmicefrompulmonaryhypertensionbyinhibitingTNF-mediatedactivationofNF-kBandAP-1.AmericanJournalofPhysiology\u0026ndash;LungCellularandMolecularPhysiology282,1209\u0026ndash;1221.\u003c/li\u003e\n \u003cli\u003eKalayarasan, S., Sriram, N., Sudhandiran, G., Diallylsulfideattenuatesbleomycin-inducedpulmonaryfibrosis:criticalroleofiNOS,NF-nB,TNF-\u0026alpha;andIL-1\u0026beta;.LifeSciences. 2008; 82:1142\u0026ndash;1153.\u003c/li\u003e\n \u003cli\u003eOluwatobi T Somade., Olubisi E Adeyi et al., Syringic and ascorbic acids prevent NDMA-induced pulmonary fibrogenesis, inflammation, apoptosis, and oxidative stress through the regulation of PI3K-Akt/PKB-mTOR-PTEN signaling pathway. Metabol Open 2022; 14;1-5.\u003c/li\u003e\n \u003cli\u003eYao Li., Yurun Zhu et al., Syringic acid inhibits IL-1\u0026beta;-induced inflammation in mice chondrocytes and ameliorates the progression of osteoarthritis via the PTEN/AKT/NF-\u0026kappa;B pathway. Journal of functional foods, 2023; 107: 105683.\u003c/li\u003e\n \u003cli\u003eGong, L., Li, X., Wang, H., Zhang, L., Chen, F., Cai, Y., Qi, X., Liu, L., Liu, Y., Xiong-fei Wu, X., Huang, C., Ren, J., 2005. EffectofFeitaionbleomycin-inducedpulmonaryfibrosisinrats.JournalofEthnopharmacology. 2005; 96: 537\u0026ndash;544.\u003c/li\u003e\n \u003cli\u003eGreen, F.H.Y., Overviewofpulmonaryfibrosis.Chest 2002; 122:334\u0026ndash;339.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bleomycin, Pulmonary fibrosis, Syringic acid, Anti-fibrotic, Anti-inflammatory, Antioxidant","lastPublishedDoi":"10.21203/rs.3.rs-4350558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4350558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Chronic administration of bleomycin (BLM), a chemotherapeutic drug, has been linked to idiopathic pulmonary fibrosis (IPF). It has been observed that syringic acid, a phenolic compound, has antiapoptotic, anti-inflammatory, and antioxidant properties. To assess syringic acid's therapeutic potential against lung fibrosis caused by BLM and determine a potential mechanism of action.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eSprague-Dawley rats were inducted into IPF after receiving 7.5 IU/kg of BLM intratracheally. Syringic acid (50 mg/kg, i.p.) was administered to rats for 14 days, after which different parameters in the lung and bronchoalveolar lavage fluid (BALF) were measured.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAltered BALF differential cell counts, elevated lung index, hydroxyproline, NO, and MDA plasma levels, and reduction in GSH, GPx, SOD, and CAT in group receiving BLM were evidence of pulmonary toxicity. Administering 50 mg/kg of syringic acid significantly reduced (p \u0026lt; 0.001) the changes brought about by BLM. The expression of TNF-α was greatly reduced by syringic acid when it was stimulated by BLM. The BLM-treated group's histological analysis revealed significant lung damage with alveolar septal thickening, interstitial infiltration, collapsing alveolar gaps, and an elevated Ashcroft and Szapiel score. Syringic acid was used to lessen these effects. The syringic acid group (p\u0026lt;0.01) markedly reduced Szapiel score, collagen deposition, lung edema, and fibrotic alterations. It also inhibited the infiltration of myofibroblasts and inflammatory cells, primarily macrophages and lymphocytes. By inhibiting the TGF-β1/NF-κB pathways, syringic acid mitigates BLM-induced IPF. This, in turn, improves the control of oxidant and pro-inflammatory markers (TNF-α) to decrease collagen deposition during pulmonary fibrosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eFinally, it is concluded that Syringic acid can protect the lung against BLM-induced pulmonary oxidative stress, inflammation and fibrosis.\u003c/p\u003e","manuscriptTitle":"Syringic Acid ameliorates Bleomycin induced Pulmonary Inflammation and Fibrosis in rats via maintenance of endogenous anti-oxidants and downregulation of pro-inflammatory markers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-13 13:54:08","doi":"10.21203/rs.3.rs-4350558/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"008fe67e-28b0-4793-b69d-1278aff95798","owner":[],"postedDate":"May 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-07T21:23:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-13 13:54:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4350558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4350558","identity":"rs-4350558","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}