Increased Expression of Desmin Contributes to the Protection of Mustard seed against iron overload- Induced cardio-toxicity in Rats

Increased Expression of Desmin Contributes to the Protection of Mustard seed against iron overload- Induced cardio-toxicity in Rats | 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 Article Increased Expression of Desmin Contributes to the Protection of Mustard seed against iron overload- Induced cardio-toxicity in Rats Benson onyeije, Silvanus Innih This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8969169/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Although iron is an essential element for life, its excess is linked to many disorders. Objective The aim of this study is to investigate the effect of mustard seed on iron induced toxicity on the heart in adult male albino rats using biochemical and immunohistochemical methods. Methods A total of 35 rats weighing between 180-250g were divided into seven groups (A, B, C, D, E, F and G) of five per group. Group A (control) was administered 1ml of distilled water, group B, C, D and E were induced with iron (II) chloride for sixty days and treated with different doses of the extract except group B for sixty days, group E was treated with standard drugs at the same time interval, while group F and G received 200mgkg and 400mg/kg of extract respectively using orogastric tube. After last day of administering drugs, the rats were left for an overnight fast and then sacrificed 24 hours later. Heart specimens were dissected from scarified rats to estimate tissue level malondialdehyde (MDA), antioxidant enzymes (SOD, CAT and GPx) and immunohistochemical staining of desmin. Results Increases in cardiac MDA and decreases in SOD, CAT, and GPx were brought on by iron overload, along with degenerative alterations in the tissues under examination and a decline in desmin expression. All of these measures showed a notable improvement following the administration of mustard seed extract. Conclusion By lowering the iron levels in heart tissue and reducing oxidative stress and inflammatory effects brought on by iron excess, mustard seed may function as an iron chelator. Biological sciences/Biochemistry Health sciences/Cardiology Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Physiology Mustard seed (Brassica nigra) Iron (II) chloride Desmin Toxicity Heart Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Iron is an essential element in biological systems because of its ability to shuttle between two oxidative states and plays a key role in cell metabolism and homeostasis, [ 20 ]. Excess iron (iron overload) or lack of iron (iron deficiency) are the two major pathophysiological states of abnormal iron metabolism, [ 4 ]. Under physiological conditions, iron transport is highly conserved and controlled by iron transporters including transferrin and its receptors via negative feedback regulatory mechanisms, [ 10 ]. Iron-overload cardiomyopathy is the most common cause of mortality in patients with secondary iron-overload and is a major co-morbidity in patients with genetic hemochromatosis, [ 22 ]. Altered iron homeostasis allows uncontrolled iron entry and deposition in different organs including the heart leading to progressive tissue damage and end-organ failure, [ 10 ]. Uncontrolled iron absorption causes transferrin saturation and increased levels of non-transferrin‐bound iron, which is highly reactive, toxic, and triggers oxidative stress, [ 16 ]. Iron‐induced oxidative stress is a key driver in the pathogenesis of myocardial tissue injury and progressive development of iron‐overload cardiomyopathy, [ 19 ]. Excess iron promotes oxidative stress via the Fenton reaction, which plays a key pathogenic role in myocardial injury and heart failure, [ 15 ]. Desmin has been defined as the constitutive subcomponent of intermediate filaments seen in skeletal, cardiac and smooth muscles. Early research revealed that the primary function of this protein was to provide structural and mechanical support. However, it was later determined that desmin, in addition to these functions, affects many biological processes including myogenesis, muscle contraction, and mitochondrial functioning, [ 2 , 7 ]. Mutations that may occur in Desmin have been reported to cause mechanical integrity deterioration as a result of misalignment of myofibrils, mitochondrial dysfunction, and myopathy in the heart and skeletal muscles, [ 2 ]. In particular, it has been stated that desmin is the main protein of the muscles and also has a critical function in the development of the heart muscle, in addition to the contraction mechanism of the muscle cells at the embryonic stage, [ 17 ]. During the literature survey performed as part of this study, no research was found regarding the localization and distribution of these proteins as a marker in iron overload myocardial degeneration. There has been a revival of attention in plant-based medicines due to the increase responsiveness of the limited ability of synthetic pharmaceutical product to control major disease and the need to determine new molecular structures as the lead compounds from other sources, comprising the plant kingdom, [ 11 ]. Mustard seed is a well-known medicinal plant in traditional medicinal system and new scientific studies have highlighted the possible use of mustard seed modern medicine, [ 14 ]. The seeds are a very good source of phosphorus, manganese, dietary fiber, magnesium, selenium, iron, calcium, protein, niacin, zinc and omega-3 fatty acids, [ 13 ]. Pharmacological activities of isolated constituents from mustard seed including antioxidant, anxiolytic, antitumor, hypoglycemic, antidiabetic, antifungicidal, antimicrobial, goitrogenic and allergenicity activities have been documented, [ 13 ] The current study uses biochemical and immunohistochemical alterations as an indicator to examine how mustard seed extract protects wistar rats from induced cardiac iron overload injury. MATERIALS AND METHOD Ethical Approval This study was reviewed and approved by the University of Benin Ethics and Research Committee with approval number CMS/REC/2024/756. Collection of plant (Mustard Seed) Mustard seed ( Brassica nigra seeds) were procured in Abavo Market in Ika South Local Government Area, Delta State and identified by the Department of Plant Biology and Biotechnology of the University of Benin with the herbarium number UBH-B539. Extraction of Mustard Seed The 3000g of mustard seeds were air dried and ground into a powder using a blender. The powdered samples were then kept in polythene bags at room temperature until they were extracted. An extraction bottle containing 1,200 grams (1200 grams) of powdered sample was filled with 4000 milliliters of 70% alcohol, agitated, and refrigerated at 40 degrees Celsius for 72 hours. Whatman No. 1 filter paper was then used to sieve and filter the mixture. The filtrates were then concentrated on water bath at 400 C and stored in the refrigerator until needed at the University of Benin in the Department of Anatomy, [28]. And a portion of the residue was dissolved in the proper amount of distilled water for use in the experiment . Iron (II) Chloride About 10g of Iron Ⅱ chloride crystals manufactured by MOLYCHEM® with catalog number 13478-10-9 was dissolved in 100 ml of distilled water to form Iron chloride solution and administered to model iron overload. Acute oral toxicity of mustard seed Acute toxicity study was done in accordance with Lorke’s method, [27] Experimental animals and protocol Adult Wistar rats weighing between 180g and 250g were used for this experiment. They were bought from the animal house of the department of Anatomy of the University of Agbor. The animals were kept in the same animal house in typical cages. Throughout the course of the research, they were given access to clean water and normal pellet animal food, as needed. Acclimatization took place over the course of two weeks (14 days). Management and experimental protocols were in accordance with recommendation of the PHS (Public Health Service), Public Health Service Policy on Human Care and Use of Laboratory Animals, 2002. Experimental Design Thirty- five adult Wistar rats weighing between 180g and 250g were used for this study. The Wistar rats were randomly divided into seven groups of five rats in each groups. Group A received 1ml of distil water Group B 2mg/kg bw of Iron (II) Chloride only Group C 2mg/kg bw of Iron (II) Chloride + 200mg/kg of mustard seed Group D 2mg/kg bw of Iron (II) Chloride + 400mg/kg of mustard seed Group E 2mg/kg bw of Iron (II) Chloride + Vitamin C Group F 200mg/kg bw of mustard seed extract only Group G 400mg/kg bw ofmustard seed extract only The course of treatment and administration was 60 days. The rats were anesthetized with a chloroform desiccator 24 hours after the previous treatment, and the heart tissues were promptly extracted and preserved in a 4% paraformaldehyde solution for the subsequent examination. Dehydration was performed in ascending grades of the ethanol and embedding in paraffin and processed for sections of 5 μm thickness. Serial sections of 5 micrometer thickness were taken from the prepared paraffin blocks. Sections were taken on slides previously coated with APES (3 amino propyl triethoxysilan; Sigma–Aldrich Chemicals, St.Louis, MO, USA) for immunohistochemistry staining. Cardiac Enzymes The antioxidant enzymes GPx, CAT and SOD, as well as the product of oxidative stress MDA, were detected using a spectrophotometric method. Preparation of heart homogenate was done using a tissue homogenizer (100 mg tissue per mL of 50 mM PBS). The homogenate was centrifuged, then the supernatant was used for biochemical analyses according to the manuals of commercially kits (Jian Cheng Biological Engineering Institute) Immunohistological Evaluation of Specimens . Using the techniques described in the previously published literature, immunohistochemical staining for Desmin was performed [23]. Sections were deparaffinized, rehydrated, and treated with methanol containing 0.03 percent hydrogen peroxide for 20 minutes before being sliced into 5 μm thick pieces. Following a 20-minute incubation period with normal serum to inhibit non-specific antibodies, heart sections were treated with anti-mouse monoclonal primary antibody against desmin (1:100, DAKO, Denmark), [24], and then anti-mouse IgG (1:500, Sigma-Aldrich), [1]. The peroxidase anti-mouse IgG (1:100, DAKO, Denmark) secondary antibody was used. Diaminobenzidine, or DAB, was used as a chromogen to visualize the reaction (Dako, Glostrup, Denmark).Slides were counterstained with Mayer’s hematoxylin and finally dehydrated, rendered transparent with xylene and cover slipped. The negative control was performed by neglecting the primary antibody. Slides were analyzed under a light microscope to identify areas with brownish color which considered as sign of a positive reaction. These procedures were carried out in the Department of Histopathology lab of the University of Benin Teaching Hospital Benin City, Nigeria. Morphometric Analysis For image analysis, the mean area % of desmin immunoreactivity was calculated using a Leica Qwin 500 image system (Cambridge, England). Ten non-overlapping fields on stained slides selected from every animal in every group were used for the measurements. Photography was conducted by Prof Innih S Orlu at the histopathology laboratory, University of Benin, Nigeria at a magnification of 100×. Statistical Analysis The results were analysed using one way analysis of variance (ANOVA), followed by the Tukey’s post-hoc test. All values are presented as the mean (M) ± standard deviation (SD). Differences between the groups were considered significant when the probability of chance (p) is less than 0.05 (p < 0.05). All the data collected from the experiment was calculated and analyzed using SPSS software version 16 (SPSS, Chicago, USA). RESULTS Oxidative Stress The results shown a significance decrease in the activities of antioxidant enzymes SOD, CAT and GPx in Fe overload group, and a significant increase in concentration of peroxidation product MDA when with the control and mustard seed treated groups.. In contrast, the activities of SOD, CAT and GPx were strengthened, and the level of MDA was decreased under the effect of mustard seed extract. We found that the degree of oxidation in the mustard seed group was decreased, and the antioxidant activity of vitamin C is between the high dose mustard seed and low dose mustard seed Immunohistochemistry staining revealed that desmin exhibited varying levels of immunoreaction in various rat cardiac cells that had been exposed to Fe overload. In the control and mustard seed treatment groups, Purkinje cells and heart muscle cells (myocytes) found in the myocardium showed a high cytoplasmic localized immunoreaction to Desmin protein (Fig. 2A, 2C, 2D, 2E, and 2F). Nonetheless, it was shown that the iron overload model group had a somewhat less severe reaction (Figure 1B). Similarly, it was shown that the standard antioxidant medication (Vitamin C group) exhibited high desmin localization. Morphometric Results Statistical analysis of the morphometric findings of desmin immunostaining in the heart showed a significant (p ˂0.05) increase in the area percentage of desmin in the control and mustard seed treated groups when compared to the Fe overload model group. While, vitamin C group (F) manifested a significant (p ˂0.05) increase in the area percentage of the desmin as compared to the Fe overload group. DISCUSSION The present study adds new data regarding the possible defensive effect of mustard seed ( Brassica nigra ) against Fe induced cardiac injury in adult wistar rats. In this studies, we found that the protective mechanisms of mustard seed against iron overload cardiac damage were at least in part due to decreased iron deposition and inhibition of oxidative stress. This study also exemplified that mustard seed extract administration can inhibit oxidative injury by increasing SOD, CAT, GPx activity and decreasing MDA concentration. Previous research has suggested that, under iron overload conditions, excessive labile irons have a propensity for inducing and generating reactive oxygen species (ROS), resulting in cell oxidative damage, [6, 25]. This indicates that oxidative stress is secondary to intracellular iron overload. Therefore, reducing iron deposition may be a basic way to prevent iron overload. In the present study, iron-overloaded rats exhibited clear signs of cardiac damage, as evidenced by altered cardiac biomarkers, histopathological abnormalities, and disrupted structural protein expression. These findings are consistent with previous reports demonstrating the vulnerability of cardiac tissue to iron-mediated oxidative stress, [21]. . The current study revealed that desmin expression was significantly upregulated in rats given mustard seed in addition to iron overload, which is one of the study's main findings. Cardiomyocytes' intracellular organization, mechanical stability, and structural integrity are all preserved by the essential intermediate filament protein desmin. Previous data had showed that there is a clear correlation between cardiomyopathy and compromised cardiac function and reduced or disordered desmin expression, [19]. It was documented that decrease in the amount of desmin in human cardiomyocytes and its focal deficits was published in the year 2004 and this was found in patients with post-infarction cardiovascular heart failure. Asangri et al. [3] affirmed that cardiac dysfunction is likely caused by low intracellular expression of desmin. Our study also showed that a decreased accumulation of desmin in cardiomyocytes was associated with more abnormal clinical parameters as compared with a group of rats treated with mustard seed. This may be a result of damage of the desmin network in cardiomyocytes, likely caused by accumulation of cytoskeleton proteins. Gradual increase of the size of cardiomyocytes and loss of proper function of contractility fibres is a well-known scenario in the development of cardiovascular heart diseases, [9]. Accumulating intermediate filaments disrupt the desmin network and consequently impair its function aimed primarily at protecting structural and functional integrity of myofibrils and being responsible for cell cohesion, [5]. A downregulation of desmin immunoreactivity was further supported by the significant decrease in its mean area percentage as compared to the control group. The cardioprotective effect of mustard seed may be attributed to its rich antioxidant and anti-inflammatory phytochemical composition , including glucosinolates, phenolic compounds, and flavonoids. Similar findings on the phytochemical of mustard seed was reported by Vinyas et al . [26]. These compounds likely mitigate iron-induced oxidative stress, thereby preventing desmin degradation and supporting its synthesis or stabilization. By maintaining desmin integrity, mustard seed treatment may preserve sarcomere alignment, mitochondrial positioning, and overall cardiomyocyte resilience. Administration of mustard seeds at various doses significantly up-regulated desmin expressivity in iron overload induced heart damage. This observations further support this mechanism, as mustard seed–treated rats showed reduced myocardial degeneration, less cellular disorganization, and improved tissue architecture compared to iron-overloaded controls. This structural preservation correlates well with enhanced desmin expression, reinforcing the role of cytoskeletal protection in attenuating cardiotoxicity. These findings were in parallel and supported by Cohort study in patients with cardiomyopathy and cardiac heart failure in which mustard seed with high phenolic compound alleviated the risk of cardiac decompensation and inflammation by reducing numerous inflammatory cytokines, [25]. Collectively, these results suggest that upregulation of desmin expression is not merely an associated finding but a functional contributor to the cardioprotective effects of mustard seed . The modulation of desmin may represent an important molecular mechanism through which mustard seed counteracts iron overload–induced cardiac injury. This highlights desmin as a potential therapeutic target and supports the use of natural dietary antioxidants as adjunct strategies in managing iron-induced cardiotoxicity. Therefore, the observed increase in desmin expression suggests a protective, compensatory response that helps preserve cytoskeletal architecture under oxidative stress conditions. The immunohistochemical findings obtained in the present study support the view that iron overload has damaging effects and causes a variety of the histological alterations in the heart. Crucially, ethanol seed extract of Brassica nigra protects against heart damage by decreasing the myocardial degradation, increase desmin expression and maintained structural integrity of the heart muscles. Though, additional studies are needed to detect further molecular mechanisms. CONCLUSION Further studies exploring upstream signaling pathways involved in desmin regulation and long-term functional outcomes would help clarify the full cardioprotective potential of mustard seed. Declarations Acknowledgements The authors would like to thank Eze Gerald in Histopathology Lab University of Benin Teaching Hospital, Benin City for preliminary results on desmin activity on iron overload induced cardiac damage and also to the Department of human Anatomy University of Delta Agbor, Nigeria Conflicts of interests The authors declare no conflict of interest Dual Publication Nil Third Party Material Nil Authors’contributions Onyeije Benson conceived and design the study, as well as methodology and data analysis. Immunohitochemical analysis was done by Innih Silvanu. He critically review and edited the manuscript. All authors read and approved the final manuscript. Availability of data and materials The datasets used and/or analysed during this current study are available from the corresponding author on reasonable request Funding No financial support was obtained from any source for this research project. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8969169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":621711152,"identity":"2ddceac2-11c5-4a2f-9322-650fe9249887","order_by":0,"name":"Benson onyeije","email":"data:image/png;base64,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","orcid":"","institution":"University of Benin","correspondingAuthor":true,"prefix":"","firstName":"Benson","middleName":"","lastName":"onyeije","suffix":""},{"id":621711153,"identity":"0584a5cc-69c5-47d7-a9e0-2147e55ea62b","order_by":1,"name":"Silvanus Innih","email":"","orcid":"","institution":"University of Benin","correspondingAuthor":false,"prefix":"","firstName":"Silvanus","middleName":"","lastName":"Innih","suffix":""}],"badges":[],"createdAt":"2026-02-25 15:09:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8969169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8969169/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106822549,"identity":"081883de-b2cf-4d15-9372-c7e7e58f8524","added_by":"auto","created_at":"2026-04-13 19:19:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92594,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of mustard seed on Activities of SOD, CAT, GPx and Concentration of MDA in the Cardiac Homogenate.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8969169/v1/9f60881a348b69c55e0b2138.png"},{"id":107480591,"identity":"8f87e8c1-74cf-4706-89d4-4f3dcc0fb37f","added_by":"auto","created_at":"2026-04-22 02:12:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":792550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlate A.\u003c/strong\u003e Rat heart control reacted with Desmin immunostain: cardiomyocyte Fascicles: strongly positive (+++), long arrow, weakly positive fascicles (+): short arrow. \u003cstrong\u003ePlate B.\u003c/strong\u003e Rat heart given FeCl\u003csub\u003e2\u003c/sub\u003e only reacted with Desmin immunostain:\u0026nbsp; most cardiomyocyte fascicles: weakly positive (+), long arrow, perivascular fascicles: negative (-), short arrow, vascular wall: strongly positive (+++). \u003cstrong\u003ePlate C.\u003c/strong\u003e Rat heart given FeCL\u003csub\u003e2\u003c/sub\u003e + 200mg/kg B. nigra reacted with Desmin immunostain: cardiomyocyte fascicles: strongly positive (+++), long arrow, some fascicles: weakly positive (+), short arrow, vascular wall: moderately positive (++), arrow head: arrow head: \u003cstrong\u003ePlate D\u003c/strong\u003e. Rat heart given FeCL2 + 400mg/kg B. nigra and stained with Desmin immunostain: remarkable number of cardiomyocyte fascicles: moderately positive (++), long arrow vascular wall: weakly positive (+), short arrow: Desmin 100 X\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8969169/v1/96fd27e838550c7d7145ab8d.png"},{"id":106960594,"identity":"4db60867-225e-451e-a0d5-ae3c91c34043","added_by":"auto","created_at":"2026-04-15 09:21:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":604029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlate E.\u003c/strong\u003e Rat heart given FeCl\u003csub\u003e2\u003c/sub\u003e + Vit. C reacted with Desmin immunostain: many cardiomyocyte fascicles: strongly positive (+++), long arrow: many fascicles: equivocal (+-), short arrow: \u003cstrong\u003ePlate F. \u003c/strong\u003eRat heart given 200mg/kg \u003cem\u003eB. nigra\u003c/em\u003e only and stained with Desmin immunostain: remarkable number of cardiomyocyte fascicles: strongly positive (+++), long arrow, some fascicles: equivocal (+-), short arrow:\u0026nbsp; \u003cstrong\u003ePlate G\u003c/strong\u003e. Rat heart given 400mg/kg \u003cem\u003eB. nigra\u003c/em\u003e only reacted with Desmin immunostain: cardiomyocyte fascicles, strongly positive (+++), long arrow, vascular wall: moderately positive (++), arrow head, some fascicles: weakly positive (+), short arrow: Desmin 100 X\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8969169/v1/9ed31bd05f10f541ed562abd.png"},{"id":106822551,"identity":"8a26de9f-dc6e-42f3-843d-3ace032395f8","added_by":"auto","created_at":"2026-04-13 19:19:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":26217,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of cell count of Desmin immunoreactivity\u003cem\u003e. Values are expressed at mean ± standard error of mean; p\u0026lt;0.05 is considered statistically significant.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSuperscript a represent statistical significant at p\u0026lt;0.05 compared to control: Superscript b represent statistical significant at p\u0026lt;0.05 compared to fecl\u003c/em\u003e\u003csub\u003e\u003cem\u003e2.\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8969169/v1/32cce20dd1b351e468caf0cc.png"},{"id":107484442,"identity":"7b6ff29e-21a1-4c5e-83a6-11eaf105c096","added_by":"auto","created_at":"2026-04-22 02:32:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2528377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8969169/v1/ba710327-aa58-4fc7-9554-d920b2b18b0a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased Expression of Desmin Contributes to the Protection of Mustard seed against iron overload- Induced cardio-toxicity in Rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIron is an essential element in biological systems because of its ability to shuttle between two oxidative states and plays a key role in cell metabolism and homeostasis, [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Excess iron (iron overload) or lack of iron (iron deficiency) are the two major pathophysiological states of abnormal iron metabolism, [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Under physiological conditions, iron transport is highly conserved and controlled by iron transporters including transferrin and its receptors via negative feedback regulatory mechanisms, [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Iron-overload cardiomyopathy is the most common cause of mortality in patients with secondary iron-overload and is a major co-morbidity in patients with genetic hemochromatosis, [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Altered iron homeostasis allows uncontrolled iron entry and deposition in different organs including the heart leading to progressive tissue damage and end-organ failure, [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Uncontrolled iron absorption causes transferrin saturation and increased levels of non-transferrin‐bound iron, which is highly reactive, toxic, and triggers oxidative stress, [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Iron‐induced oxidative stress is a key driver in the pathogenesis of myocardial tissue injury and progressive development of iron‐overload cardiomyopathy, [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Excess iron promotes oxidative stress via the Fenton reaction, which plays a key pathogenic role in myocardial injury and heart failure, [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDesmin has been defined as the constitutive subcomponent of intermediate filaments seen in skeletal, cardiac and smooth muscles. Early research revealed that the primary function of this protein was to provide structural and mechanical support. However, it was later determined that desmin, in addition to these functions, affects many biological processes including myogenesis, muscle contraction, and mitochondrial functioning, [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Mutations that may occur in Desmin have been reported to cause mechanical integrity deterioration as a result of misalignment of myofibrils, mitochondrial dysfunction, and myopathy in the heart and skeletal muscles, [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In particular, it has been stated that desmin is the main protein of the muscles and also has a critical function in the development of the heart muscle, in addition to the contraction mechanism of the muscle cells at the embryonic stage, [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. During the literature survey performed as part of this study, no research was found regarding the localization and distribution of these proteins as a marker in iron overload myocardial degeneration.\u003c/p\u003e \u003cp\u003eThere has been a revival of attention in plant-based medicines due to the increase responsiveness of the limited ability of synthetic pharmaceutical product to control major disease and the need to determine new molecular structures as the lead compounds from other sources, comprising the plant kingdom, [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mustard seed is a well-known medicinal plant in traditional medicinal system and new scientific studies have highlighted the possible use of mustard seed modern medicine, [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The seeds are a very good source of phosphorus, manganese, dietary fiber, magnesium, selenium, iron, calcium, protein, niacin, zinc and omega-3 fatty acids, [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Pharmacological activities of isolated constituents from mustard seed including antioxidant, anxiolytic, antitumor, hypoglycemic, antidiabetic, antifungicidal, antimicrobial, goitrogenic and allergenicity activities have been documented, [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe current study uses biochemical and immunohistochemical alterations as an indicator to examine how mustard seed extract protects wistar rats from induced cardiac iron overload injury.\u003c/p\u003e"},{"header":"MATERIALS AND METHOD","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the University of Benin Ethics and Research Committee with approval number CMS/REC/2024/756.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of plant (Mustard Seed)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMustard seed (\u003cem\u003eBrassica nigra\u003c/em\u003e seeds) were procured in Abavo Market in Ika South Local Government Area, Delta State and identified by the Department of Plant Biology and Biotechnology of the University of Benin with the herbarium number UBH-B539.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction of Mustard Seed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3000g of mustard seeds were air dried and ground into a powder using a blender. The powdered samples were then kept in polythene bags at room temperature until they were extracted. An extraction bottle containing 1,200 grams (1200 grams) of powdered sample was filled with 4000 milliliters of 70% alcohol, agitated, and refrigerated at 40 degrees Celsius for 72 hours. Whatman No. 1 filter paper was then used to sieve and filter the mixture. The filtrates were then concentrated on water bath at 400\u003csup\u003eC\u003c/sup\u003e and stored in the refrigerator until needed at the University of Benin in the Department of Anatomy, [28]. And a portion of the residue was dissolved in the proper amount of distilled water for use in the experiment\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIron (II) Chloride\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAbout 10g of Iron Ⅱ chloride crystals manufactured by MOLYCHEM® with catalog number 13478-10-9 was dissolved in 100 ml of distilled water to form Iron chloride solution and administered to model iron overload.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcute oral toxicity of mustard seed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcute toxicity study was done in accordance with Lorke’s method, [27]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental animals and protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Adult Wistar rats weighing between 180g and 250g were used for this experiment. They were bought from the animal house of the department of Anatomy of the University of Agbor. The animals were kept in the same animal house in typical cages. Throughout the course of the research, they were given access to clean water and normal pellet animal food, as needed. Acclimatization took place over the course of two weeks (14 days).\u0026nbsp;Management and experimental protocols were in accordance with recommendation of the PHS (Public Health Service), Public Health Service Policy on Human Care and Use of Laboratory Animals, 2002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty- five adult Wistar rats weighing between 180g and 250g were used for this study. The Wistar rats were randomly divided into seven groups of five rats in each groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup A\u003c/strong\u003e received 1ml of distil water\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup B\u003c/strong\u003e 2mg/kg bw of Iron (II) Chloride only\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup C\u003c/strong\u003e 2mg/kg bw of Iron (II) Chloride + 200mg/kg of mustard seed\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup D\u003c/strong\u003e 2mg/kg bw of Iron (II) Chloride + 400mg/kg of mustard seed\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup E\u003c/strong\u003e 2mg/kg bw of Iron (II) Chloride + Vitamin C\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup F\u003c/strong\u003e 200mg/kg bw of mustard seed extract only\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup G\u003c/strong\u003e 400mg/kg bw ofmustard seed extract only\u003c/p\u003e\n\u003cp\u003eThe course of treatment and administration was 60 days. The rats were anesthetized with a chloroform desiccator 24 hours after the previous treatment, and the heart tissues were promptly extracted and preserved in a 4% paraformaldehyde solution for the subsequent examination.\u003c/p\u003e\n\u003cp\u003eDehydration was performed in ascending grades of the ethanol and embedding in paraffin and processed for sections of 5 μm thickness. Serial sections of 5 micrometer thickness were taken from the prepared paraffin blocks. Sections were taken on slides previously coated with APES (3 amino propyl triethoxysilan; Sigma–Aldrich Chemicals, St.Louis, MO, USA) for immunohistochemistry staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCardiac Enzymes\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/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antioxidant enzymes GPx, CAT and SOD, as well as the product of oxidative stress MDA, were detected using a spectrophotometric method. Preparation of heart homogenate was done using a tissue homogenizer (100 mg tissue per mL of 50 mM PBS). The homogenate was centrifuged, then the supernatant was used for biochemical analyses according to the manuals of commercially kits (Jian Cheng Biological Engineering Institute)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Immunohistological Evaluation of Specimens\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eUsing the techniques described in the previously published literature, immunohistochemical staining for Desmin was performed [23]. Sections were deparaffinized, rehydrated, and treated with methanol containing 0.03 percent hydrogen peroxide for 20 minutes before being sliced into 5 μm thick pieces. Following a 20-minute incubation period with normal serum to inhibit non-specific antibodies, heart sections were treated with anti-mouse monoclonal primary antibody against desmin (1:100, DAKO, Denmark), [24], and then anti-mouse IgG (1:500, Sigma-Aldrich), [1]. The peroxidase anti-mouse IgG (1:100, DAKO, Denmark) secondary antibody was used. Diaminobenzidine, or DAB, was used as a chromogen to visualize the reaction (Dako, Glostrup, Denmark).Slides were counterstained with Mayer’s hematoxylin and finally dehydrated, rendered transparent with xylene and cover slipped. The negative control was performed by neglecting the primary antibody. Slides were analyzed under a light microscope to identify areas with brownish color which considered as sign of a positive reaction. These procedures were carried out in the Department of Histopathology lab of the University of Benin Teaching Hospital Benin City, Nigeria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphometric Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor image analysis, the mean area % of desmin immunoreactivity was calculated using a Leica Qwin 500 image system (Cambridge, England). Ten non-overlapping fields on stained slides selected from every animal in every group were used for the measurements.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Photography was conducted by Prof Innih S Orlu at the histopathology laboratory, University of Benin, Nigeria\u0026nbsp;at a magnification of 100×.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The results were analysed using one way analysis of variance (ANOVA), followed by the Tukey’s post-hoc test. All values are presented as the mean (M) ± standard deviation (SD). Differences between the groups were considered significant when the probability of chance (p) is less than 0.05 (p \u0026lt; 0.05). All the data collected from the experiment was calculated and analyzed using SPSS software version 16 (SPSS, Chicago, USA).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eOxidative Stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results shown a significance decrease in the activities of antioxidant enzymes SOD, CAT and GPx in Fe overload group, and a significant increase in concentration of peroxidation product MDA when with the control and mustard seed treated groups.. In contrast, the activities of SOD, CAT and GPx were strengthened, and the level of MDA was decreased under the effect of mustard seed extract. We found that the degree of oxidation in the mustard seed group was decreased, and the antioxidant activity of vitamin C is between the high dose mustard seed and low dose mustard seed\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry staining revealed that desmin exhibited varying levels of immunoreaction in various rat cardiac cells that had been exposed to Fe overload. In the control and mustard seed treatment groups, Purkinje cells and heart muscle cells (myocytes) found in the myocardium showed a high cytoplasmic localized immunoreaction to Desmin protein (Fig. 2A, 2C, 2D, 2E, and 2F). Nonetheless, it was shown that the iron overload model group had a somewhat less severe reaction (Figure 1B). Similarly, it was shown that the standard antioxidant medication (Vitamin C group) exhibited high desmin localization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphometric Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis of the morphometric findings of desmin immunostaining in the heart showed a significant (p ˂0.05) increase in the area percentage of desmin in the control and mustard seed treated groups when compared to the Fe overload model group. While, vitamin C group (F) manifested a significant (p ˂0.05) increase in the area percentage of the desmin as compared to the Fe overload group.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study adds new data regarding the possible defensive effect of mustard seed (\u003cem\u003eBrassica nigra\u003c/em\u003e) against Fe induced cardiac injury in adult wistar rats.\u0026nbsp;In this studies, we found that the protective mechanisms of mustard seed against iron overload cardiac damage were at least in part due to decreased iron deposition and inhibition of oxidative stress. This study also exemplified that mustard seed extract administration can inhibit oxidative injury by increasing SOD, CAT, GPx activity and decreasing MDA concentration. Previous research has suggested that, under iron overload conditions, excessive labile irons have a propensity for inducing and generating reactive oxygen species (ROS), resulting in cell oxidative damage, [6, \u0026nbsp;25]. This indicates that oxidative stress is secondary to intracellular iron overload. Therefore, reducing iron deposition may be a basic way to prevent iron overload.\u0026nbsp;In the present study, iron-overloaded rats exhibited clear signs of cardiac damage, as evidenced by altered cardiac biomarkers, histopathological abnormalities, and disrupted structural protein expression. These findings are consistent with previous reports demonstrating the vulnerability of cardiac tissue to iron-mediated oxidative stress, [21]. \u0026nbsp; .\u003c/p\u003e\n\u003cp\u003eThe current study revealed that desmin expression was significantly upregulated in rats given mustard seed in addition to iron overload, which is one of the study's main findings. Cardiomyocytes' intracellular organization, mechanical stability, and structural integrity are all preserved by the essential intermediate filament protein desmin. Previous data had showed that there is a clear correlation between cardiomyopathy and compromised cardiac function and reduced or disordered desmin expression, [19]. It was documented that\u0026nbsp;decrease in the amount of desmin in human cardiomyocytes and its focal deficits was published in the year 2004 and this was found in patients with post-infarction cardiovascular heart failure. Asangri \u003cem\u003eet al.\u003c/em\u003e [3] affirmed that cardiac dysfunction is likely caused by low intracellular expression of desmin. Our study also showed that a decreased accumulation of desmin in cardiomyocytes was associated with more abnormal clinical parameters as compared with a group of rats treated with mustard seed. \u0026nbsp;This may be a result of damage of the desmin network in cardiomyocytes, likely caused by accumulation of cytoskeleton proteins.\u003c/p\u003e\n\u003cp\u003eGradual increase of the size of cardiomyocytes and loss of proper function of contractility fibres is a well-known scenario in the development of cardiovascular heart diseases, [9]. Accumulating intermediate filaments disrupt the desmin network and consequently impair its function aimed primarily at protecting structural and functional integrity of myofibrils and being responsible for cell cohesion, [5].\u0026nbsp;A downregulation of desmin immunoreactivity was further supported by the significant decrease in its mean area percentage as compared to the control group.\u003c/p\u003e\n\u003cp\u003eThe cardioprotective effect of mustard seed may be attributed to its\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003erich antioxidant and anti-inflammatory phytochemical composition\u003c/strong\u003e\u003c/strong\u003e, including glucosinolates, phenolic compounds, and flavonoids. Similar findings on the phytochemical of mustard seed was reported by Vinyas \u003cem\u003eet al\u003c/em\u003e. [26]. \u0026nbsp;These compounds likely mitigate iron-induced oxidative stress, thereby preventing desmin degradation and supporting its synthesis or stabilization. By maintaining desmin integrity, mustard seed treatment may preserve sarcomere alignment, mitochondrial positioning, and overall cardiomyocyte resilience.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdministration of mustard seeds at various doses significantly up-regulated desmin expressivity in iron overload induced heart damage. This observations further support this mechanism, as mustard seed–treated rats showed reduced myocardial degeneration, less cellular disorganization, and improved tissue architecture compared to iron-overloaded controls. This structural preservation correlates well with enhanced desmin expression, reinforcing the role of cytoskeletal protection in attenuating cardiotoxicity. \u0026nbsp;These findings were in parallel and supported by Cohort study in patients with cardiomyopathy and cardiac heart failure in which mustard seed with high phenolic compound alleviated the risk of cardiac decompensation and inflammation by reducing numerous inflammatory cytokines, [25]. Collectively, these results suggest that\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eupregulation of desmin expression is not merely an associated finding but a functional contributor to the cardioprotective effects of mustard seed\u003c/strong\u003e.\u0026nbsp;\u003c/strong\u003eThe modulation of desmin may represent an important molecular mechanism through which mustard seed counteracts iron overload–induced cardiac injury. This highlights desmin as a potential therapeutic target and supports the use of natural dietary antioxidants as adjunct strategies in managing iron-induced cardiotoxicity. Therefore, the observed increase in desmin expression suggests a protective, compensatory response that helps preserve cytoskeletal architecture under oxidative stress conditions.\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical findings obtained in the present study support the view that iron overload has damaging effects and causes a variety of the histological alterations in the heart. Crucially, ethanol seed extract of \u003cem\u003eBrassica nigra\u003c/em\u003e protects against heart damage by decreasing the myocardial degradation, increase desmin expression and maintained structural integrity of the heart muscles. Though, additional studies are needed to detect further molecular mechanisms.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eFurther studies exploring upstream signaling pathways involved in desmin regulation and long-term functional outcomes would help clarify the full cardioprotective potential of mustard seed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Eze Gerald in Histopathology Lab University of Benin Teaching Hospital, Benin City for preliminary results on desmin activity on iron overload induced cardiac damage and also to the\u0026nbsp;Department of human Anatomy University of Delta Agbor, Nigeria\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThird Party Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnyeije Benson conceived and design the study, as well as methodology and data analysis. Immunohitochemical analysis was done by Innih Silvanu. He critically review and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during this current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo financial support was obtained from any source for this research project. The study was conducted without external funding or institutional grants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdEl-Moniem, M. et al. The ameliorative potential of Hyphaene thebaica on streptozotocin-induced diabetic nephropathy. \u003cem\u003eFolia Morphol. 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Res.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 252\u0026ndash;260 (2019).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mustard seed (Brassica nigra), Iron (II) chloride, Desmin, Toxicity, Heart","lastPublishedDoi":"10.21203/rs.3.rs-8969169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8969169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlthough iron is an essential element for life, its excess is linked to many disorders.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe aim of this study is to investigate the effect of mustard seed on iron induced toxicity on the heart in adult male albino rats using biochemical and immunohistochemical methods.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 35 rats weighing between 180-250g were divided into seven groups (A, B, C, D, E, F and G) of five per group. Group A (control) was administered 1ml of distilled water, group B, C, D and E were induced with iron (II) chloride for sixty days and treated with different doses of the extract except group B for sixty days, group E was treated with standard drugs at the same time interval, while group F and G received 200mgkg and 400mg/kg of extract respectively using orogastric tube. After last day of administering drugs, the rats were left for an overnight fast and then sacrificed 24 hours later. Heart specimens were dissected from scarified rats to estimate tissue level malondialdehyde (MDA), antioxidant enzymes (SOD, CAT and GPx) and immunohistochemical staining of desmin.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIncreases in cardiac MDA and decreases in SOD, CAT, and GPx were brought on by iron overload, along with degenerative alterations in the tissues under examination and a decline in desmin expression. All of these measures showed a notable improvement following the administration of mustard seed extract.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eBy lowering the iron levels in heart tissue and reducing oxidative stress and inflammatory effects brought on by iron excess, mustard seed may function as an iron chelator.\u003c/p\u003e","manuscriptTitle":"Increased Expression of Desmin Contributes to the Protection of Mustard seed against iron overload- Induced cardio-toxicity in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 19:19:06","doi":"10.21203/rs.3.rs-8969169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T11:02:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T01:17:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T14:07:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336882978123179517333644879606347647201","date":"2026-04-12T14:10:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T18:38:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111540018829421720554634937499369744280","date":"2026-04-07T17:51:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241759217697786738114329109698084964660","date":"2026-04-07T12:46:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T09:19:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T09:11:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-19T11:54:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-17T00:21:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-16T18:35:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59e2bf42-7c30-4778-878f-12c4d79c3411","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66159058,"name":"Biological sciences/Biochemistry"},{"id":66159059,"name":"Health sciences/Cardiology"},{"id":66159060,"name":"Biological sciences/Drug discovery"},{"id":66159061,"name":"Health sciences/Medical research"},{"id":66159062,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-27T11:10:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 19:19:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8969169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8969169","identity":"rs-8969169","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}