Therapeutic Potential of Fucoidan from a Persian Gulf Alga (Sargassum tenerrimum) in Rheumatoid Arthritis: In Vivo Evaluation in a Rat Model

Therapeutic Potential of Fucoidan from a Persian Gulf Alga (Sargassum tenerrimum) in Rheumatoid Arthritis: In Vivo Evaluation in a Rat Model | 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 Therapeutic Potential of Fucoidan from a Persian Gulf Alga (Sargassum tenerrimum) in Rheumatoid Arthritis: In Vivo Evaluation in a Rat Model Leila Mahmoudzadeh, Seyyed Meysam Abtahi Froushani, Seyede Soraya Mahmoudi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7981987/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Inflammopharmacology → Version 1 posted 10 You are reading this latest preprint version Abstract Fucoidan, due to its anti-inflammatory and immune-modulating effects, shows promise for treating rheumatoid arthritis (RA). This study tested fucoidan from the Persian Gulf algae Sargassum tenerrimum on adjuvant-induced RA in Wistar rats. The isolated fucoidan demonstrated excellent scavenging capability (83.66 ± 0.35%) on 2,2-diphenyl-1-picrylhydrazyl radicals at a dosage of 6 mg/mL. RA was induced in rats via Freund's adjuvant injection. From days 5 to 23, rats (n = 10/group) received daily placebo, fucoidan (50, 100, 150 mg/kg), or prednisolone (10 mg/kg). Fucoidan effectively reduced inflammation and alleviated symptoms associated with adjuvant-induced RA in a dose-dependent manner. Hematoxylin and eosin staining strongly supported the previous results regarding fucoidan's effectiveness, especially at 150 mg/kg, in reducing inflammation and repairing joint structure. Safranin O staining confirmed that better joint and cartilage health was linked to higher fucoidan doses. Immunohistochemical analysis showed that fucoidan treatment significantly lowered levels of the inflammatory cytokines IL-1β and TNF-α in the joint tissue of RA rats. Moreover, fucoidan treatment led to a dose-dependent reduction in inflammatory biochemical parameters such as myeloperoxidase, nitric oxide, malondialdehyde, and C-reactive protein. In the joint,tissue. Fucoidan reduced the activity of inflammatory genes (T-bet, RORγt), leading to decreased differentiation of Th1 and Th17 cells, while enhancing the activity of GATA3 and FOXP3 genes, which promoted Th2 and Treg cell polarization. In contrast to prednisolone, fucoidan elevated the levels of Nrf2 and HO-1 in the plantar joint.Fucoidan from S. tenerrimum has strong antioxidant and immunomodulatory benefits, making it a promising option for managing complex inflammation like RA. Fucoidan Brown algae seaweeds Rheumatoid arthritis Autoimmunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction RA is a chronic autoimmune disease causing joint inflammation, pain, swelling, and stiffness. It damages cartilage and bone, leading to deformities and impaired function. The cause is unknown but involves genetics, environmental factors, and hormones triggering an immune response. Symptoms include fatigue and mild fevers (Ciofoaia et al. 2022 ; Etemadi et al. 2022 ). RA affects 0.5-1% globally, more women, and increases with age. Untreated RA causes joint damage, declining physical function, deformities, and disability (Ciofoaia et al. 2022 ). RA is mainly managed with corticosteroids, nonsteroidal anti-inflammatory agents (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), biologics, and JAK inhibitors. Current treatments have side effects and aren't always effective. Corticosteroids can cause bone loss and heart problems. Long-term drug use increases risks (Singh 2022 ). DMARDs and biologics raise infection and malignancy risks. Up to 40% don't respond to biologics like anti-TNF-α agents (Singh 2022 ; Narváez et al. 2024 ). Natural NSAIDs may offer therapeutic alternatives. Rat models like adjuvant-induced arthritis are key for RA research. Injecting Freund's complete adjuvant (FCA) triggers arthritis, mimicking human RA (Etemadi et al. 2022 ; Mirzaaghasi and Froushani 2023). FCA contains inactivated Mycobacterium tuberculosis in mineral oil. This model shows swelling, cartilage damage, impaired joint function, and lymphocyte infiltration, making it reliable for evaluating RA treatments and other inflammatory conditions (Etemadi et al. 2022 ; Zeng et al. 2022 ). Algae show potential for treating autoimmune diseases due to their bioactive compounds like antioxidants, anti-inflammatory substances, and immunomodulators, which reduce oxidative stress and inflammation (Linares-Maurizi et al. 2023 ). Brown algae (Phaeophyceae) are a major macroalgae group with brown pigments, mainly fucoxanthin (Silberfeld et al. 2014 ). They contain bioactive compounds beneficial for autoimmune and inflammatory conditions, including phlorotannin and polysaccharides like fucoidan, laminarin, and porphyrin, which have anti-inflammatory and immunomodulatory effects (Kadam et al. 2014 ; Ersoydan and Rustemeyer 2024 ). Algae farming is eco-friendly, using less land and water than regular farming. Some algae compounds may be absorbed better by the body than synthetic versions, which is key for better treatment (Gurau et al. 2025 ). Fucoidan, a sulfated polysaccharide predominantly obtained from brown algae, demonstrates a broad spectrum of biological activities, encompassing anti-inflammatory, immunomodulatory, antiviral, antidiabetic, anticancer, antioxidant, and anticoagulant effects. The structure of fucoidan, which can vary based on the algae source and extraction techniques, influences its bioactivity and therapeutic efficacy (Alea and Meyer 2013 ; Pacheco et al. 2020 ). Pretreatment with Fucoidan extracted from Costaria costata reduced dendritic cell activation, pro-inflammatory cytokine secretion (IL-1β, IL-6, TNF-α), and lung inflammation (Zhang et al. 2024 ).Fucoidan extracted from five algae species ( Padina pavonica , Stoechospermum marginatum , Spatolossum macrodontum , Dictyota bartayresiana , and Turbinaria decurrens ) showed inhibition of nitric oxide production by RAW 264.7 macrophage cell lines with increasing concentrations of fucoidan (Raj et al. 2024 ). Studies suggest that fucoidan can impede the differentiation of osteoclasts, which play a vital role in bone resorption and the inflammation associated with RA. It modulates signaling pathways, including Akt/GSK3β/PTEN and NFATc1, leading to decreased osteoclast activity and the inflammatory bone loss linked to the disease (Lu et al. 2019 ; Apostolova et al. 2020 ). F. vesiculosus fucoidan has anticancer, anti-inflammatory, and antioxidant effects. It inhibits COX-1/2, p38 MAPK, hyaluronidase, and protein denaturation, while also stabilizing RBC membranes (Zhang et al. 2024 ) .It was reported that oral administration of Fucoidan extracted from Costaria costata alleviated LPS-induced sepsis, highlighting its potential as a therapeutic agent for inflammatory diseases (Zhang et al. 2024 ). Due to its anti-inflammatory and immunomodulatory characteristics, Fucoidan's promising therapeutic effects in the context of RA provide reassurance and confidence in its potential. These findings support the investigation of various doses of fucoidan for treating RA induced by CFA in Wistar rats, as it may provide a natural alternative to conventional therapies. In this study, we examined the effects of fucoidan extracted from the Brown algae Sargassum tenerrimum , collected from the Persian Gulf region, in a rat model of rheumatoid arthritis induced by FCA. Material and Methods Chemicals GIBCO/Life Technologies Inc. (USA) provided cell culture media and FBS. PeproTech EC, Ltd. (UK) supplied ELISA kits. TAKAPOUZIST (Iran) provided RNX-Plus. TAKARA (China) provided SYBR Premix Ex TaqII and cDNA kits. SibZist Fan Co (Iran) provided myeloperoxidase and nitric oxide assay kits. Elabscience (China) supplied CRP, COX-2, NF-κB, Nrf-2, and HO-1 ELISA kits. Sigma-Aldrich (USA) provided hematoxylin-eosin staining kits and other reagents. Seaweed preparation and Fucoidan extraction procedure Seaweed preparation and Fucoidan extraction procedure Sargassum tenerrimum was collected from Chabahar, Iran (25°16′36.6″N; 60°40′10.0″E) in December 2023. The seaweed was rinsed, dried (50°C, 4 hours), and powdered. The powder was suspended in McIlvaine's buffer (pH 3–7) and extracted at 60–100°C for 3 hours. After filtration, CaCl2·2H2O (3% w/v) was added (1:1) and incubated overnight at 4°C to precipitate alginate, which was collected by vacuum filtration. Ethanol was added to the filtrate and stored overnight at 4°C to remove salts, followed by centrifugation (6000 rpm, 15 minutes) to harvest the crude polysaccharide. Freeze-drying yielded cream-colored fucoidan. (Fig. 1 c suppl) (Hifney et al. 2016 ; Lim et al. 2016 ). The yield of fucoidan was determined by employing the subsequent equation: Fucoidan yield = [dry wt. of obtained fucoidan / dry wt. of sample] × 100 Fucoidan Characterization For Fourier transform infrared spectrometry (FT-IR) analysis, a pellet of acid-digested fucoidan and KBr (1 mg/100 mg) was prepared under 10,000 psi and analyzed from 600–4000 cm − 1 (Zhao et al. 2021 ). DPPH Free-Radical Scavenging Activity: The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging was tested as described before (Marudhupandi, Kumar et al. 2014 ). Fucoidan solutions (1–6 mg mL-1) were mixed with DPPH in methanol and DMSO (0.5 mM), incubated for 30 min in the dark, and absorbance was read at 517 nm (MeOH blank). Butylated hydroxyanisole (BHA) was the reference. Scavenging capacity was calculated using the equation below: Scavenging activity (%) = (1 - Asample / Acontrol) × 100 A control is the absorbance of DPPH in methanol without the sample, while A sample is the absorbance of DPPH in methanol with the sample. Induction of RA and animal groups Sixty male Wistar rats (8 weeks old, 150 ± 10 g) from the Razi Institute in Iran were housed in a controlled environment (23°C ± 1, 12-hour light/dark cycle) with unrestricted access to food and water. Ethical guidelines from the National Institutes of Health Guide were adhered to, and all procedures were approved by the Faculty Ethics Committee. Proper housing is essential for animal welfare and the validity of research (Mlynarik et al. 2004 ). Rheumatoid arthritis (RA) was induced in rats via an intradermal injection of 0.1 mL Complete Freund's Adjuvant (CFA), containing 10 mg/mL killed Mycobacterium, at the base of the tail. Disease severity was assessed by measuring the volume of the contralateral hind paw at two-day intervals. A scoring system was employed to evaluate the condition: 0 = normal paw; 1 = erythema of the toe; 2 = erythema and swelling of the paws; 3 = swelling of the ankle; and 4 = total swelling of the entire leg, resulting in an inability to flex. Assessments were conducted weekly by three observers. Body weight was monitored bi-daily following immunization (Ghaffary and Froushani 2020; Hajizadeh et al. 2021 ). The study comprised six groups of rats (n = 10 per group). Group 1 (Control) consisted of healthy, untreated rats. Group 2 (RA group) had RA induced but received no therapeutic intervention. Group 3 (RA + Fuc50) had RA induced and was treated daily with 50 mg/kg fucoidan via oral gavage. Group 4 (RA + Fuc100) had RA induced and received 100 mg/kg fucoidan daily via oral gavage. Group 5 (RA + Fuc150) had RA induced and was treated with 150 mg/kg fucoidan daily via oral gavage. Group 6 (RA + Pred) had RA induced and was administered 10 mg/kg prednisolone daily. All animals were euthanized 20 days post-disease induction, and samples were collected for subsequent analysis. Tissue Preparation and staining Histopathology relies on tissue fixation and embedding to preserve cell structure. Tissue was fixed in 10% formalin (24–72 hours), dehydrated with alcohols, cleared with xylene, and embedded in paraffin. 5-micrometer sections were stained with hematoxylin and eosin (H&E) and Safranin O-fast green for diagnosis (Rahawy et al. 2021 ). Immunofluorescence Protocol Immunofluorescence Protocol Samples were boiled in TBS 1X and then washed three times with PBS. A 0.3% Triton solution was applied for 30 minutes, followed by a PBS wash. To block secondary antibody reactions, 10% goat serum was used for 45 minutes. The primary antibody (1:100 in PBS) was applied and refrigerated (2–8°C) for 24 hours. After four PBS washes, the secondary antibody (1:150) was incubated in the dark at 37°C for 1.5 hours. Following three washes, DAPI was applied for 20 minutes and then washed with PBS. Finally, glycerol/PBS was added, and coverslips were placed for fluorescent imaging using an Olympus microscope to confirm the markers (Kajimura et al. 2016 ). Biochemical assays On day 28, the animals were deeply anesthetized, and blood samples were collected to obtain serum for various tests, including the Myeloperoxidase (MPO) activity assay. Serum MPO activity was assessed using a method analogous to one previously detailed. MPO activity was quantified by measuring the absorbance difference and comparing it to a horseradish peroxidase standard curve (Froushani and Mashhouri 2019; Jafari- Khataylou et al. 2023). The nitric oxide level of serum was determined by comparing it to the standard curve obtained through the Griess method, following the manufacturing protocol (Jahantigh et al. 2023 ). The sandwich ELISA (Enzyme-Linked Immunosorbent Assay) was used to measure C-reactive protein (CRP) levels according to the manufacturer's guidelines (Bassuk et al. 2004 ). The left hind paws were removed, rinsed with cold saline, and homogenized in 0.01 M Tris-HCl buffer (pH 7.4). The homogenate was then centrifuged at 10,000×g for 15 minutes at 4°C (Pal et al. 2018 ). The resulting supernatant was used to measure the levels of Nrf2, HO-1, and NF-κB by ELISA kits.. Molecular assays Real-time PCR Total mRNA was extracted from right hind paws using RNX-Plus and used to synthesize cDNA. GATA3, T-bet, RORγt, and FoxP3 mRNA levels were measured by PCR with SYBR Green, using HPRT as a reference. Primers are listed in Table 1 . Results are expressed as 2 −ΔΔCt (mean fold change (Lin et al. 2015 ; Massalska et al. 2020 ; Poursamimi et al. 2022 ). Table 1 Forward and Reverse primers used in Real-Time PCR Genes Forward primes Reverse primer GATA3 5'-TCA TTA AGC CCA AGC GAA GG-3' 5'-GTC CCC ATT GGC ATT CCT C-3' T.bet 5'-CGG CTG CAT ATC GTT GAG GT-3' 5'-GTC CCC ATT GGC ATT CCT C-3' RORɣt 5-GCA GCG CTC CAA CAT CTT CT-3' 5'-ACG TAC TGA ATG GCC TCG GT-3' FOXP3 5'-CAC CTG GCT GGG AAA ATG G-3' 5'-GGA GCC CTT GTC GGA TGA-3.' GAPDH 5′-CAA AGC CAG AGT CCT TCA GA-3' 5′-GAT GGT CTT GGT CCT TAG CC -3' Statistical Analysis The normality of the data was assessed utilizing the Shapiro-Wilk test. Subsequently, a one-way analysis of variance (ANOVA) was conducted, accompanied by Tukey's post hoc test, to explore the results further. The outcomes were reported as means ± standard deviation (SD), with a significance level of p < 0.05. Results Fucoidan evaluation and characterization Air-dried Sargassum tenerrimum samples showed a moisture content of approximately 7.93%. Carbohydrates made up the largest portion of the seaweed's dry weight (58.13%±1.45), followed by ash (24.95%±1.85), crude protein (16.08%±0.78), and crude fat (0.67%±0.03). The fucoidan extraction process yielded approximately 9.25 grams of fucoidan from 110 grams of dried and ground brown algae, representing an 8.4 percent yield. The FTIR method was employed to verify the successful separation of fucoidan (Fig. 1 d suppl). It is demonstrated that the absorption band at 3411 cm⁻¹ is associated with the O-H stretching vibration found in carbohydrates. The bands at 2928 cm⁻¹ and 1625 cm⁻¹ correspond to the C-6 group of fucose or galactose, as well as C-H stretching vibrations of the pyranoid ring and OH bending vibrations, respectively, which are indicative of polysaccharide absorption. The 1625 cm⁻¹ band also reflects the C = C vibration of uronic acid. The bands at 1557 cm⁻¹ and 1433 cm⁻¹ signify the asymmetric and symmetric stretching vibrations of carboxylate groups from uronic acids. The combination of peaks at 1557 cm⁻¹ and 1432 cm⁻¹ strongly indicates the presence of uronic acids or carboxylate functionalities, commonly found alongside sulfated fucose residues in fucoidan. The band at 1126 cm⁻¹ suggests S = O stretching from sulfates, which is particularly relevant to fucoidan, known for its high content of sulfated fucose residues. Lastly, the peaks at 854 cm⁻¹ are linked to C-O-S bending vibrations in sulfate groups attached to sugar units, especially at the C-4 position of fucose residues in fucoidan, highlighting the characteristic nature of sulfated polysaccharides in this compound (Fig. 1 d supplementary) (Isnansetyo et al. 2017 ; Palanisamy et al. 2017 ). Figure 1 illustrates the DPPH radical scavenging activities of fucoidan. The intact fucoidan exhibited a notable scavenging capability (85.65 ± 4.35) against DPPH radicals at a concentration of 4 mg mL − 1 . Clinical evaluations Inflammatory responses in the joint environment serve as a critical clinical indicator in RA and its animal models. Therapeutic regimens were initiated on the fifth day post-immunization upon detecting an arthritis index of ≥ 1 in each rat (Golbahari and Froushani 2019). According to Fig. 2 a, the course of the disease was milder in the treatment groups, especially in groups RA + Pred and RA + Fuc150. Figures 1 b illustrate that the group treated with prednisolone exhibited the lowest arthritis index, followed by the group receiving fucoidan at 150 mg/kg (RA + Fuc150). The RA + Fuc50 and RA + Fuc100 groups did not show a statistically significant difference in average arthritis index. Notably, on the final assessment day, the prednisolone group exhibited the lowest severity of arthritis index compared to the other groups. Treatment with fucoidan was also effective in reducing the severity index on the last day in a dose-dependent manner (Figs. 1 b). Also, RA caused weight loss, but treated animals exhibited better weight gain than untreated RA rats. The prednisolone group had the lowest severity of weight loss among all groups, while fucoidan treatment also effectively reduced the severity of weight loss in a dose-dependent manner (Figs. 1 b). Overall, the study indicates that fucoidan can enhance the clinical signs of RA in a rat model, with higher doses (150 mg/kg) demonstrating greater effectiveness, although prednisolone remained the most effective treatment overall (Fig. 2 b). Histopathological assays H&E histopathological examination of joints affected by RA revealed distinct differences between the control and RA groups (Fig. 3 ). Control rats exhibited normal articular cartilage with no evidence of mononuclear cell infiltration in the synovium. In contrast, the knee joints of the RA group displayed significant cartilage erosion, synovial hyperplasia, and marked mononuclear cell infiltration within the synovial membrane. Additionally, there was extensive bone damage accompanied by a substantial infiltration of inflammatory cells, including lymphocytes, macrophages, and congested vessels (Fig. 3 ). Rats treated with prednisolone demonstrated joint structures resembling those of the control group, restoring normal histopathology. The groups receiving 50 mg/kg and 100 mg/kg of fucoidan showed reduced inflammatory responses, although these effects were less pronounced than those observed with prednisolone. Notably, the group administered 150 mg/kg of fucoidan exhibited a significant decrease in joint inflammation and lymphocyte accumulation. The findings suggest that daily administration of 150 mg/kg of fucoidan and prednisolone resulted in favorable outcomes concerning joint pathology in RA (Fig. 3 ). Figure 4 illustrates saffronin O staining, which aids in assessing the extent of inflammation and the structural alterations in the synovium associated with RA. As shown in Fig. 4 a, the RA group exhibits significant infiltration of inflammatory cells and reduced cartilage thickness, indicating structural changes and active inflammation compared to the control group. Treatment with fucoidan led to a dose-dependent reduction in inflammation within the joint tissue, decreased infiltration of inflammatory cells, and increased cartilage thickness. These improvements in joint and cartilage conditions are directly related to the dose of fucoidan. Figure 4 b illustrates that all studied groups show statistically significant differences, with the most favorable outcomes observed in the prednisolone group, followed by the RA + Fuc 150 group. Immunohistochemical evaluation Figure 5 illustrates the immunohistochemical (IHC) reaction of IL-1β in mouse knee joint tissue.The highest expression of IL-1β was observed in the RA group, while the fucoidan-treated groups showed a significant dose-dependent decrease in IL-1β expression. Specifically, the lowest expression of IL-1β was recorded in the RA group treated with fucoidan at 150 mg/kg. However, the greatest decrease in IL-1β expression was in the prednisolone-treated group (Fig. 5 ). Figure 6 shows the IHC reaction associated with the inflammatory cytokine TNF-α in mouse knee joint tissue. The results show that the RA group showed the highest expression of TNF-α, while a significant decrease was observed in the treated groups. This decrease in IHC reaction was dose-dependent among the groups receiving fucoidan, and the lowest reactivity was recorded in the RA + fuc150 group. Prednisone treatment was effective in reducing the expression of TNF-α, such that the expression level of the cytokine was not significantly different from that of healthy mice (Fig. 6 ). Biochemical assays In this study, serum levels of biochemical factors CRP, MPO, and NO were evaluated in RA rats as inflammatory markers. Data in Table 2 indicated that the activity levels of these inflammatory markers were significantly elevated in the RA group. In contrast, a dose-dependent decrease in these levels was observed in the groups treated with fucoidan, with the most notable reduction at 150 mg/kg. Furthermore, the group receiving prednisolone showed lower levels of these inflammatory markers compared to the other treatment group. Statistically, prednisolone treatment was as effective in reducing CRP levels as the levels in healthy mice (Table 2 ). Table 2 Biochemical modifications in the sera of rats with RA. Inflammatory Enzymes/Groups Control RA RA + Fuc50 RA + Fuc100 RA + Fuc150 RA + Pred MPO enzyme activity (ng/ml) 10.82 ± 0.63 a 67.56 ± 0.34 b 53.58 ± 0.86 c 44.88 ± 0.78 d 29.60 ± 0.17 e 20.34 ± 0.23 f NO enzyme activity (ϻM) 20.54 ± 0.89 a 80.1 ± 1.5 b 73.3 ± 1.26 c 61.67 ± 0.9 d 43.95 ± 0.39 e 31.67 ± 0.95 f CRP (mg/ml) 0.07 ± 0.004 a 1.6 ± 0.03 b 1.1 ± 0.028 c 0.7 ± 0.053 d 0.2 ± 0.023 e 0.08 ± 0.061 a Nrf2 (pg/ml) 240.28 ± 15.3 a 68.43 ± 10.48 b 73.1 ± 11.76 b 94.26 ± 9.38 c 121.60 ± 11.1 d 70.01 ± 6.93 b HO-1 (pg/ml) 1.68 ± 0.12 a 0.28 ± 0.13 b 0.33 ± 0.18 b 0.45 ± 0.16 c 0.6 ± 0.12 d 0.35 ± 0.15 b NF κb (pg/ml) 0.47 ± 0.11 a 2.09 ± 0.23 b 1.1 ± 0.028 c 0.65 ± 0.15 d 0.9 ± 0.12 e 0.51 ± 0.191 a The results were presented as mean ± standard deviation, and using different letters indicated statistical significance at a p < 0.05. (RA., Rheumatoid arthritis; Fuc., Fucoidan; Pred., Prednisolone). Data analysis showed that Nrf2 and HO-1 levels were significantly lower, while NF-κB levels were higher in the RA rats compared to the control group (Table 3). However, treatment with fucoidan (100 and 150 mg/kg) significantly increased Nrf-2 and HO-1 levels in the joint tissues of RA rats. Fucoidan at 50 mg/kg also increased Nrf2 and HO-1 levels, though these results were not statistically significant. NF-κB levels were significantly decreased in fucoidan-treated RA rats in a dose-dependent manner. While prednisolone treatment did not significantly improve Nrf2 and HO-1 levels, NF-κB levels were reduced more significantly in CFA-injected rats than in fucoidan-treated RA rats(Table 3). Molecular assays via Real-Time PCR In the RA group, an increase in mRNA expression of T-bet and RORɣt genes was noted alongside a decrease in mRNA expression of GATA3 and Foxp3 genes. As anticipated, prednisolone, a standard anti-inflammatory, proved to be more effective than the other drugs in reversing this condition. Treatment with fucoidan also led to a dose-dependent increase in the expression of mRNAs GATA3 and Foxp3, while decreasing the expression of T-bet and RORɣt. Discussion Uncontrolled inflammation driven by type 1 and type 3 immune responses (Th1/Th17) in RA and its experimental models progressively damages joint tissues and impairs organ function, prompting the search for safe and effective anti-inflammatory agents that can be administered orally (Etemadi et al. 2022 ). The rationale for prioritizing natural anti-inflammatory compounds arises from their multi-target mechanisms, favorable safety profiles, and cost-effectiveness, thereby addressing limitations inherent in conventional pharmaceuticals (Jamtsho et al. 2024 ; Kang et al. 2025 ). Prior research indicates that natural products such as curcumin and fish oil offer a multifaceted approach to rheumatoid arthritis by simultaneously reducing inflammation, modulating immune responses, and promoting tissue resolution, thus targeting both symptomatic manifestations and underlying causes (Kou et al. 2023 ). Herein, our findings definitively establish the therapeutic potential of fucoidan extracted from Persian Gulf Sargassum tenerrimum in alleviating inflammation and modulating immune responses within a rat model of rheumatoid arthritis. Former studies have shown that S. lomentaria fucoidanfucoidan can alleviate symptoms of IBD by modulating gut microbiota and reducing intestinal inflammation (Tang et al. 2024 ), and suppress T cell activation and TNF-α in models of multiple sclerosis (Kim et al. 2010 ). Algal polysaccharides are active natural compounds with potential uses. Brown algae are a large source of important polysaccharides, including fucoidan, which has been studied extensively (Zhang et al. 2024 ). This polysaccharide has been isolated from various seaweed species using different methods. Prior research has shown that the amounts of total carbohydrates, protein, and sulfate in fucoidan from S. tenerrimum can vary, differing from the values found in the current study (Ashayerizadeh et al. 2020 ). A crucial consideration is that fucoidan derived from different sources may exhibit variable immunomodulatory functions. Prior investigations have posited that high molecular weight fucoidans may exacerbate the severity of arthritis, whereas low molecular weight variants have the capacity to reduce inflammation and Th1-mediated immune responses (Park et al. 2010 ). These differences may arise due to factors like geographical location, harvest season, and environmental conditions, all of which can affect the chemical composition of seaweeds(Apostolova et al. 2020 ). Consequently, to plan for the potential applications of fucoidan, it is important to assess fucoidans from algae in specific geographical areas. As per IUPAC nomenclature and terminology guidelines, a sulfated polysaccharide with an L-fucose (6-deoxy-L-galactose) content ranging from 20% to 60% can be classified as fucoidan (Ashayerizadeh et al. 2020 ). The fucoidan derived from Persian Gulf Sargassum tenerrimum demonstrated a significant abundance of sulfated fucose. his was confirmed through FTIR analysis. The quantification of DPPH free radical scavenging is a widely used technique for evaluating the antioxidant capabilities of extracts and natural compounds isolated from marine flora. An alcoholic solution of DPPH forms a stable radical at ambient temperature (Baliyan et al. 2022 ). Upon the addition of algal extract, the solution undergoes a color change from blue-purple to yellow, facilitated by the acceptance of electrons or hydrogen atoms; the measurement of this color transition serves as the fundamental basis of the method. In the present study, the highest DPPH free radical scavenging activity of fucoidan from S. tenerrimum collected from the Persian Gulf was observed in the fucoidan treatment at a concentration of 4 mg/ml (85.65 ± 4.35). A previous study reported that fucoidan from S. tenerrimum collected from the Mandapam coast of Tamil Nadu, India, effectively scavenges DPPH free radicals, showing activity between 64.66% and 83.66% at concentrations of 1–6 mg/mL for both intact and fractionated forms (Marudhupandi et al. 2014 ). Therefore, despite the apparent similarity between the two species of brown algae, this ability is greater in fucoidan extracted from the Persian Gulf. The fucoidan extracted from S. polycystum demonstrates maximum DPPH radical scavenging activity of 61.2 ± 0.33% at 1000 µg/mL (Palanisamy et al. 2017 ). However, it has been noted in the past that the antioxidant efficacy of fucoidan is strongly influenced by sulfate content, extraction method, and concentration (Yue et al. 2025 ). These properties make fucoidan extracted from Persian Gulf seaweed a promising natural antioxidant for the pharmaceutical and food industries. For precise applications, further studies on the standardization and optimization of extraction parameters are recommended. The processes of inflammation and oxidative stress are interconnected (Abtahi Froushani and Esmaili Gourvarchin Galeh 2014 ). Unlike conventional drugs used in the treatment of RA, natural compounds such as fucoidan may offer the added benefit of antioxidant properties (Ashayerizadeh et al. 2020 ), enhancing their direct anti-inflammatory effects. The in vitro results (DPPH free radical scavenging assay) of this study clearly demonstrated the antioxidant potential of fucoidan derived from the Persian Gulf Sargassum tenerrimum . Malondialdehyde (MDA) serum levels serve as a key biomarker of oxidative stress in RA, reflecting lipid peroxidation and contributing to disease pathogenesis through various mechanisms (Jahantigh et al. 2023 ). Our data indicated that the fucoidan used in this study has the potential to reduce oxidative stress and serum levels of MDA in RA rats. Oxidative stress activates the Nrf2/HO-1 pathway, which is crucial in RA by enhancing antioxidant enzyme production and regulating inflammatory gene expression (Chadha et al. 2020 ; Ngo and Duennwald 2022 ). Nrf2 induces antioxidant and detoxifying genes such as HO-1, which breaks down heme into protective molecules. Activation of Nrf2 reduces joint damage in RA animal models (Chadha et al. 2020 ). Nrf2 activators like dimethyl fumarate and isoliquiritigenin demonstrate anti-arthritic effects by decreasing oxidative stress and inflammation (Izumi and Koyama 2024 ). According to our results, RA rats had lower Nrf2 and HO-1 than controls. Fucoidan (100, 150 mg/kg) significantly increased these levels in RA rat joints, but 50 mg/kg did not significantly increase them. As expected,Ddexamethasone did not improve Nrf2 or HO-1 levels.Fucoidan derived from Sargassum wightii has been shown to mitigate oxidative stress in diabetic rats via activation of the Nrf2/HO-1 signaling pathway (Puhari et al. 2023 ). Furthermore, fucoidan supplementation has been demonstrated to enhance antioxidant capacity in piglets by modulating the Keap1/Nrf2 signaling pathway and mitochondrial function (Yin et al. 2024 ). In human keratinocytes, fucoidan has exhibited protective effects against oxidative stress through the upregulation of Nrf2 and its downstream targets (Zayed et al. 2023 ). The integration of clinical and histopathological findings provides insight into the disease progression of CFA-induced RA and is crucial for evaluating the effectiveness of therapeutic interventions (Takahashi et al. 2024 ). The histopathological schema of adjuvant-induced arthritis in rats is characterized by a progressive inflammatory process primarily affecting the joints. Synovial hyperplasia, inflammatory cell infiltration, and increased vascularity become more pronounced (Morvaridi et al. 2025 ). The results of our study showed that fucoidan is dose-dependently effective in improving histopathological appearance, as well as clinical indicators and weight gain. H&E data from our investigation indicated that daily administration of 150 mg/kg fucoidan and prednisolone produced the most favorable outcomes in managing RA-related joint inflammation and leukocyte infiltration. The inflammatory environment caused by RA leads to the breakdown of cartilage and underlying bone in the joint area (Morvaridi et al. 2025 ). Interestingly, fucoidan has been shown to inhibit osteoclastogenesis, a key factor in bone loss in RA, by regulating signaling pathways such as Akt/GSK3β/PTEN/NFATc1, thereby reducing inflammatory bone loss (Lu et al. 2019 ). Safranin O staining provides both visual and quantitative evidence of the dose-dependent potential of fucoidan as an anti-inflammatory and cartilage-protective agent in rheumatoid arthritis. In rheumatoid arthritis, the pro-inflammatory cytokines IL-1β and TNF-α are highly expressed in synovial tissue, particularly near cartilage and bone. They drive inflammation, contributing to depression, sickness behavior, pain, and weight loss, with their levels rising as arthritis progresses (Yang et al. 2019 ). Biological agents, such as anti-TNF therapies (e.g., etanercept, infliximab) or IL-1 blockers, have been developed to mitigate their effects and improve outcomes in RA patients, making them significant in research and clinical contexts (Law and Taylor 2019 ). Our ICH results indicated that fucoidan reduced the expression of inflammatory cytokines IL-1β and TNF-α in inflamed joints dose-dependently. Previous data showed that fucoidan in Sargassum horneri extracts reduced TNF-α-driven inflammation in RPE cells, indicating potential use in macular degeneration (Lee et al. 2023 ). Research on synovial cells supports that fucoidan reduces joint inflammation caused by IL-1β. It appears fucoidan's anti-inflammatory benefits may occur by blocking the NF-κB pathway and the mitogen-activated protein kinases (MAPKs) cascade (Park et al. 2011 ). NF-κB regulates inflammatory genes and promotes synovial hyperplasia. Its activation is seen in RA patient synovial tissues and RA animal models (Makarov 2001 ). In this study, treatment with fucoidan reduced NF-κB levels, resulting in the suppression of several downstream inflammatory mediators. Myeloperoxidase (MPO), primarily in neutrophils and monocytes, produces HOCl from H₂O₂ and chloride to kill pathogens, but excessive MPO/ROS damages tissues. In chronic inflammation, high MPO levels overwhelm antioxidants. MPO-driven stress is linked to conditions like RA, suggesting potential treatment targets (Golbahari and Abtahi Froushani 2019 ; Moases Ghaffary and Abtahi Froushani 2020 ). MPO-derived ROS activates inflammatory pathways such as NF-κB, worsening inflammation and MPO release in a harmful cycle (Mittal et al. 2014 ). Elevated MPO in blood or tissues often correlates with oxidative stress severity, serving as a diagnostic marker for diseases like RA (Golbahari and Abtahi Froushani 2019 ). In the present study, following an increase in serum MPO levels in RA rats, a dose-dependent decrease in serum MPO levels was observed in the fucoidan-treated groups, with the most significant decrease observed at a dose of 150 mg/kg. Fucoidan reduces oxidative stress by boosting antioxidant activity and lowering ROS and lipid peroxidation. It enhances SOD, catalase, and GPx via the Nrf2/ERK pathway and directly inhibits MPO enzyme, reducing neuroinflammation and improving memory (Ryu and Chung 2016 ). Fucoidan eases pancreatitis by lowering MPO activity in the pancreas and lungs, indicating less neutrophil infiltration. It also inhibits MAPK and NF-κB signaling, which reduces MPO expression, ROS output, and inflammatory cytokines like TNF-α, IL-6, and IL-1β, all tied to MPO-related oxidative stress (Carvalho et al. 2013). Another study demonstrated that fucoidan extracted from Undaria pinnatifida effectively inhibited reactive oxygen species (ROS) production in vitro and reduced cell death in zebrafish models exposed to oxidative stress (Oh et al. 2020 ). Additionally, fucoidan fractions from Sargassum tenerrimum used in this study showed vigorous antioxidant activity, with their antioxidant capacity linked to their sulfate content (Marudhupandi et al. 2014 ). In adjuvant induced arthritis, elevated blood NO strongly correlates with disease severity. NO levels increase during inflammation in adjuvant induced arthritis models, reflecting joint swelling and histological damage. Similarly, RA patients with severe inflammation exhibit high NO. This correlation suggests that monitoring blood NO could aid in assessing therapeutic efficacy in adjuvant induced arthritis (Golbahari and Abtahi Froushani 2019 ; Etemadi et al. 2022 ). In this study, an elevation of serum NO levels was noted in RA rats. Subsequently, fucoidan treatment led to a reduction in these levels, with the extent of the decrease correlating with the dosage administered. The most notable reduction was seen with a dosage of 150 mg/kg. In inflammatory contexts (e.g., LPS-activated microglia or macrophages), fucoidan suppresses inducible NOS (iNOS) expression, thereby reducing excessive NO production. Fucoidan also decreases superoxide (O₂⁻) and NO overproduction, limiting peroxynitrite formation (Cui et al. 2010 ). By enhancing antioxidant enzymes through the Nrf2/ERK pathway, fucoidan lowers oxidative stress, indirectly reducing reactive nitrogen species (RNS) generation (Zayed et al. 2023 ). CRP, a liver protein increased by inflammation (e.g., IL-1, TNF-α), reflects systemic inflammation and plays a key role in adjuvant-induced arthritis. Elevated CRP in RA correlates with joint swelling and damage. CRP exacerbates inflammation by activating the complement system and recruiting immune cells. It also stimulates cytokine production, sustaining the inflammatory response. CRP binds to damaged joint cells, marking them for clearance and contributing to cartilage and bone erosion (Moases Ghaffary and Abtahi Froushani 2020 ; Morvaridiet al. 2025 ). RA rats in the current investigation showed increased serum CRP, which fucoidan reduced in a dose-dependent manner. In mice with thioacetamide-induced liver injury, fucoidan significantly reduced serum CRP levels and pro-inflammatory cytokines by inhibiting the NF-κB and MAPK pathways (Tsai et al. 2021 ). Similarly, in LPS-induced lung inflammation and arthritis models, fucoidan lowered CRP levels, demonesterating its systemic anti-inflammatory effects (Zhu et al. 2020 ). The trigger and driver of inflammation in the joints in rheumatoid arthritis involve T-lymphocyte responses. Depending on the conditions and microenvironment, T lymphocytes polarize in different ways, which affect the fate and course of an autoimmune disease (Etemadi et al. 2022 ). Type 1 immunity involves innate and adaptive responses mediated by T-bet expressing ILC-1, NKT, γδ-T, and Th1 lymphocytes that produce the key cytokine IFN-γ. Dysregulation of type 1 immunity can lead to delayed-type hypersensitivity and organ-specific autoimmunity (Nabekura and Shibuya 2021 ). Type 2 immunity is characterized by GATA-3 + ILC-2, NKT, γδ-T, and Th2 lymphocytes that produce the signature cytokines IL-4, IL-5, and IL-13. Uncontrolled type 2 responses can cause allergic diseases and suppress inflammatory arthritis (Kim et al. 2024 ). Type 3 immunity, mediated by RORγt expressing lymphocytes, is increasingly recognized in the pathogenesis of RA, mainly through IL-17 production. IL-17 stimulates synovial joint cells, including stromal cells, chondrocytes, fibroblasts, and macrophages, to secrete pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α (Woś and Tabarkiewicz 2021 ). Meanwhile, FoxP3-Treg lymphocytes are crucial for maintaining self-tolerance by inhibiting effector T cells (Khandpur et al. 2013 ; Annunziato et al. 2015 ; Etemadi et al. 2022 ). It is clear that RA shifts the balance towards pro-inflammatory Th1/Th17 responses, and prednisolone effectively reverses this shift (Mirzaaghasi and Froushani 2023). According to our findings, fucoidan reduced the expression of inflammatory genes T-bet and RORγt in a dose-dependent manner, leading to a decrease in polarization towards the Th1 and Th17 subsets. Conversely, it increased the expression of GATA3 and FOXP3 genes, resulting in enhanced polarization towards Th2 and Treg cells. These findings provide a molecular mechanism to explain the observed anti-inflammatory effects of fucoidan in the RA model. By modulating the expression of key transcription factors, fucoidan helps to re-establish immune homeostasis and reduce inflammation. Notably, long-term use of conventional NSAIDs can lead to gastrointestinal ulcers, renal toxicity, and cardiovascular risks. More importantly, NSAIDs suppress symptoms without addressing the underlying immune dysregulation (Wongrakpanich et al. 2018 ; Bindu et al. 2020 ). Here, we clearly demonstrated that fucoidan extracted from Sargassum tenerrimum , as a natural NSAID, has the ability to modulate and regulate immune responses in an experimental model of RA. Conclusion Fucoidan extracted from Persian Gulf Sargassum tenerrimum , characterized by a high content of sulfated fucose and confirmed via FTIR, showed strong antioxidant activity (DPPH scavenging ~ 85.65%). In a rat model of adjuvant-induced arthritis, fucoidan had dose-dependent anti-inflammatory and joint-protective effects. The 150 mg/kg dose showed the greatest benefits, even surpassing prednisolone in some aspects. Overall, fucoidan's potential to treat rheumatoid arthritis appears to stem from antioxidant, anti-inflammatory, and immune-regulating actions. These actions work together to lower oxidative stress, reduce inflammatory signals, and restore balance to the immune system. While prednisolone is still more powerful, a high dose of fucoidan (150 mg/kg) presents a hopeful natural supplement with multiple benefits and potentially fewer side effects compared to corticosteroids. Further research is warranted to explore higher dosages exceeding 150 mg/kg and to investigate the precise mechanisms underlying fucoidan’s action. Declarations Conflict of interest The author(s) declare no competing interests. Acknowledgements The authors would like to thank the staffs of the Veterinary Faculty of Urmia University. Funding This research was supported by Urmia University, Urmia,Iran. Data availability Enquiries about data availability should be directed to the authors. Author Contribution Leila Mahmoudzadeh: Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Writing – original draft. Seyyed Meysam Abtahi Froushani: Methodology, Funding acquisition, Conceptualization, Writing – review & editing. Seyede Soraya Mahmoudi: Data curation, Visualization. References Abtahi Froushani SM, Esmaili Gourvarchin Galeh H (2014) New insight into the immunomodulatory mechanisms of Tretinoin in NMRI mice. Iranian Journal of Basic Medical Sciences 17 :632-637. Alea MT, Meyer AS (2013). Fucoidans from brown seaweeds: an update on structures, extraction techniques and use of enzymes as tools for structural elucidation. RSC Adv 3 :8131-8141. Annunziato F, Romagnani C, Romagnani S (2015). The 3 major types of innate and adaptive cell-mediated effector immunity. J Allergy Clin Immunol 135 :626-635. Apostolova E, Lukova P, Baldzhieva A, Katsarov P, Nikolova M, Iliev L, Peychev L, Trica B, Oancea F, Delattre C, Kokova V (2020). Immunomodulatory and Anti-Inflammatory Effects of Fucoidan: A Review. Polymers (Basel) 12 :2338. Apostolova E, Lukova P, Baldzhieva A, Katsarov P, Nikolova M, Iliev I, Peychev L, Trica B, Oancea F, Delattre C, Kokova V (2020). Immunomodulatory and Anti-Inflammatory Effects of Fucoidan: A Review. Polymers (Basel) 12 :2338. Ashayerizadeh O, Dastar B, Pourashouri P (2020). Study of antioxidant and antibacterial activities of depolymerized fucoidans extracted from Sargassum tenerrimum. International Journal of Biological Macromolecules 151 :1259-1266. Baliyan S, Mukherjee R, Priyadarshini A, Vibhuti A, Gupta A, Pandey RP, Chang CM (2022). Determination of Antioxidants by DPPH Radical Scavenging Activity and Quantitative Phytochemical Analysis of Ficus religiosa. Molecules 27 :1326. Bassuk SS, Rifai N, Ridker PM (2004). High-sensitivity C-reactive protein: clinical importance. Curr Probl Cardiol 29 :439-493. Bindu S, Mazumder S, Bandyopadhyay U (2020). Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective. Biochem Pharmacol 180:114147. Carvalho A, Sousa R, Franco A, Costa J, Neves L, Ribeiro R, Sutton R, Criddle D, Soares P, Souza M (2014). Protective Effects of Fucoidan, a P- and L-Selectin Inhibitor, in Murine Acute Pancreatitis. Pancreas 43:82-87. Chadha S, Behl T, Kumar A, Khullar G, Arora S (2020). Role of Nrf2 in rheumatoid arthritis. Current Research in Translational Medicine 68:171-181. Ciofoaia E I, Pillarisetty A, Constantinescu F (2022). Health disparities in rheumatoid arthritis. Ther Adv Musculoskelet Dis 14 : 1759720x221137127. Cui Y Q, Zhang L J, Zhang T, Luo D Z, Jia Y J, Guo Z X, Zhang Q B, Wang X, Wang X M (2010). Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide-activated primary microglia. Clin Exp Pharmacol Physiol 37 :422-428. Ersoydan S, Rustemeyer T (2024). Investigating the Anti-Inflammatory Activity of Various Brown Algae Species. Mar Drugs 22:457. Etemadi S, Abtahi Froushani SM, Hashemi Asl SM, Mahmoudian A (2022). Combined atorvastatin and pentoxifylline in ameliorating inflammation induced by complete Freund's adjuvant. Inflammopharmacology 30:935-944. Abtahi Froushani SM, Mashhouri S (2019). The Effect of Mesenchymal Stem Cells Pulsed with 17 Beta-Estradiol in an Ameliorating Rat Model of Ulcerative Colitis. Zahedan Journal of Research in Medical Sciences 21:33-45. Ghaffary E M, Abtahi Froushani SM (2020). Immunomodulatory benefits of mesenchymal stem cells treated with Caffeine in adjuvant-induced arthritis. Life Sci 246:117420. Golbahari S, Abtahi Froushani SM (2019). Synergistic benefits of Nicotine and Thymol in alleviating experimental rheumatoid arthritis. Life Sci 239:117037. Gurau S, Imran M, Ray R L (2025). Algae: A cutting-edge solution for enhancing soil health and accelerating carbon sequestration – A review. Environmental Technology & Innovation 37:103980. Hajizadeh A, Abtahi Froushani SM, Tehrani AA, Azizi S, Hashemi S R B (2021). Effects of Naringenin on Experimentally Induced Rheumatoid Arthritis in Wistar Rats. Archives of Razi Institute 76:903-912. Hifney A F, Fawzy M A, Abdel-Gawad KM, Gomaa M (2016). Industrial optimization of fucoidan extraction from Sargassum sp. and its potential antioxidant and emulsifying activities. Food Hydrocolloids 54:77-88. Isnansetyo A, Lutfia FNL, Nursid M, Trijoko, Susidarti RA (2017). Cytotoxicity of Fucoidan from Three Tropical Brown Algae Against Breast and Colon Cancer Cell Lines. Pharmacognosy Journal 9:14-20. Izumi Y, Koyama Y (2024). Nrf2-Independent Anti-Inflammatory Effects of Dimethyl Fumarate: Challenges and Prospects in Developing Electrophilic Nrf2 Activators for Neurodegenerative Diseases. Antioxidants (Basel) 13:1527. Jafari- Khataylou, Kazemi-Darabadi YS, Ahmadi Afshar S (2023). Effects of adenosine N1-Oxide and pioglitazone on inflammatory and antioxidant state in sepsis caused by experimental cecal puncture in rat. Veterinary Research Forum 14:381-387. Jahantigh M, Abtahi Froushani SM, Afzale Ahangaran N (2023). Benefits of bone marrow-derived mesenchymal stem cells primed with estradiol in alleviating collagen-induced arthritis. Iranian Journal of Basic Medical Sciences 26:400-407. Jamtsho T, Yeshi K, Perry MJ, Loukas A, Wangchuk P (2024). Approaches, Strategies and Procedures for Identifying Anti-Inflammatory Drug Lead Molecules from Natural Products. Pharmaceuticals (Basel) 17:283. Kadam S U, Tiwari B K, O'Donnell C P (2014). Extraction, structure and biofunctional activities of laminarin from brown algae. International Journal of Food Science & Technology 50:24-31. Kajimura J, Ito R, Manley N R, Hale L P (2016). Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues. J Histochem Cytochem 64:112-124. Kang N, Zhao J, Gao P, Lu Y, Chen Z, Li X, Khan I A, Yang S, Xu Q, Liu Y (2025). Innovative Strategies in Natural Product Drug Discovery: The Case of Anemoside B4. Engineering. Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, Gizinski A, Yalavarthi S, Knight J S, Friday S, Li S, Patel R M, Subramanian V, Thompson P, Chen P, Fox D A, Pennathur S, Kaplan M J (2013). NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med 5:140-178. Kim H, Moon C, Park E J, Jee Y, Ahn M, Wie M B, Shin T (2010). Amelioration of experimental autoimmune encephalomyelitis in Lewis rats treated with fucoidan. Phytother Res 24:399-403. Kim H Y, Jeong D, Kim J H, Chung D H (2024). Innate Type-2 Cytokines: From Immune Regulation to Therapeutic Targets. Immune Netw 24:e6. Kou H, Huang L, Jin M, He Q, Zhang R, Ma J (2023). Effect of curcumin on rheumatoid arthritis: a systematic review and meta-analysis. Front Immunol 14: 1121655. Law S T, Taylor P C (2019). Role of biological agents in treatment of rheumatoid arthritis. Pharmacol Res 150:104497. Lee S, Lee E J, Lee G M, Yun J H, Yoo W (2023). Inhibitory effect of fucoidan on TNF-α-induced inflammation in human retinal pigment epithelium cells. Front Nutr 10:1162934. Lim S J, Aida W M W, Maskat M Y, Latip J, Badri K H, Hassan O, Yamin B M (2016). Characterisation of fucoidan extracted from Malaysian Sargassum binderi. Food Chem 209:267-273. Lin Z W, Wu L X, Xie Y, Ou X, Tian P K, Liu X P, Min J, Wang J, Chen R F, Chen Y J, Liu C, Ye H, Ou Q J (2015). The Expression Levels of Transcription Factors T-bet, GATA-3, RORγt and FOXP3 in Peripheral Blood Lymphocyte (PBL) of Patients with Liver Cancer and their Significance. International Journal of Medical Sciences 12:7-16. Linares-Maurizi A, Reversat G, Awad R, Bultel-Poncé V, Oger C, Galano J M, Balas L, Durbec A, Bertrand-Michel J, Durand T, Pradelles R, Vigor C (2023). Bioactive Oxylipins Profile in Marine Microalgae. Mar Drugs 21:136. Lu SH, Hsia YJ, Shih KC, Chou TC (2019). Fucoidan Prevents RANKL-Stimulated Osteoclastogenesis and LPS-Induced Inflammatory Bone Loss via Regulation of Akt/GSK3β/PTEN/NFATc1 Signaling Pathway and Calcineurin Activity. Mar Drugs 17:345. Makarov SS (2001). NF-kappa B in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res 3:200-206. Marudhupandi T, Kumar TT, Senthil SL, Devi K N (2014). In vitro antioxidant properties of fucoidan fractions from Sargassum tenerrimum. Pak J Biol Sci 17:402-407. Marudhupandi T, Kumar TTA, Senthil SL, Devi KN (2014). In vitro antioxidant properties of fucoidan fractions from Sargassum tenerrimum. Pak J Biol Sci 17:402-407. Massalska M, Radzikowska A, Kuca-Warnawin E, Plebanczyk M, Prochorec-Sobieszek M, Skalska U, Kurowska W, Maldyk P, Kontny E, Gober HJ, Maslinski W (2020). CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired. Cells 9:549. Mirzaaghasi S, Abtahi Froushani SM (2023). Immunomodulatory Effects of Combined Nicotinic Acid and Prednisolone in Adjuvant-induced Arthritis. Antiinflamm Antiallergy Agents Med Chem 22:104-112. Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB (2014). Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal 20:1126-1167. Mlynarik M, Johansson BB, Jezova D (2004). Enriched environment influences adrenocortical response to immune challenge and glutamate receptor gene expression in rat hippocampus. Ann N Y Acad Sci 1018: 273-280. Moases Ghaffary E, Abtahi Froushani SM (2020). Immunomodulatory benefits of mesenchymal stem cells treated with Caffeine in adjuvant-induced arthritis. Life Sciences 246: 117420. Morvaridi A, Abtahi Froushani SM, Farshid AA (2025). Benefits of combining piperine with prednisolone in an experimental model of rheumatoid arthritis. Vet Res Forum 16:117-124. Nabekura T, Shibuya A (2021). Type 1 innate lymphoid cells: Soldiers at the front line of immunity. Biomed J 44:115-122. Narváez J, Aguilar-Coll M, Roig-Kim M, Maymó-Paituvi P, Palacios-Olid J, Nolla JM, LLop D (2024). Janus kinase inhibitors in rheumatoid arthritis-associated interstitial lung disease: A systematic review and meta-analysis. Autoimmun Rev 23(10):103636. Ngo V, Duennwald ML (2022). Nrf2 and Oxidative Stress: A General Overview of Mechanisms and Implications in Human Disease. Antioxidants (Basel) 11:2345. Oh JY, Kim EA, Kang SI, Yang HW, Ryu B, Wang L, Lee JS, Jeon YJ (2020). Protective Effects of Fucoidan Isolated from Celluclast-Assisted Extract of Undaria pinnatifida Sporophylls against AAPH-Induced Oxidative Stress In Vitro and In Vivo Zebrafish Model. Molecules 25:2361. Pacheco D, Poza SG, Cotas J, Gonçalves AMM (2020). Fucoidan - A valuable source from the ocean to pharmaceutical. Frontiers in Drug, Chemistry and Clinical Research 3:1-4. Pal R, Chaudhary MJ, Tiwari PC, Nath R, Pant KK (2018). Pharmacological and biochemical studies on protective effects of mangiferin and its interaction with nitric oxide (NO) modulators in adjuvant-induced changes in arthritic parameters, inflammatory, and oxidative biomarkers in rats. Inflammopharmacology. Palanisamy S, Vinosha M, Marudhupandi T, Rajasekar P, Prabhu NM (2017). Isolation of fucoidan from Sargassum polycystum brown algae: Structural characterization, in vitro antioxidant and anticancer activity. Int J Biol Macromol 102:405-412. Park HY, Han MH, Park C, Jin CY, Kim GY, Choi IW, Kim ND, Nam TJ, Kwon TK, Choi YH (2011). Anti-inflammatory effects of fucoidan through inhibition of NF-κB, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells. Food Chem Toxicol 49:1745-1752. Park SB, Chun KR, Kim JK, Suk K, Jung YM, Lee WH (2010). The differential effect of high and low molecular weight fucoidans on the severity of collagen-induced arthritis in mice. Phytother Res 24:1384-1391. Poursamimi J, Shariati-Sarabi Z, Tavakkol-Afshari J, Mohammadi M (2022). A Significant Increase in the Gene Expression of GATA-3 Following the Treatment of Osteoarthritis Patients with Crocin. Iran J Allergy Asthma Immunol 21:35-43. Puhari SSM, Yuvaraj S, Vasudevan V, Ramprasath T, Arunkumar K, Amutha C, Selvam GS (2023). Fucoidan from Sargassum wightii reduces oxidative stress through upregulating Nrf2/HO-1 signaling pathway in alloxan-induced diabetic cardiomyopathy rats. Mol Biol Rep 50:8855-8866. Rahawy AM, Al-Mahmood SS, Basim H, Rahawi AM (2021). Standard techniques for formalin-fixed paraffin-embedded tissue: A Pathologist's perspective. Iraqi Journal of Veterinary Sciences 35. Raj PP, Gopal RK, Sanniyasi E (2024). Investigating the anti-inflammatory and anti-arthritis effects of fucoidan from a brown seaweed." Current Research in Biotechnology 7 :100220. Ryu MJ, Chung HS (2016). Fucoidan reduces oxidative stress by regulating the gene expression of HO‑1 and SOD‑1 through the Nrf2/ERK signaling pathway in HaCaT cells. Mol Med Rep 14:3255-3260. Silberfeld T, Rousseau F, Reviers B (2014). An Updated Classification of Brown Algae (Ochrophyta, Phaeophyceae). Cryptogamie Algologie 35:117-156. Singh J A (2022). Treatment Guidelines in Rheumatoid Arthritis. Rheum Dis Clin North Am 48:679-689. Takahashi I, Takeda K, Toyama T, Matsuzaki T, Kuroki H, Hoso M (2024). Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model. Sci Rep 14:10568. Tang S, Wang L, Ren X, Song S, Ai C (2024). Fucoidan Alleviates Colitis and Metabolic Disorder by Protecting the Intestinal Barrier, Suppressing the MAPK/NF-κB Pathways, and Regulating the Gut Microbiota." Journal of Food Biochemistry 2024:7955190. Tsai M Y, Yang WC, Lin CF, Wang CM, Liu HY, Lin CS, Lin JW, Lin WL, Lin TC, Fan PS, Hung KH, Lu YW, Chang GR (2021). The Ameliorative Effects of Fucoidan in Thioacetaide-Induced Liver Injury in Mice. Molecules 26:1937. Wongrakpanich S, Wongrakpanich A, Melhado K, Rangaswami J (2018). A Comprehensive Review of Non-Steroidal Anti-Inflammatory Drug Use in The Elderly. Aging Dis 9:143-150. Woś I, Tabarkiewicz J (2021). Effect of interleukin-6, -17, -21, -22, and -23 and STAT3 on signal transduction pathways and their inhibition in autoimmune arthritis. Immunologic Research 69:26-42. Yang J, Wang J, Liang X, Zhao H, Lu J, Ma Q, Jing B, F. Tian F (2019). IL‑1β increases the expression of inflammatory factors in synovial fluid‑derived fibroblast‑like synoviocytes via activation of the NF‑κB‑mediated ERK‑STAT1 signaling pathway. Mol Med Rep 20:4993-5001. Yin C, Bi Q, Chen W, Wang C, Castiglioni B, Li Y, Sun W, Pi Y, Bontempo V, Li X, Jiang X (2024). Fucoidan Supplementation Improves Antioxidant Capacity via Regulating the Keap1/Nrf2 Signaling Pathway and Mitochondrial Function in Low-Weaning Weight Piglets. Antioxidants (Basel) 13:407. Yue Q, Liu Y, Li F, Hong T, Guo S, Cai M, Zhao L, Su L, Zhang S, Zhao C, Li K (2025). Antioxidant and anticancer properties of fucoidan isolated from Saccharina Japonica brown algae. Sci Rep 15:8962. Zayed A, Al-Saedi DA, Mensah EO, Kanwugu ON, Adadi P, Ulber R (2023). Fucoidan's Molecular Targets: A Comprehensive Review of Its Unique and Multiple Targets Accounting for Promising Bioactivities Supported by In Silico Studies. Mar Drugs 22:29-39. Zeng Z, Yan K, Liu W (2022). Specneuzhenide Ameliorate Complete Freund Adjuvant Induced Arthritis in Rats: Involvement of NF-κB and HO-1/Nrf-2 Pathway. Journal of Oleo Science 71:551-561. Zhang W, Lee PCW, Jin JO (2024). Anti-Inflammatory Effect of Fucoidan from Costaria costata Inhibited Lipopolysaccharide-Induced Inflammation in Mice. Mar Drugs 22:401. Zhao M, Garcia-Vaquero M, Przyborska J, Sivagnanam SP, Tiwari B (2021). The development of analytical methods for the purity determination of fucoidan extracted from brown seaweed species. International Journal of Biological Macromolecules 173:90-98. Zhu DZ, Wang YT, Zhuo YL, Zhu KJ, Wang XZ, Liu AJ (2020). Fucoidan inhibits LPS-induced acute lung injury in mice through regulating GSK-3β-Nrf2 signaling pathway. Arch Pharm Res 43:646-654. Additional Declarations No competing interests reported. Supplementary Files Highlights.docx supplfig.tiff Figure 1 suppl. (a) shows marine brown seaweed, specifically Sargassum tentorium . (b) The seaweeds were rinsed with tap water to remove epiphytes. (c) cream color, odorless powder that was identified as fucoidan. (d) The FTIR method was employed to verify the successful separation of fucoidan. Graphicalabstract.jpg Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Inflammopharmacology → Version 1 posted Editorial decision: Revision requested 26 Dec, 2025 Reviews received at journal 24 Dec, 2025 Reviewers agreed at journal 13 Dec, 2025 Reviews received at journal 18 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers invited by journal 31 Oct, 2025 Editor assigned by journal 31 Oct, 2025 Submission checks completed at journal 31 Oct, 2025 First submitted to journal 29 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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-7981987","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542775693,"identity":"3f8436af-2160-44bb-8603-16bde7411ce9","order_by":0,"name":"Leila Mahmoudzadeh","email":"","orcid":"","institution":"Urmia University","correspondingAuthor":false,"prefix":"","firstName":"Leila","middleName":"","lastName":"Mahmoudzadeh","suffix":""},{"id":542775694,"identity":"81f0d604-2bb9-4c42-94db-70d4984fa441","order_by":1,"name":"Seyyed Meysam Abtahi Froushani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYJACZiBOYGNvAFIGEJEDhLUkALXwHCBVC4NEApGOkp92gPFz4Q+7PD7JN2afbhTYMfC3H2A8XIFHi8HtBGbpGQnJxWzSOcazcwySGSTOJDAcPINPi3QCgzRPAnNiG1ALc44BMChuMDAcbMDnsNkJzL95EuoT2yTPgLTUM8gT0sJwO4ENaMvhxDYJHpCWwwwGhLQY3E5ss56RdjyxjSetGKjlOI/hmcQGAg5LPny7wKY6cX774c3MOX+q5eSOHz78Ea/DGBhRpXkwREbBKBgFo2AUkA4A2YxGbV8p7VEAAAAASUVORK5CYII=","orcid":"","institution":"Urmia University","correspondingAuthor":true,"prefix":"","firstName":"Seyyed","middleName":"Meysam Abtahi","lastName":"Froushani","suffix":""},{"id":542775695,"identity":"b4b69f54-ff5c-4d62-b62e-fbed86307827","order_by":2,"name":"Seyede Soraya Mahmoudi","email":"","orcid":"","institution":"Urmia University","correspondingAuthor":false,"prefix":"","firstName":"Seyede","middleName":"Soraya","lastName":"Mahmoudi","suffix":""}],"badges":[],"createdAt":"2025-10-29 16:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7981987/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7981987/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10787-026-02165-x","type":"published","date":"2026-02-28T15:58:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95801761,"identity":"9f808180-1ef4-47b8-8d1d-a4000dce43cf","added_by":"auto","created_at":"2025-11-13 08:26:07","extension":"png","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29777,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/dfcd974cfd88ef88b3325955.png"},{"id":95753694,"identity":"6aa8e1aa-eee1-4624-82ac-adebd7eb4e6f","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61870,"visible":true,"origin":"","legend":"","description":"","filename":"Article.inflam.docx","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/c520eb3ebeee9f15d6dd9cf5.docx"},{"id":95753697,"identity":"092493fd-f93c-4d2d-b980-031f426ceb61","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2182606,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/f2836e501db0e82fb9ae6157.tiff"},{"id":95801899,"identity":"0d457455-8525-4c2b-baf8-e6ea5ab085ac","added_by":"auto","created_at":"2025-11-13 08:26:25","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18444,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/452c31d1a4fd612480a0edde.docx"},{"id":95753711,"identity":"6ac41eb1-84d4-43db-83f1-9e791b105adb","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4032206,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/18bbc461737dc7989a42b65e.tiff"},{"id":95802347,"identity":"a596501f-b34c-4977-b69b-8bf0f415a66d","added_by":"auto","created_at":"2025-11-13 08:27:30","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13818,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/3ef249c576fadad25e5c612b.docx"},{"id":95801943,"identity":"9730106d-b6db-4460-a14f-e1cb5c57b398","added_by":"auto","created_at":"2025-11-13 08:26:29","extension":"tiff","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4553806,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/be00c8bad3bbc026c6b158c1.tiff"},{"id":95801864,"identity":"74e69797-8668-4a14-ad0e-01fbe8194438","added_by":"auto","created_at":"2025-11-13 08:26:21","extension":"tiff","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2901806,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/0481666a6b5ce01a6dc8a0d3.tiff"},{"id":95753708,"identity":"be9e2892-4990-4306-8cf1-57e757334c37","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"json","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5483,"visible":true,"origin":"","legend":"","description":"","filename":"b99cb72176194c67b232f983381eb972.json","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/f39f30107db8a2758be0b70d.json"},{"id":95753712,"identity":"8556f950-bd36-4a30-b6b3-eb436bed8af7","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"jpg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342326,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/479b175c9162cbf13cc5b77f.jpg"},{"id":95753707,"identity":"46e8ff85-5348-4b02-8e53-a1dc9f0896dd","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14654,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/49a6bc94f6ef3ea69200a80b.docx"},{"id":95753722,"identity":"6659ad8d-eede-4ed9-8a8a-3b6f55e37813","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"tiff","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2928206,"visible":true,"origin":"","legend":"","description":"","filename":"supplfig.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/b0a13e90b708e8c2b28c6070.tiff"},{"id":95753709,"identity":"c43b937f-da04-455e-8476-0ad015da4465","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168317,"visible":true,"origin":"","legend":"","description":"","filename":"b99cb72176194c67b232f983381eb9721enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/9d345aa4fd4c4cd3097a27bb.xml"},{"id":95801830,"identity":"ae53d016-6af3-40f1-a0ee-6322c1227ac5","added_by":"auto","created_at":"2025-11-13 08:26:17","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29777,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/b199b584492a61d7b603f5d1.png"},{"id":95753714,"identity":"cfd97c84-df5a-4ebd-af9f-e5f829ff9528","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"tiff","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2182606,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/312afacf3598a1b5edfb02d0.tiff"},{"id":95801561,"identity":"aa092f5e-7c8c-46ab-8472-15c578b20642","added_by":"auto","created_at":"2025-11-13 08:25:41","extension":"tiff","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4032206,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/7dd489401347845b58b97d4c.tiff"},{"id":95801954,"identity":"8c0b2e89-a5c8-4f29-9c4c-528d0f6a5486","added_by":"auto","created_at":"2025-11-13 08:26:31","extension":"tiff","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4553806,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/472ea5b69374e82b7121e2ed.tiff"},{"id":95800172,"identity":"f23ea4e8-5eea-4131-a494-238dd74e51ac","added_by":"auto","created_at":"2025-11-13 08:21:46","extension":"tiff","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1398846,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/8480f03342410350492e449e.tiff"},{"id":95753719,"identity":"80adfbbb-158a-4762-ba4b-203f650c4a2a","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"tiff","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":655360,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/2a7bf16866a9132c999d41f6.tiff"},{"id":95753724,"identity":"c463a6a8-50a4-4e5a-b82f-1939274cecff","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"tiff","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2901806,"visible":true,"origin":"","legend":"","description":"","filename":"Figure7.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/6510953b7fed80d322f96cfc.tiff"},{"id":95753716,"identity":"9f3b1898-20d2-4756-9bd3-54876d97e580","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27687,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/6697203787ddb6c5eebade15.png"},{"id":95753721,"identity":"00d0db3c-cd19-4fe4-9309-4c06fa9add6c","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48277,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/4f9e6bb895a6af808d6ac33d.png"},{"id":95753723,"identity":"3ff8c48f-9dbb-4b62-b26d-28d1985ba533","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342212,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/db26cc4f7e11b2fb6ee179ae.png"},{"id":95753715,"identity":"26af4f26-2c47-4c65-b1f1-784d9951d4c7","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":261986,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/b1c3a01a8c264e5b9e5fff2b.png"},{"id":95801772,"identity":"910c555e-54d4-480a-b9b5-f291e8a9ba99","added_by":"auto","created_at":"2025-11-13 08:26:07","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53753,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/46dd1c80234ac0c4aacf3603.png"},{"id":95801937,"identity":"bec296e3-4a07-4304-98cb-c7a9189f2e3d","added_by":"auto","created_at":"2025-11-13 08:26:29","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168240,"visible":true,"origin":"","legend":"","description":"","filename":"b99cb72176194c67b232f983381eb9721structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/ea7b02adeeb8bdba2fe70eeb.xml"},{"id":95753727,"identity":"0df2bd36-132c-4e0b-933a-e8f5f2abef13","added_by":"auto","created_at":"2025-11-12 16:16:11","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174852,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/513c14e7ecf58cc6ad9ecca6.html"},{"id":95753691,"identity":"61499b2a-7f75-4332-b46b-7060153c799f","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66655,"visible":true,"origin":"","legend":"\u003cp\u003eDPPH radical scavenging activity of fucoidan extracted from the brown seaweed \u003cem\u003eSargassum tenerrimum\u003c/em\u003e. Data are presented as mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/6f6ec914cdd0d39fe3254b6c.png"},{"id":95753693,"identity":"4b7e43a7-b8d5-470a-890e-80781fb5e509","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1449769,"visible":true,"origin":"","legend":"\u003cp\u003eClinical features of rheumatoid arthritis (RA) rats after fucoidan/prednisolone treatment. A dose-dependent reduction in RA features was seen, with prednisolone and 150 mg fucoidan showing the best response. (A) Arthritis index and paw swelling, (B) average arthritis index, final arthritis index, and mean body weight. Data are mean ± S.D., with statistical significance indicated (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/9d45e8bd985ebdc97e62f9c8.png"},{"id":95753701,"identity":"624cd4e5-641c-4942-83c1-9d7aa2151e62","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8559627,"visible":true,"origin":"","legend":"\u003cp\u003eHind paw histology (H\u0026amp;E) after arthritis induction. The control group was normal. The RA group showed bone/joint damage, inflammation, and congested vessels. Fucoidan decreased joint damage and inflammation in a dose-dependent manner. Prednisolone and Fuc 150 reduced inflammation and lymphocyte accumulation (arrows indicate changes).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/fa08478a74354ceeca5d45ef.png"},{"id":95800442,"identity":"fcd6163b-123a-4687-88b3-402a3cfe24cd","added_by":"auto","created_at":"2025-11-13 08:22:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8604322,"visible":true,"origin":"","legend":"\u003cp\u003eJoint tissue histology (Safranin O). (a) RA group showed reduced cartilage thickness (lower staining). Fucoidan groups had increased staining, showing better cartilage and less inflammation. (b) Safranin O staining intensity. Colorimetric chart shows staining differences. Significance at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/1da0195a57bab6a3d046ea72.png"},{"id":95753696,"identity":"fe6c296b-00cf-4e24-a944-f783bbcabdea","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1507049,"visible":true,"origin":"","legend":"\u003cp\u003eIL-1β immunohistochemistry (IHC) in rat knee synovial tissue: (a) IHC images (green = antibody, blue = DAPI). (b) Chart of groups studied. Significance at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/834f90b1db7a84dd0d018a92.png"},{"id":95802379,"identity":"45a36233-63c4-488a-8a11-eadb522b2e0a","added_by":"auto","created_at":"2025-11-13 08:27:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1643635,"visible":true,"origin":"","legend":"\u003cp\u003eTNFα immunohistochemistry (IHC) in rat knee synovial tissue: (a) IHC images (green = antibody, blue = DAPI). (b) Chart of groups studied. Significance at p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure61.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/8e18ebc1f7b8a78a49804fac.png"},{"id":95753702,"identity":"3c4eb845-b227-46fc-bb6a-20b0afda6ec2","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1597178,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression of Th0 differentiation transcription factors (Real-Time PCR, mRNA fold change in log2). Data are mean ± standard deviation, with letters indicating statistical significance (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/6879d224feb538c84a791622.png"},{"id":103765567,"identity":"f2aeb831-753c-4f75-972e-296d873c3400","added_by":"auto","created_at":"2026-03-02 16:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21866125,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/ecdf1cfe-4aa1-400a-8aba-fe9060837223.pdf"},{"id":95753695,"identity":"08790701-f786-47ac-9937-b2db2bb84067","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14654,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/8a8c9220710bd8c587003f3d.docx"},{"id":95801763,"identity":"55c6c9dc-b957-47e3-b34b-dad799b0a71f","added_by":"auto","created_at":"2025-11-13 08:26:07","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2928206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1 suppl.\u003c/strong\u003e (a) shows marine brown seaweed, specifically \u003cem\u003eSargassum tentorium\u003c/em\u003e. (b) The seaweeds were rinsed with tap water to remove epiphytes. (c) cream color, odorless powder that was identified as fucoidan. (d) The FTIR method was employed to verify the successful separation of fucoidan.\u003c/p\u003e","description":"","filename":"supplfig.tiff","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/63cbbd88d3740da1e8e6103b.tiff"},{"id":95753699,"identity":"44c6012b-aab3-4cf4-947e-44dc765b268f","added_by":"auto","created_at":"2025-11-12 16:16:10","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":342326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7981987/v1/08c0199231b48be6da1b4f61.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic Potential of Fucoidan from a Persian Gulf Alga (Sargassum tenerrimum) in Rheumatoid Arthritis: In Vivo Evaluation in a Rat Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRA is a chronic autoimmune disease causing joint inflammation, pain, swelling, and stiffness. It damages cartilage and bone, leading to deformities and impaired function. The cause is unknown but involves genetics, environmental factors, and hormones triggering an immune response. Symptoms include fatigue and mild fevers (Ciofoaia et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). RA affects 0.5-1% globally, more women, and increases with age. Untreated RA causes joint damage, declining physical function, deformities, and disability (Ciofoaia et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). RA is mainly managed with corticosteroids, nonsteroidal anti-inflammatory agents (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), biologics, and JAK inhibitors. Current treatments have side effects and aren't always effective. Corticosteroids can cause bone loss and heart problems. Long-term drug use increases risks (Singh \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). DMARDs and biologics raise infection and malignancy risks. Up to 40% don't respond to biologics like anti-TNF-α agents (Singh \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Narv\u0026aacute;ez et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Natural NSAIDs may offer therapeutic alternatives.\u003c/p\u003e\u003cp\u003eRat models like adjuvant-induced arthritis are key for RA research. Injecting Freund's complete adjuvant (FCA) triggers arthritis, mimicking human RA (Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mirzaaghasi and Froushani 2023). FCA contains inactivated Mycobacterium tuberculosis in mineral oil. This model shows swelling, cartilage damage, impaired joint function, and lymphocyte infiltration, making it reliable for evaluating RA treatments and other inflammatory conditions (Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zeng et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlgae show potential for treating autoimmune diseases due to their bioactive compounds like antioxidants, anti-inflammatory substances, and immunomodulators, which reduce oxidative stress and inflammation (Linares-Maurizi et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Brown algae (Phaeophyceae) are a major macroalgae group with brown pigments, mainly fucoxanthin (Silberfeld et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). They contain bioactive compounds beneficial for autoimmune and inflammatory conditions, including phlorotannin and polysaccharides like fucoidan, laminarin, and porphyrin, which have anti-inflammatory and immunomodulatory effects (Kadam et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ersoydan and Rustemeyer \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Algae farming is eco-friendly, using less land and water than regular farming. Some algae compounds may be absorbed better by the body than synthetic versions, which is key for better treatment (Gurau et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFucoidan, a sulfated polysaccharide predominantly obtained from brown algae, demonstrates a broad spectrum of biological activities, encompassing anti-inflammatory, immunomodulatory, antiviral, antidiabetic, anticancer, antioxidant, and anticoagulant effects. The structure of fucoidan, which can vary based on the algae source and extraction techniques, influences its bioactivity and therapeutic efficacy (Alea and Meyer \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pacheco et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pretreatment with Fucoidan extracted from \u003cem\u003eCostaria costata\u003c/em\u003e reduced dendritic cell activation, pro-inflammatory cytokine secretion (IL-1β, IL-6, TNF-α), and lung inflammation (Zhang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Fucoidan extracted from five algae species (\u003cem\u003ePadina pavonica\u003c/em\u003e, \u003cem\u003eStoechospermum marginatum\u003c/em\u003e, \u003cem\u003eSpatolossum macrodontum\u003c/em\u003e, \u003cem\u003eDictyota bartayresiana\u003c/em\u003e, and \u003cem\u003eTurbinaria decurrens\u003c/em\u003e) showed inhibition of nitric oxide production by RAW 264.7 macrophage cell lines with increasing concentrations of fucoidan (Raj et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies suggest that fucoidan can impede the differentiation of osteoclasts, which play a vital role in bone resorption and the inflammation associated with RA. It modulates signaling pathways, including Akt/GSK3β/PTEN and NFATc1, leading to decreased osteoclast activity and the inflammatory bone loss linked to the disease (Lu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Apostolova et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eF. vesiculosus\u003c/em\u003e fucoidan has anticancer, anti-inflammatory, and antioxidant effects. It inhibits COX-1/2, p38 MAPK, hyaluronidase, and protein denaturation, while also stabilizing RBC membranes (Zhang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) .It was reported that oral administration of Fucoidan extracted from \u003cem\u003eCostaria costata\u003c/em\u003e alleviated LPS-induced sepsis, highlighting its potential as a therapeutic agent for inflammatory diseases (Zhang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDue to its anti-inflammatory and immunomodulatory characteristics, Fucoidan's promising therapeutic effects in the context of RA provide reassurance and confidence in its potential. These findings support the investigation of various doses of fucoidan for treating RA induced by CFA in Wistar rats, as it may provide a natural alternative to conventional therapies. In this study, we examined the effects of fucoidan extracted from the Brown algae \u003cem\u003eSargassum tenerrimum\u003c/em\u003e, collected from the Persian Gulf region, in a rat model of rheumatoid arthritis induced by FCA.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals\u003c/h2\u003e\u003cp\u003eGIBCO/Life Technologies Inc. (USA) provided cell culture media and FBS. PeproTech EC, Ltd. (UK) supplied ELISA kits. TAKAPOUZIST (Iran) provided RNX-Plus. TAKARA (China) provided SYBR Premix Ex TaqII and cDNA kits. SibZist Fan Co (Iran) provided myeloperoxidase and nitric oxide assay kits. Elabscience (China) supplied CRP, COX-2, NF-κB, Nrf-2, and HO-1 ELISA kits. Sigma-Aldrich (USA) provided hematoxylin-eosin staining kits and other reagents.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSeaweed preparation and Fucoidan extraction procedure\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eSeaweed preparation and Fucoidan extraction procedure\u003c/div\u003e\u003cp\u003e\u003cem\u003eSargassum tenerrimum\u003c/em\u003e was collected from Chabahar, Iran (25\u0026deg;16\u0026prime;36.6\u0026Prime;N; 60\u0026deg;40\u0026prime;10.0\u0026Prime;E) in December 2023. The seaweed was rinsed, dried (50\u0026deg;C, 4 hours), and powdered. The powder was suspended in McIlvaine's buffer (pH 3\u0026ndash;7) and extracted at 60\u0026ndash;100\u0026deg;C for 3 hours. After filtration, CaCl2\u0026middot;2H2O (3% w/v) was added (1:1) and incubated overnight at 4\u0026deg;C to precipitate alginate, which was collected by vacuum filtration. Ethanol was added to the filtrate and stored overnight at 4\u0026deg;C to remove salts, followed by centrifugation (6000 rpm, 15 minutes) to harvest the crude polysaccharide. Freeze-drying yielded cream-colored fucoidan. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec suppl) (Hifney et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The yield of fucoidan was determined by employing the subsequent equation:\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFucoidan yield = [dry wt. of obtained fucoidan / dry wt. of sample] \u0026times; 100\u003c/p\u003e\n\u003ch3\u003eFucoidan Characterization\u003c/h3\u003e\n\u003cp\u003eFor Fourier transform infrared spectrometry (FT-IR) analysis, a pellet of acid-digested fucoidan and KBr (1 mg/100 mg) was prepared under 10,000 psi and analyzed from 600\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Zhao et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDPPH Free-Radical Scavenging Activity:\u003c/h3\u003e\n\u003cp\u003eThe 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging was tested as described before (Marudhupandi, Kumar et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Fucoidan solutions (1\u0026ndash;6 mg mL-1) were mixed with DPPH in methanol and DMSO (0.5 mM), incubated for 30 min in the dark, and absorbance was read at 517 nm (MeOH blank). Butylated hydroxyanisole (BHA) was the reference. Scavenging capacity was calculated using the equation below:\u003c/p\u003e\u003cp\u003eScavenging activity (%) = (1 - Asample / Acontrol) \u0026times; 100\u003c/p\u003e\u003cp\u003eA\u003csub\u003econtrol\u003c/sub\u003e is the absorbance of DPPH in methanol without the sample, while A\u003csub\u003esample\u003c/sub\u003e is the absorbance of DPPH in methanol with the sample.\u003c/p\u003e\n\u003ch3\u003eInduction of RA and animal groups\u003c/h3\u003e\n\u003cp\u003eSixty male Wistar rats (8 weeks old, 150\u0026thinsp;\u0026plusmn;\u0026thinsp;10 g) from the Razi Institute in Iran were housed in a controlled environment (23\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1, 12-hour light/dark cycle) with unrestricted access to food and water. Ethical guidelines from the National Institutes of Health Guide were adhered to, and all procedures were approved by the Faculty Ethics Committee. Proper housing is essential for animal welfare and the validity of research (Mlynarik et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRheumatoid arthritis (RA) was induced in rats via an intradermal injection of 0.1 mL Complete Freund's Adjuvant (CFA), containing 10 mg/mL killed Mycobacterium, at the base of the tail. Disease severity was assessed by measuring the volume of the contralateral hind paw at two-day intervals. A scoring system was employed to evaluate the condition: 0\u0026thinsp;=\u0026thinsp;normal paw; 1\u0026thinsp;=\u0026thinsp;erythema of the toe; 2\u0026thinsp;=\u0026thinsp;erythema and swelling of the paws; 3\u0026thinsp;=\u0026thinsp;swelling of the ankle; and 4\u0026thinsp;=\u0026thinsp;total swelling of the entire leg, resulting in an inability to flex. Assessments were conducted weekly by three observers. Body weight was monitored bi-daily following immunization (Ghaffary and Froushani 2020; Hajizadeh et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe study comprised six groups of rats (n\u0026thinsp;=\u0026thinsp;10 per group). Group 1 (Control) consisted of healthy, untreated rats. Group 2 (RA group) had RA induced but received no therapeutic intervention. Group 3 (RA\u0026thinsp;+\u0026thinsp;Fuc50) had RA induced and was treated daily with 50 mg/kg fucoidan via oral gavage. Group 4 (RA\u0026thinsp;+\u0026thinsp;Fuc100) had RA induced and received 100 mg/kg fucoidan daily via oral gavage. Group 5 (RA\u0026thinsp;+\u0026thinsp;Fuc150) had RA induced and was treated with 150 mg/kg fucoidan daily via oral gavage. Group 6 (RA\u0026thinsp;+\u0026thinsp;Pred) had RA induced and was administered 10 mg/kg prednisolone daily. All animals were euthanized 20 days post-disease induction, and samples were collected for subsequent analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eTissue Preparation and staining\u003c/h2\u003e\u003cp\u003eHistopathology relies on tissue fixation and embedding to preserve cell structure. Tissue was fixed in 10% formalin (24\u0026ndash;72 hours), dehydrated with alcohols, cleared with xylene, and embedded in paraffin. 5-micrometer sections were stained with hematoxylin and eosin (H\u0026amp;E) and Safranin O-fast green for diagnosis (Rahawy et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence Protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eImmunofluorescence Protocol\u003c/div\u003e\u003cp\u003eSamples were boiled in TBS 1X and then washed three times with PBS. A 0.3% Triton solution was applied for 30 minutes, followed by a PBS wash. To block secondary antibody reactions, 10% goat serum was used for 45 minutes. The primary antibody (1:100 in PBS) was applied and refrigerated (2\u0026ndash;8\u0026deg;C) for 24 hours. After four PBS washes, the secondary antibody (1:150) was incubated in the dark at 37\u0026deg;C for 1.5 hours. Following three washes, DAPI was applied for 20 minutes and then washed with PBS. Finally, glycerol/PBS was added, and coverslips were placed for fluorescent imaging using an Olympus microscope to confirm the markers (Kajimura et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBiochemical assays\u003c/h3\u003e\n\u003cp\u003eOn day 28, the animals were deeply anesthetized, and blood samples were collected to obtain serum for various tests, including the Myeloperoxidase (MPO) activity assay. Serum MPO activity was assessed using a method analogous to one previously detailed. MPO activity was quantified by measuring the absorbance difference and comparing it to a horseradish peroxidase standard curve (Froushani and Mashhouri 2019; Jafari- Khataylou et al. 2023).\u003c/p\u003e\u003cp\u003eThe nitric oxide level of serum was determined by comparing it to the standard curve obtained through the Griess method, following the manufacturing protocol (Jahantigh et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The sandwich ELISA (Enzyme-Linked Immunosorbent Assay) was used to measure C-reactive protein (CRP) levels according to the manufacturer's guidelines (Bassuk et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe left hind paws were removed, rinsed with cold saline, and homogenized in 0.01 M Tris-HCl buffer (pH 7.4). The homogenate was then centrifuged at 10,000\u0026times;g for 15 minutes at 4\u0026deg;C (Pal et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The resulting supernatant was used to measure the levels of Nrf2, HO-1, and NF-κB by ELISA kits..\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMolecular assays Real-time PCR\u003c/h2\u003e\u003cp\u003eTotal mRNA was extracted from right hind paws using RNX-Plus and used to synthesize cDNA. GATA3, T-bet, RORγt, and FoxP3 mRNA levels were measured by PCR with SYBR Green, using HPRT as a reference. Primers are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Results are expressed as 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e (mean fold change (Lin et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Massalska et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Poursamimi et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eForward and Reverse primers used in Real-Time PCR\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward primes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse primer\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGATA3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-TCA TTA AGC CCA AGC GAA GG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GTC CCC ATT GGC ATT CCT C-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT.bet\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-CGG CTG CAT ATC GTT GAG GT-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GTC CCC ATT GGC ATT CCT C-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRORɣt\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5-GCA GCG CTC CAA CAT CTT CT-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ACG TAC TGA ATG GCC TCG GT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFOXP3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-CAC CTG GCT GGG AAA ATG G-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-GGA GCC CTT GTC GGA TGA-3.'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CAA AGC CAG AGT CCT TCA GA-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GAT GGT CTT GGT CCT TAG CC -3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe normality of the data was assessed utilizing the Shapiro-Wilk test. Subsequently, a one-way analysis of variance (ANOVA) was conducted, accompanied by Tukey's post hoc test, to explore the results further. The outcomes were reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), with a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFucoidan evaluation and characterization\u003c/h2\u003e\u003cp\u003eAir-dried \u003cem\u003eSargassum tenerrimum\u003c/em\u003e samples showed a moisture content of approximately 7.93%. Carbohydrates made up the largest portion of the seaweed's dry weight (58.13%\u0026plusmn;1.45), followed by ash (24.95%\u0026plusmn;1.85), crude protein (16.08%\u0026plusmn;0.78), and crude fat (0.67%\u0026plusmn;0.03). The fucoidan extraction process yielded approximately 9.25 grams of fucoidan from 110 grams of dried and ground brown algae, representing an 8.4 percent yield.\u003c/p\u003e\u003cp\u003eThe FTIR method was employed to verify the successful separation of fucoidan (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed suppl). It is demonstrated that the absorption band at 3411 cm⁻\u0026sup1; is associated with the O-H stretching vibration found in carbohydrates. The bands at 2928 cm⁻\u0026sup1; and 1625 cm⁻\u0026sup1; correspond to the C-6 group of fucose or galactose, as well as C-H stretching vibrations of the pyranoid ring and OH bending vibrations, respectively, which are indicative of polysaccharide absorption. The 1625 cm⁻\u0026sup1; band also reflects the C\u0026thinsp;=\u0026thinsp;C vibration of uronic acid. The bands at 1557 cm⁻\u0026sup1; and 1433 cm⁻\u0026sup1; signify the asymmetric and symmetric stretching vibrations of carboxylate groups from uronic acids. The combination of peaks at 1557 cm⁻\u0026sup1; and 1432 cm⁻\u0026sup1; strongly indicates the presence of uronic acids or carboxylate functionalities, commonly found alongside sulfated fucose residues in fucoidan. The band at 1126 cm⁻\u0026sup1; suggests S\u0026thinsp;=\u0026thinsp;O stretching from sulfates, which is particularly relevant to fucoidan, known for its high content of sulfated fucose residues. Lastly, the peaks at 854 cm⁻\u0026sup1; are linked to C-O-S bending vibrations in sulfate groups attached to sugar units, especially at the C-4 position of fucose residues in fucoidan, highlighting the characteristic nature of sulfated polysaccharides in this compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed supplementary) (Isnansetyo et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Palanisamy et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the DPPH radical scavenging activities of fucoidan. The intact fucoidan exhibited a notable scavenging capability (85.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35) against DPPH radicals at a concentration of 4 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eClinical evaluations\u003c/h2\u003e\u003cp\u003eInflammatory responses in the joint environment serve as a critical clinical indicator in RA and its animal models. Therapeutic regimens were initiated on the fifth day post-immunization upon detecting an arthritis index of \u0026ge;\u0026thinsp;1 in each rat (Golbahari and Froushani 2019). According to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the course of the disease was milder in the treatment groups, especially in groups RA\u0026thinsp;+\u0026thinsp;Pred and RA\u0026thinsp;+\u0026thinsp;Fuc150. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb illustrate that the group treated with prednisolone exhibited the lowest arthritis index, followed by the group receiving fucoidan at 150 mg/kg (RA\u0026thinsp;+\u0026thinsp;Fuc150). The RA\u0026thinsp;+\u0026thinsp;Fuc50 and RA\u0026thinsp;+\u0026thinsp;Fuc100 groups did not show a statistically significant difference in average arthritis index. Notably, on the final assessment day, the prednisolone group exhibited the lowest severity of arthritis index compared to the other groups. Treatment with fucoidan was also effective in reducing the severity index on the last day in a dose-dependent manner (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Also, RA caused weight loss, but treated animals exhibited better weight gain than untreated RA rats. The prednisolone group had the lowest severity of weight loss among all groups, while fucoidan treatment also effectively reduced the severity of weight loss in a dose-dependent manner (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Overall, the study indicates that fucoidan can enhance the clinical signs of RA in a rat model, with higher doses (150 mg/kg) demonstrating greater effectiveness, although prednisolone remained the most effective treatment overall (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eHistopathological assays\u003c/h2\u003e\u003cp\u003eH\u0026amp;E histopathological examination of joints affected by RA revealed distinct differences between the control and RA groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Control rats exhibited normal articular cartilage with no evidence of mononuclear cell infiltration in the synovium. In contrast, the knee joints of the RA group displayed significant cartilage erosion, synovial hyperplasia, and marked mononuclear cell infiltration within the synovial membrane. Additionally, there was extensive bone damage accompanied by a substantial infiltration of inflammatory cells, including lymphocytes, macrophages, and congested vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRats treated with prednisolone demonstrated joint structures resembling those of the control group, restoring normal histopathology. The groups receiving 50 mg/kg and 100 mg/kg of fucoidan showed reduced inflammatory responses, although these effects were less pronounced than those observed with prednisolone. Notably, the group administered 150 mg/kg of fucoidan exhibited a significant decrease in joint inflammation and lymphocyte accumulation. The findings suggest that daily administration of 150 mg/kg of fucoidan and prednisolone resulted in favorable outcomes concerning joint pathology in RA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates saffronin O staining, which aids in assessing the extent of inflammation and the structural alterations in the synovium associated with RA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the RA group exhibits significant infiltration of inflammatory cells and reduced cartilage thickness, indicating structural changes and active inflammation compared to the control group. Treatment with fucoidan led to a dose-dependent reduction in inflammation within the joint tissue, decreased infiltration of inflammatory cells, and increased cartilage thickness. These improvements in joint and cartilage conditions are directly related to the dose of fucoidan. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb illustrates that all studied groups show statistically significant differences, with the most favorable outcomes observed in the prednisolone group, followed by the RA\u0026thinsp;+\u0026thinsp;Fuc 150 group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical evaluation\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the immunohistochemical (IHC) reaction of IL-1β in mouse knee joint tissue.The highest expression of IL-1β was observed in the RA group, while the fucoidan-treated groups showed a significant dose-dependent decrease in IL-1β expression. Specifically, the lowest expression of IL-1β was recorded in the RA group treated with fucoidan at 150 mg/kg. However, the greatest decrease in IL-1β expression was in the prednisolone-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the IHC reaction associated with the inflammatory cytokine TNF-α in mouse knee joint tissue. The results show that the RA group showed the highest expression of TNF-α, while a significant decrease was observed in the treated groups. This decrease in IHC reaction was dose-dependent among the groups receiving fucoidan, and the lowest reactivity was recorded in the RA\u0026thinsp;+\u0026thinsp;fuc150 group. Prednisone treatment was effective in reducing the expression of TNF-α, such that the expression level of the cytokine was not significantly different from that of healthy mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eBiochemical assays\u003c/h2\u003e\u003cp\u003eIn this study, serum levels of biochemical factors CRP, MPO, and NO were evaluated in RA rats as inflammatory markers. Data in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicated that the activity levels of these inflammatory markers were significantly elevated in the RA group. In contrast, a dose-dependent decrease in these levels was observed in the groups treated with fucoidan, with the most notable reduction at 150 mg/kg. Furthermore, the group receiving prednisolone showed lower levels of these inflammatory markers compared to the other treatment group. Statistically, prednisolone treatment was as effective in reducing CRP levels as the levels in healthy mice (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBiochemical modifications in the sera of rats with RA.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInflammatory Enzymes/Groups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRA\u0026thinsp;+\u0026thinsp;Fuc50\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRA\u0026thinsp;+\u0026thinsp;Fuc100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRA\u0026thinsp;+\u0026thinsp;Fuc150\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRA\u0026thinsp;+\u0026thinsp;Pred\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMPO enzyme activity (ng/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e67.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e29.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNO enzyme activity (ϻM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e73.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e61.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRP (mg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.061\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNrf2 (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e240.28\u0026thinsp;\u0026plusmn;\u0026thinsp;15.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68.43\u0026thinsp;\u0026plusmn;\u0026thinsp;10.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e73.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e94.26\u0026thinsp;\u0026plusmn;\u0026thinsp;9.38\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e121.60\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e70.01\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHO-1 (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNF\u003csub\u003eκb\u003c/sub\u003e (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.191\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eThe results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and using different letters indicated statistical significance at a p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. (RA., Rheumatoid arthritis; Fuc., Fucoidan; Pred., Prednisolone).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData analysis showed that Nrf2 and HO-1 levels were significantly lower, while NF-κB levels were higher in the RA rats compared to the control group (Table\u0026nbsp;3). However, treatment with fucoidan (100 and 150 mg/kg) significantly increased Nrf-2 and HO-1 levels in the joint tissues of RA rats. Fucoidan at 50 mg/kg also increased Nrf2 and HO-1 levels, though these results were not statistically significant. NF-κB levels were significantly decreased in fucoidan-treated RA rats in a dose-dependent manner. While prednisolone treatment did not significantly improve Nrf2 and HO-1 levels, NF-κB levels were reduced more significantly in CFA-injected rats than in fucoidan-treated RA rats(Table\u0026nbsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMolecular assays via Real-Time PCR\u003c/h2\u003e\u003cp\u003eIn the RA group, an increase in mRNA expression of T-bet and RORɣt genes was noted alongside a decrease in mRNA expression of GATA3 and Foxp3 genes. As anticipated, prednisolone, a standard anti-inflammatory, proved to be more effective than the other drugs in reversing this condition. Treatment with fucoidan also led to a dose-dependent increase in the expression of mRNAs GATA3 and Foxp3, while decreasing the expression of T-bet and RORɣt.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUncontrolled inflammation driven by type 1 and type 3 immune responses (Th1/Th17) in RA and its experimental models progressively damages joint tissues and impairs organ function, prompting the search for safe and effective anti-inflammatory agents that can be administered orally (Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The rationale for prioritizing natural anti-inflammatory compounds arises from their multi-target mechanisms, favorable safety profiles, and cost-effectiveness, thereby addressing limitations inherent in conventional pharmaceuticals (Jamtsho et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Prior research indicates that natural products such as curcumin and fish oil offer a multifaceted approach to rheumatoid arthritis by simultaneously reducing inflammation, modulating immune responses, and promoting tissue resolution, thus targeting both symptomatic manifestations and underlying causes (Kou et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Herein, our findings definitively establish the therapeutic potential of fucoidan extracted from Persian Gulf \u003cem\u003eSargassum tenerrimum\u003c/em\u003e in alleviating inflammation and modulating immune responses within a rat model of rheumatoid arthritis. Former studies have shown that \u003cem\u003eS. lomentaria\u003c/em\u003e fucoidanfucoidan can alleviate symptoms of IBD by modulating gut microbiota and reducing intestinal inflammation (Tang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and suppress T cell activation and TNF-α in models of multiple sclerosis (Kim et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlgal polysaccharides are active natural compounds with potential uses. Brown algae are a large source of important polysaccharides, including fucoidan, which has been studied extensively (Zhang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This polysaccharide has been isolated from various seaweed species using different methods. Prior research has shown that the amounts of total carbohydrates, protein, and sulfate in fucoidan from \u003cem\u003eS. tenerrimum\u003c/em\u003e can vary, differing from the values found in the current study (Ashayerizadeh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A crucial consideration is that fucoidan derived from different sources may exhibit variable immunomodulatory functions. Prior investigations have posited that high molecular weight fucoidans may exacerbate the severity of arthritis, whereas low molecular weight variants have the capacity to reduce inflammation and Th1-mediated immune responses (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These differences may arise due to factors like geographical location, harvest season, and environmental conditions, all of which can affect the chemical composition of seaweeds(Apostolova et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, to plan for the potential applications of fucoidan, it is important to assess fucoidans from algae in specific geographical areas.\u003c/p\u003e\u003cp\u003eAs per IUPAC nomenclature and terminology guidelines, a sulfated polysaccharide with an L-fucose (6-deoxy-L-galactose) content ranging from 20% to 60% can be classified as fucoidan (Ashayerizadeh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The fucoidan derived from Persian Gulf \u003cem\u003eSargassum tenerrimum\u003c/em\u003e demonstrated a significant abundance of sulfated fucose. his was confirmed through FTIR analysis.\u003c/p\u003e\u003cp\u003eThe quantification of DPPH free radical scavenging is a widely used technique for evaluating the antioxidant capabilities of extracts and natural compounds isolated from marine flora. An alcoholic solution of DPPH forms a stable radical at ambient temperature (Baliyan et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Upon the addition of algal extract, the solution undergoes a color change from blue-purple to yellow, facilitated by the acceptance of electrons or hydrogen atoms; the measurement of this color transition serves as the fundamental basis of the method. In the present study, the highest DPPH free radical scavenging activity of fucoidan from \u003cem\u003eS. tenerrimum\u003c/em\u003e collected from the Persian Gulf was observed in the fucoidan treatment at a concentration of 4 mg/ml (85.65\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35). A previous study reported that fucoidan from \u003cem\u003eS. tenerrimum\u003c/em\u003e collected from the Mandapam coast of Tamil Nadu, India, effectively scavenges DPPH free radicals, showing activity between 64.66% and 83.66% at concentrations of 1\u0026ndash;6 mg/mL for both intact and fractionated forms (Marudhupandi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, despite the apparent similarity between the two species of brown algae, this ability is greater in fucoidan extracted from the Persian Gulf. The fucoidan extracted from \u003cem\u003eS. polycystum\u003c/em\u003e demonstrates maximum DPPH radical scavenging activity of 61.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33% at 1000 \u0026micro;g/mL (Palanisamy et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, it has been noted in the past that the antioxidant efficacy of fucoidan is strongly influenced by sulfate content, extraction method, and concentration (Yue et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These properties make fucoidan extracted from Persian Gulf seaweed a promising natural antioxidant for the pharmaceutical and food industries. For precise applications, further studies on the standardization and optimization of extraction parameters are recommended.\u003c/p\u003e\u003cp\u003eThe processes of inflammation and oxidative stress are interconnected (Abtahi Froushani and Esmaili Gourvarchin Galeh \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Unlike conventional drugs used in the treatment of RA, natural compounds such as fucoidan may offer the added benefit of antioxidant properties (Ashayerizadeh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), enhancing their direct anti-inflammatory effects. The in vitro results (DPPH free radical scavenging assay) of this study clearly demonstrated the antioxidant potential of fucoidan derived from the Persian Gulf \u003cem\u003eSargassum tenerrimum\u003c/em\u003e. Malondialdehyde (MDA) serum levels serve as a key biomarker of oxidative stress in RA, reflecting lipid peroxidation and contributing to disease pathogenesis through various mechanisms (Jahantigh et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our data indicated that the fucoidan used in this study has the potential to reduce oxidative stress and serum levels of MDA in RA rats.\u003c/p\u003e\u003cp\u003eOxidative stress activates the Nrf2/HO-1 pathway, which is crucial in RA by enhancing antioxidant enzyme production and regulating inflammatory gene expression (Chadha et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ngo and Duennwald \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nrf2 induces antioxidant and detoxifying genes such as HO-1, which breaks down heme into protective molecules. Activation of Nrf2 reduces joint damage in RA animal models (Chadha et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nrf2 activators like dimethyl fumarate and isoliquiritigenin demonstrate anti-arthritic effects by decreasing oxidative stress and inflammation (Izumi and Koyama \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). According to our results, RA rats had lower Nrf2 and HO-1 than controls. Fucoidan (100, 150 mg/kg) significantly increased these levels in RA rat joints, but 50 mg/kg did not significantly increase them. As expected,Ddexamethasone did not improve Nrf2 or HO-1 levels.Fucoidan derived from \u003cem\u003eSargassum wightii\u003c/em\u003e has been shown to mitigate oxidative stress in diabetic rats via activation of the Nrf2/HO-1 signaling pathway (Puhari et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, fucoidan supplementation has been demonstrated to enhance antioxidant capacity in piglets by modulating the Keap1/Nrf2 signaling pathway and mitochondrial function (Yin et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In human keratinocytes, fucoidan has exhibited protective effects against oxidative stress through the upregulation of Nrf2 and its downstream targets (Zayed et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe integration of clinical and histopathological findings provides insight into the disease progression of CFA-induced RA and is crucial for evaluating the effectiveness of therapeutic interventions (Takahashi et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The histopathological schema of adjuvant-induced arthritis in rats is characterized by a progressive inflammatory process primarily affecting the joints. Synovial hyperplasia, inflammatory cell infiltration, and increased vascularity become more pronounced (Morvaridi et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The results of our study showed that fucoidan is dose-dependently effective in improving histopathological appearance, as well as clinical indicators and weight gain. H\u0026amp;E data from our investigation indicated that daily administration of 150 mg/kg fucoidan and prednisolone produced the most favorable outcomes in managing RA-related joint inflammation and leukocyte infiltration. The inflammatory environment caused by RA leads to the breakdown of cartilage and underlying bone in the joint area (Morvaridi et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Interestingly, fucoidan has been shown to inhibit osteoclastogenesis, a key factor in bone loss in RA, by regulating signaling pathways such as Akt/GSK3β/PTEN/NFATc1, thereby reducing inflammatory bone loss (Lu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Safranin O staining provides both visual and quantitative evidence of the dose-dependent potential of fucoidan as an anti-inflammatory and cartilage-protective agent in rheumatoid arthritis.\u003c/p\u003e\u003cp\u003eIn rheumatoid arthritis, the pro-inflammatory cytokines IL-1β and TNF-α are highly expressed in synovial tissue, particularly near cartilage and bone. They drive inflammation, contributing to depression, sickness behavior, pain, and weight loss, with their levels rising as arthritis progresses (Yang et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Biological agents, such as anti-TNF therapies (e.g., etanercept, infliximab) or IL-1 blockers, have been developed to mitigate their effects and improve outcomes in RA patients, making them significant in research and clinical contexts (Law and Taylor \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our ICH results indicated that fucoidan reduced the expression of inflammatory cytokines IL-1β and TNF-α in inflamed joints dose-dependently. Previous data showed that fucoidan in \u003cem\u003eSargassum horneri\u003c/em\u003e extracts reduced TNF-α-driven inflammation in RPE cells, indicating potential use in macular degeneration (Lee et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research on synovial cells supports that fucoidan reduces joint inflammation caused by IL-1β. It appears fucoidan's anti-inflammatory benefits may occur by blocking the NF-κB pathway and the mitogen-activated protein kinases (MAPKs) cascade (Park et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). NF-κB regulates inflammatory genes and promotes synovial hyperplasia. Its activation is seen in RA patient synovial tissues and RA animal models (Makarov \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In this study, treatment with fucoidan reduced NF-κB levels, resulting in the suppression of several downstream inflammatory mediators.\u003c/p\u003e\u003cp\u003eMyeloperoxidase (MPO), primarily in neutrophils and monocytes, produces HOCl from H₂O₂ and chloride to kill pathogens, but excessive MPO/ROS damages tissues. In chronic inflammation, high MPO levels overwhelm antioxidants. MPO-driven stress is linked to conditions like RA, suggesting potential treatment targets (Golbahari and Abtahi Froushani \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Moases Ghaffary and Abtahi Froushani \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MPO-derived ROS activates inflammatory pathways such as NF-κB, worsening inflammation and MPO release in a harmful cycle (Mittal et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Elevated MPO in blood or tissues often correlates with oxidative stress severity, serving as a diagnostic marker for diseases like RA (Golbahari and Abtahi Froushani \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the present study, following an increase in serum MPO levels in RA rats, a dose-dependent decrease in serum MPO levels was observed in the fucoidan-treated groups, with the most significant decrease observed at a dose of 150 mg/kg. Fucoidan reduces oxidative stress by boosting antioxidant activity and lowering ROS and lipid peroxidation. It enhances SOD, catalase, and GPx via the Nrf2/ERK pathway and directly inhibits MPO enzyme, reducing neuroinflammation and improving memory (Ryu and Chung \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Fucoidan eases pancreatitis by lowering MPO activity in the pancreas and lungs, indicating less neutrophil infiltration. It also inhibits MAPK and NF-κB signaling, which reduces MPO expression, ROS output, and inflammatory cytokines like TNF-α, IL-6, and IL-1β, all tied to MPO-related oxidative stress (Carvalho et al. 2013). Another study demonstrated that fucoidan extracted from \u003cem\u003eUndaria pinnatifida\u003c/em\u003e effectively inhibited reactive oxygen species (ROS) production in vitro and reduced cell death in zebrafish models exposed to oxidative stress (Oh et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, fucoidan fractions from \u003cem\u003eSargassum tenerrimum\u003c/em\u003e used in this study showed vigorous antioxidant activity, with their antioxidant capacity linked to their sulfate content (Marudhupandi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn adjuvant induced arthritis, elevated blood NO strongly correlates with disease severity. NO levels increase during inflammation in adjuvant induced arthritis models, reflecting joint swelling and histological damage. Similarly, RA patients with severe inflammation exhibit high NO. This correlation suggests that monitoring blood NO could aid in assessing therapeutic efficacy in adjuvant induced arthritis (Golbahari and Abtahi Froushani \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, an elevation of serum NO levels was noted in RA rats. Subsequently, fucoidan treatment led to a reduction in these levels, with the extent of the decrease correlating with the dosage administered. The most notable reduction was seen with a dosage of 150 mg/kg. In inflammatory contexts (e.g., LPS-activated microglia or macrophages), fucoidan suppresses inducible NOS (iNOS) expression, thereby reducing excessive NO production. Fucoidan also decreases superoxide (O₂⁻) and NO overproduction, limiting peroxynitrite formation (Cui et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). By enhancing antioxidant enzymes through the Nrf2/ERK pathway, fucoidan lowers oxidative stress, indirectly reducing reactive nitrogen species (RNS) generation (Zayed et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCRP, a liver protein increased by inflammation (e.g., IL-1, TNF-α), reflects systemic inflammation and plays a key role in adjuvant-induced arthritis. Elevated CRP in RA correlates with joint swelling and damage. CRP exacerbates inflammation by activating the complement system and recruiting immune cells. It also stimulates cytokine production, sustaining the inflammatory response. CRP binds to damaged joint cells, marking them for clearance and contributing to cartilage and bone erosion (Moases Ghaffary and Abtahi Froushani \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Morvaridiet al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). RA rats in the current investigation showed increased serum CRP, which fucoidan reduced in a dose-dependent manner. In mice with thioacetamide-induced liver injury, fucoidan significantly reduced serum CRP levels and pro-inflammatory cytokines by inhibiting the NF-κB and MAPK pathways (Tsai et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, in LPS-induced lung inflammation and arthritis models, fucoidan lowered CRP levels, demonesterating its systemic anti-inflammatory effects (Zhu et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe trigger and driver of inflammation in the joints in rheumatoid arthritis involve T-lymphocyte responses. Depending on the conditions and microenvironment, T lymphocytes polarize in different ways, which affect the fate and course of an autoimmune disease (Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Type 1 immunity involves innate and adaptive responses mediated by T-bet expressing ILC-1, NKT, γδ-T, and Th1 lymphocytes that produce the key cytokine IFN-γ. Dysregulation of type 1 immunity can lead to delayed-type hypersensitivity and organ-specific autoimmunity (Nabekura and Shibuya \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Type 2 immunity is characterized by GATA-3\u003csup\u003e+\u003c/sup\u003e ILC-2, NKT, γδ-T, and Th2 lymphocytes that produce the signature cytokines IL-4, IL-5, and IL-13. Uncontrolled type 2 responses can cause allergic diseases and suppress inflammatory arthritis (Kim et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Type 3 immunity, mediated by RORγt expressing lymphocytes, is increasingly recognized in the pathogenesis of RA, mainly through IL-17 production. IL-17 stimulates synovial joint cells, including stromal cells, chondrocytes, fibroblasts, and macrophages, to secrete pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α (Woś and Tabarkiewicz \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Meanwhile, FoxP3-Treg lymphocytes are crucial for maintaining self-tolerance by inhibiting effector T cells (Khandpur et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Annunziato et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Etemadi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is clear that RA shifts the balance towards pro-inflammatory Th1/Th17 responses, and prednisolone effectively reverses this shift (Mirzaaghasi and Froushani 2023). According to our findings, fucoidan reduced the expression of inflammatory genes T-bet and RORγt in a dose-dependent manner, leading to a decrease in polarization towards the Th1 and Th17 subsets. Conversely, it increased the expression of GATA3 and FOXP3 genes, resulting in enhanced polarization towards Th2 and Treg cells. These findings provide a molecular mechanism to explain the observed anti-inflammatory effects of fucoidan in the RA model. By modulating the expression of key transcription factors, fucoidan helps to re-establish immune homeostasis and reduce inflammation. Notably, long-term use of conventional NSAIDs can lead to gastrointestinal ulcers, renal toxicity, and cardiovascular risks. More importantly, NSAIDs suppress symptoms without addressing the underlying immune dysregulation (Wongrakpanich et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bindu et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Here, we clearly demonstrated that fucoidan extracted from \u003cem\u003eSargassum tenerrimum\u003c/em\u003e, as a natural NSAID, has the ability to modulate and regulate immune responses in an experimental model of RA.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFucoidan extracted from Persian Gulf \u003cem\u003eSargassum tenerrimum\u003c/em\u003e, characterized by a high content of sulfated fucose and confirmed via FTIR, showed strong antioxidant activity (DPPH scavenging\u0026thinsp;~\u0026thinsp;85.65%). In a rat model of adjuvant-induced arthritis, fucoidan had dose-dependent anti-inflammatory and joint-protective effects. The 150 mg/kg dose showed the greatest benefits, even surpassing prednisolone in some aspects. Overall, fucoidan's potential to treat rheumatoid arthritis appears to stem from antioxidant, anti-inflammatory, and immune-regulating actions. These actions work together to lower oxidative stress, reduce inflammatory signals, and restore balance to the immune system. While prednisolone is still more powerful, a high dose of fucoidan (150 mg/kg) presents a hopeful natural supplement with multiple benefits and potentially fewer side effects compared to corticosteroids. Further research is warranted to explore higher dosages exceeding 150 mg/kg and to investigate the precise mechanisms underlying fucoidan\u0026rsquo;s action.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflict of interest The author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the staffs of the\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eVeterinary Faculty of Urmia University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Urmia University, Urmia,Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eEnquiries about data availability should be directed \u003cstrong\u003eto the authors.\u003c/strong\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLeila Mahmoudzadeh: Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Writing \u0026ndash; original draft. Seyyed Meysam Abtahi Froushani: Methodology, Funding acquisition, Conceptualization, Writing \u0026ndash; review \u0026amp; editing. Seyede Soraya Mahmoudi: Data curation, Visualization.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbtahi Froushani SM, Esmaili Gourvarchin Galeh H (2014) New insight into the immunomodulatory mechanisms of Tretinoin in NMRI mice. Iranian Journal of Basic Medical Sciences \u003cstrong\u003e17\u003c/strong\u003e:632-637.\u003c/li\u003e\n \u003cli\u003eAlea MT, Meyer AS (2013). Fucoidans from brown seaweeds: an update on structures, extraction techniques and use of enzymes as tools for structural elucidation. RSC Adv \u003cstrong\u003e3\u003c/strong\u003e:8131-8141.\u003c/li\u003e\n \u003cli\u003eAnnunziato F, Romagnani C, Romagnani S (2015). The 3 major types of innate and adaptive cell-mediated effector immunity. J Allergy Clin Immunol \u003cstrong\u003e135\u003c/strong\u003e:626-635.\u003c/li\u003e\n \u003cli\u003eApostolova E, Lukova P, Baldzhieva A, Katsarov P, Nikolova M, Iliev L, Peychev L, Trica B, Oancea F, Delattre C, Kokova V (2020). Immunomodulatory and Anti-Inflammatory Effects of Fucoidan: A Review. Polymers (Basel) \u003cstrong\u003e12\u003c/strong\u003e:2338.\u003c/li\u003e\n \u003cli\u003eApostolova E, Lukova P, Baldzhieva A, Katsarov P, Nikolova M, Iliev I, Peychev L, Trica B, Oancea F, Delattre C, Kokova V (2020). Immunomodulatory and Anti-Inflammatory Effects of Fucoidan: A Review. Polymers (Basel) \u003cstrong\u003e12\u003c/strong\u003e:2338.\u003c/li\u003e\n \u003cli\u003eAshayerizadeh O, Dastar B, Pourashouri P (2020). Study of antioxidant and antibacterial activities of depolymerized fucoidans extracted from Sargassum tenerrimum. International Journal of Biological Macromolecules \u003cstrong\u003e151\u003c/strong\u003e:1259-1266.\u003c/li\u003e\n \u003cli\u003eBaliyan S, Mukherjee R, Priyadarshini A, Vibhuti A, Gupta A, Pandey RP, Chang CM (2022). Determination of Antioxidants by DPPH Radical Scavenging Activity and Quantitative Phytochemical Analysis of Ficus religiosa. Molecules \u003cstrong\u003e27\u003c/strong\u003e:1326.\u003c/li\u003e\n \u003cli\u003eBassuk SS, Rifai N, Ridker PM (2004). High-sensitivity C-reactive protein: clinical importance. Curr Probl Cardiol \u003cstrong\u003e29\u003c/strong\u003e:439-493.\u003c/li\u003e\n \u003cli\u003eBindu S, Mazumder S, Bandyopadhyay U (2020). Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective. Biochem Pharmacol 180:114147.\u003c/li\u003e\n \u003cli\u003eCarvalho A, Sousa R, Franco A, Costa J, Neves L, Ribeiro R, Sutton R, Criddle D, Soares P, Souza M (2014). Protective Effects of Fucoidan, a P- and L-Selectin Inhibitor, in Murine Acute Pancreatitis. Pancreas 43:82-87.\u003c/li\u003e\n \u003cli\u003eChadha S, Behl T, Kumar A, Khullar G, Arora S (2020). Role of Nrf2 in rheumatoid arthritis. Current Research in Translational Medicine 68:171-181.\u003c/li\u003e\n \u003cli\u003eCiofoaia E I, Pillarisetty A, Constantinescu F (2022). Health disparities in rheumatoid arthritis. Ther Adv Musculoskelet Dis \u003cstrong\u003e14\u003c/strong\u003e: 1759720x221137127.\u003c/li\u003e\n \u003cli\u003eCui Y Q, Zhang L J, Zhang T, Luo D Z, Jia Y J, Guo Z X, Zhang Q B, Wang X, Wang X M (2010). Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide-activated primary microglia. Clin Exp Pharmacol Physiol \u003cstrong\u003e37\u003c/strong\u003e:422-428.\u003c/li\u003e\n \u003cli\u003eErsoydan S, Rustemeyer T (2024). Investigating the Anti-Inflammatory Activity of Various Brown Algae Species. Mar Drugs 22:457.\u003c/li\u003e\n \u003cli\u003eEtemadi S, Abtahi Froushani SM, Hashemi Asl SM, Mahmoudian A (2022). Combined atorvastatin and pentoxifylline in ameliorating inflammation induced by complete Freund\u0026apos;s adjuvant. Inflammopharmacology 30:935-944.\u003c/li\u003e\n \u003cli\u003eAbtahi Froushani SM, Mashhouri S (2019). The Effect of Mesenchymal Stem Cells Pulsed with 17 Beta-Estradiol in an Ameliorating Rat Model of Ulcerative Colitis. Zahedan Journal of Research in Medical Sciences 21:33-45.\u003c/li\u003e\n \u003cli\u003eGhaffary E M, Abtahi Froushani SM (2020). Immunomodulatory benefits of mesenchymal stem cells treated with Caffeine in adjuvant-induced arthritis. Life Sci 246:117420.\u003c/li\u003e\n \u003cli\u003eGolbahari S, Abtahi Froushani SM (2019). Synergistic benefits of Nicotine and Thymol in alleviating experimental rheumatoid arthritis. Life Sci 239:117037.\u003c/li\u003e\n \u003cli\u003eGurau S, Imran M, Ray R L (2025). Algae: A cutting-edge solution for enhancing soil health and accelerating carbon sequestration \u0026ndash; A review. Environmental Technology \u0026amp; Innovation 37:103980.\u003c/li\u003e\n \u003cli\u003eHajizadeh A, Abtahi Froushani SM, Tehrani AA, Azizi S, Hashemi S R B (2021). Effects of Naringenin on Experimentally Induced Rheumatoid Arthritis in Wistar Rats. Archives of Razi Institute 76:903-912.\u003c/li\u003e\n \u003cli\u003eHifney A F, Fawzy M A, Abdel-Gawad KM, Gomaa M (2016). Industrial optimization of fucoidan extraction from Sargassum sp. and its potential antioxidant and emulsifying activities. Food Hydrocolloids 54:77-88.\u003c/li\u003e\n \u003cli\u003eIsnansetyo A, Lutfia FNL, Nursid M, Trijoko, Susidarti RA (2017). Cytotoxicity of Fucoidan from Three Tropical Brown Algae Against Breast and Colon Cancer Cell Lines. Pharmacognosy Journal 9:14-20.\u003c/li\u003e\n \u003cli\u003eIzumi Y, Koyama Y (2024). Nrf2-Independent Anti-Inflammatory Effects of Dimethyl Fumarate: Challenges and Prospects in Developing Electrophilic Nrf2 Activators for Neurodegenerative Diseases. Antioxidants (Basel) 13:1527.\u003c/li\u003e\n \u003cli\u003eJafari- Khataylou, Kazemi-Darabadi YS, Ahmadi Afshar S (2023). Effects of adenosine N1-Oxide and pioglitazone on inflammatory and antioxidant state in sepsis caused by experimental cecal puncture in rat. Veterinary Research Forum 14:381-387.\u003c/li\u003e\n \u003cli\u003eJahantigh M, Abtahi Froushani SM, Afzale Ahangaran N (2023). Benefits of bone marrow-derived mesenchymal stem cells primed with estradiol in alleviating collagen-induced arthritis. Iranian Journal of Basic Medical Sciences 26:400-407.\u003c/li\u003e\n \u003cli\u003eJamtsho T, Yeshi K, Perry MJ, Loukas A, Wangchuk P (2024). Approaches, Strategies and Procedures for Identifying Anti-Inflammatory Drug Lead Molecules from Natural Products. Pharmaceuticals (Basel) 17:283.\u003c/li\u003e\n \u003cli\u003eKadam S U, Tiwari B K, O\u0026apos;Donnell C P (2014). Extraction, structure and biofunctional activities of laminarin from brown algae. International Journal of Food Science \u0026amp; Technology 50:24-31.\u003c/li\u003e\n \u003cli\u003eKajimura J, Ito R, Manley N R, Hale L P (2016). Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues. J Histochem Cytochem 64:112-124.\u003c/li\u003e\n \u003cli\u003eKang N, Zhao J, Gao P, Lu Y, Chen Z, Li X, Khan I A, Yang S, Xu Q, Liu Y (2025). Innovative Strategies in Natural Product Drug Discovery: The Case of Anemoside B4. Engineering.\u003c/li\u003e\n \u003cli\u003eKhandpur R, Carmona-Rivera C, Vivekanandan-Giri A, Gizinski A, Yalavarthi S, Knight J S, Friday S, Li S, Patel R M, Subramanian V, Thompson P, Chen P, Fox D A, Pennathur S, Kaplan M J (2013). NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med 5:140-178.\u003c/li\u003e\n \u003cli\u003eKim H, Moon C, Park E J, Jee Y, Ahn M, Wie M B, Shin T (2010). Amelioration of experimental autoimmune encephalomyelitis in Lewis rats treated with fucoidan. Phytother Res 24:399-403.\u003c/li\u003e\n \u003cli\u003eKim H Y, Jeong D, Kim J H, Chung D H (2024). Innate Type-2 Cytokines: From Immune Regulation to Therapeutic Targets. Immune Netw 24:e6.\u003c/li\u003e\n \u003cli\u003eKou H, Huang L, Jin M, He Q, Zhang R, Ma J (2023). Effect of curcumin on rheumatoid arthritis: a systematic review and meta-analysis. Front Immunol 14: 1121655.\u003c/li\u003e\n \u003cli\u003eLaw S T, Taylor P C (2019). Role of biological agents in treatment of rheumatoid arthritis. Pharmacol Res 150:104497.\u003c/li\u003e\n \u003cli\u003eLee S, Lee E J, Lee G M, Yun J H, Yoo W (2023). Inhibitory effect of fucoidan on TNF-\u0026alpha;-induced inflammation in human retinal pigment epithelium cells. Front Nutr 10:1162934.\u003c/li\u003e\n \u003cli\u003eLim S J, Aida W M W, Maskat M Y, Latip J, Badri K H, Hassan O, Yamin B M (2016). Characterisation of fucoidan extracted from Malaysian Sargassum binderi. Food Chem 209:267-273.\u003c/li\u003e\n \u003cli\u003eLin Z W, Wu L X, Xie Y, Ou X, Tian P K, Liu X P, Min J, Wang J, Chen R F, Chen Y J, Liu C, Ye H, Ou Q J (2015). The Expression Levels of Transcription Factors T-bet, GATA-3, ROR\u0026gamma;t and FOXP3 in Peripheral Blood Lymphocyte (PBL) of Patients with Liver Cancer and their Significance. International Journal of Medical Sciences 12:7-16.\u003c/li\u003e\n \u003cli\u003eLinares-Maurizi A, Reversat G, Awad R, Bultel-Ponc\u0026eacute; V, Oger C, Galano J M, Balas L, Durbec A, Bertrand-Michel J, Durand T, Pradelles R, Vigor C (2023). Bioactive Oxylipins Profile in Marine Microalgae. Mar Drugs 21:136.\u003c/li\u003e\n \u003cli\u003eLu SH, Hsia YJ, Shih KC, Chou TC (2019). Fucoidan Prevents RANKL-Stimulated Osteoclastogenesis and LPS-Induced Inflammatory Bone Loss via Regulation of Akt/GSK3\u0026beta;/PTEN/NFATc1 Signaling Pathway and Calcineurin Activity. Mar Drugs 17:345.\u003c/li\u003e\n \u003cli\u003eMakarov SS (2001). NF-kappa B in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res 3:200-206.\u003c/li\u003e\n \u003cli\u003eMarudhupandi T, Kumar TT, Senthil SL, Devi K N (2014). In vitro antioxidant properties of fucoidan fractions from Sargassum tenerrimum. Pak J Biol Sci 17:402-407.\u003c/li\u003e\n \u003cli\u003eMarudhupandi T, Kumar TTA, Senthil SL, Devi KN (2014). In vitro antioxidant properties of fucoidan fractions from Sargassum tenerrimum. Pak J Biol Sci 17:402-407.\u003c/li\u003e\n \u003cli\u003eMassalska M, Radzikowska A, Kuca-Warnawin E, Plebanczyk M, Prochorec-Sobieszek M, Skalska U, Kurowska W, Maldyk P, Kontny E, Gober HJ, Maslinski W (2020). CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired. Cells 9:549.\u003c/li\u003e\n \u003cli\u003eMirzaaghasi S, Abtahi Froushani SM (2023). Immunomodulatory Effects of Combined Nicotinic Acid and Prednisolone in Adjuvant-induced Arthritis. Antiinflamm Antiallergy Agents Med Chem 22:104-112.\u003c/li\u003e\n \u003cli\u003eMittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB (2014). Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal 20:1126-1167.\u003c/li\u003e\n \u003cli\u003eMlynarik M, Johansson BB, Jezova D (2004). Enriched environment influences adrenocortical response to immune challenge and glutamate receptor gene expression in rat hippocampus. Ann N Y Acad Sci 1018: 273-280.\u003c/li\u003e\n \u003cli\u003eMoases Ghaffary E, Abtahi Froushani SM (2020). Immunomodulatory benefits of mesenchymal stem cells treated with Caffeine in adjuvant-induced arthritis. Life Sciences 246: 117420.\u003c/li\u003e\n \u003cli dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMorvaridi A, Abtahi Froushani SM, Farshid AA (2025). Benefits of combining piperine with prednisolone in an experimental model of rheumatoid arthritis. Vet Res Forum 16:117-124.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eNabekura T, Shibuya A (2021). Type 1 innate lymphoid cells: Soldiers at the front line of immunity. Biomed J 44:115-122.\u003c/li\u003e\n \u003cli\u003eNarv\u0026aacute;ez J, Aguilar-Coll M, Roig-Kim M, Maym\u0026oacute;-Paituvi P, Palacios-Olid J, Nolla JM, LLop\u003csup\u003e\u0026nbsp;\u003c/sup\u003eD (2024). Janus kinase inhibitors in rheumatoid arthritis-associated interstitial lung disease: A systematic review and meta-analysis. Autoimmun Rev 23(10):103636.\u003c/li\u003e\n \u003cli\u003eNgo V, Duennwald ML (2022). Nrf2 and Oxidative Stress: A General Overview of Mechanisms and Implications in Human Disease. Antioxidants (Basel) 11:2345.\u003c/li\u003e\n \u003cli\u003eOh JY, Kim EA, Kang SI, Yang HW, Ryu B, Wang L, Lee JS, Jeon YJ (2020). Protective Effects of Fucoidan Isolated from Celluclast-Assisted Extract of Undaria pinnatifida Sporophylls against AAPH-Induced Oxidative Stress In Vitro and In Vivo Zebrafish Model. Molecules 25:2361.\u003c/li\u003e\n \u003cli\u003ePacheco D, Poza SG, Cotas J, Gon\u0026ccedil;alves AMM (2020). Fucoidan - A valuable source from the ocean to pharmaceutical. Frontiers in Drug, Chemistry and Clinical Research 3:1-4.\u003c/li\u003e\n \u003cli\u003ePal R, Chaudhary MJ, Tiwari PC, Nath R, Pant KK (2018). Pharmacological and biochemical studies on protective effects of mangiferin and its interaction with nitric oxide (NO) modulators in adjuvant-induced changes in arthritic parameters, inflammatory, and oxidative biomarkers in rats. Inflammopharmacology.\u003c/li\u003e\n \u003cli\u003ePalanisamy S, Vinosha M, Marudhupandi T, Rajasekar P, Prabhu NM (2017). Isolation of fucoidan from Sargassum polycystum brown algae: Structural characterization, in vitro antioxidant and anticancer activity. Int J Biol Macromol 102:405-412.\u003c/li\u003e\n \u003cli\u003ePark HY, Han MH, Park C, Jin CY, Kim GY, Choi IW, Kim ND, Nam TJ, Kwon TK, Choi YH (2011). Anti-inflammatory effects of fucoidan through inhibition of NF-\u0026kappa;B, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells. Food Chem Toxicol 49:1745-1752.\u003c/li\u003e\n \u003cli\u003ePark SB, Chun KR, Kim JK, Suk K, Jung YM, Lee WH (2010). The differential effect of high and low molecular weight fucoidans on the severity of collagen-induced arthritis in mice. Phytother Res 24:1384-1391.\u003c/li\u003e\n \u003cli\u003ePoursamimi J, Shariati-Sarabi Z, Tavakkol-Afshari J, Mohammadi M (2022). A Significant Increase in the Gene Expression of GATA-3 Following the Treatment of Osteoarthritis Patients with Crocin. Iran J Allergy Asthma Immunol 21:35-43.\u003c/li\u003e\n \u003cli\u003ePuhari SSM, Yuvaraj S, Vasudevan V, Ramprasath T, Arunkumar K, Amutha C, Selvam GS (2023). Fucoidan from Sargassum wightii reduces oxidative stress through upregulating Nrf2/HO-1 signaling pathway in alloxan-induced diabetic cardiomyopathy rats. Mol Biol Rep 50:8855-8866.\u003c/li\u003e\n \u003cli\u003eRahawy AM, Al-Mahmood SS, Basim H, Rahawi AM (2021). Standard techniques for formalin-fixed paraffin-embedded tissue: A Pathologist\u0026apos;s perspective. Iraqi Journal of Veterinary Sciences 35.\u003c/li\u003e\n \u003cli\u003eRaj PP, Gopal RK, Sanniyasi E (2024). Investigating the anti-inflammatory and anti-arthritis effects of fucoidan from a brown seaweed.\u0026quot; Current Research in Biotechnology \u003cstrong\u003e7\u003c/strong\u003e:100220.\u003c/li\u003e\n \u003cli\u003eRyu MJ, Chung HS (2016). Fucoidan reduces oxidative stress by regulating the gene expression of HO‑1 and SOD‑1 through the Nrf2/ERK signaling pathway in HaCaT cells. Mol Med Rep 14:3255-3260.\u003c/li\u003e\n \u003cli\u003eSilberfeld T, Rousseau F, Reviers B (2014). An Updated Classification of Brown Algae (Ochrophyta, Phaeophyceae). Cryptogamie Algologie 35:117-156.\u003c/li\u003e\n \u003cli\u003eSingh J A (2022). Treatment Guidelines in Rheumatoid Arthritis. Rheum Dis Clin North Am 48:679-689.\u003c/li\u003e\n \u003cli\u003eTakahashi I, Takeda K, Toyama T, Matsuzaki T, Kuroki H, Hoso M (2024). Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model. Sci Rep 14:10568.\u003c/li\u003e\n \u003cli\u003eTang S, Wang L, Ren X, Song S, Ai C (2024). Fucoidan Alleviates Colitis and Metabolic Disorder by Protecting the Intestinal Barrier, Suppressing the MAPK/NF-\u0026kappa;B Pathways, and Regulating the Gut Microbiota.\u0026quot; Journal of Food Biochemistry 2024:7955190.\u003c/li\u003e\n \u003cli\u003eTsai M Y, Yang WC, Lin CF, Wang CM, Liu HY, Lin CS, Lin JW, Lin WL, Lin TC, Fan PS, Hung KH, Lu YW, Chang GR (2021). The Ameliorative Effects of Fucoidan in Thioacetaide-Induced Liver Injury in Mice. Molecules 26:1937.\u003c/li\u003e\n \u003cli\u003eWongrakpanich S, Wongrakpanich A, Melhado K, Rangaswami J (2018). A Comprehensive Review of Non-Steroidal Anti-Inflammatory Drug Use in The Elderly. Aging Dis 9:143-150.\u003c/li\u003e\n \u003cli\u003eWoś I, Tabarkiewicz J (2021). Effect of interleukin-6, -17, -21, -22, and -23 and STAT3 on signal transduction pathways and their inhibition in autoimmune arthritis. Immunologic Research 69:26-42.\u003c/li\u003e\n \u003cli\u003eYang J, Wang J, Liang X, Zhao H, Lu J, Ma Q, Jing B, F. Tian F (2019). IL‑1\u0026beta; increases the expression of inflammatory factors in synovial fluid‑derived fibroblast‑like synoviocytes via activation of the NF‑\u0026kappa;B‑mediated ERK‑STAT1 signaling pathway. Mol Med Rep 20:4993-5001.\u003c/li\u003e\n \u003cli\u003eYin C, Bi Q, Chen W, Wang C, Castiglioni B, Li Y, Sun W, Pi Y, Bontempo V, Li X, Jiang X (2024). Fucoidan Supplementation Improves Antioxidant Capacity via Regulating the Keap1/Nrf2 Signaling Pathway and Mitochondrial Function in Low-Weaning Weight Piglets. Antioxidants (Basel) 13:407.\u003c/li\u003e\n \u003cli\u003eYue Q, Liu Y, Li F, Hong T, Guo S, Cai M, Zhao L, Su L, Zhang S, Zhao C, Li K (2025). Antioxidant and anticancer properties of fucoidan isolated from Saccharina Japonica brown algae. Sci Rep 15:8962.\u003c/li\u003e\n \u003cli\u003eZayed A, Al-Saedi DA, Mensah EO, Kanwugu ON, Adadi P, Ulber R (2023). Fucoidan\u0026apos;s Molecular Targets: A Comprehensive Review of Its Unique and Multiple Targets Accounting for Promising Bioactivities Supported by In Silico Studies. Mar Drugs 22:29-39.\u003c/li\u003e\n \u003cli\u003eZeng Z, Yan K, Liu W (2022). Specneuzhenide Ameliorate Complete Freund Adjuvant Induced Arthritis in Rats: Involvement of NF-\u0026kappa;B and HO-1/Nrf-2 Pathway. Journal of Oleo Science 71:551-561.\u003c/li\u003e\n \u003cli\u003eZhang W, Lee PCW, Jin JO (2024). Anti-Inflammatory Effect of Fucoidan from Costaria costata Inhibited Lipopolysaccharide-Induced Inflammation in Mice. Mar Drugs 22:401.\u003c/li\u003e\n \u003cli\u003eZhao M, Garcia-Vaquero M, Przyborska J, Sivagnanam SP, Tiwari B (2021). The development of analytical methods for the purity determination of fucoidan extracted from brown seaweed species. International Journal of Biological Macromolecules 173:90-98.\u003c/li\u003e\n \u003cli\u003eZhu DZ, Wang YT, Zhuo YL, Zhu KJ, Wang XZ, Liu AJ (2020). Fucoidan inhibits LPS-induced acute lung injury in mice through regulating GSK-3\u0026beta;-Nrf2 signaling pathway. Arch Pharm Res 43:646-654.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fucoidan, Brown algae, seaweeds, Rheumatoid arthritis, Autoimmunity","lastPublishedDoi":"10.21203/rs.3.rs-7981987/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7981987/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFucoidan, due to its anti-inflammatory and immune-modulating effects, shows promise for treating rheumatoid arthritis (RA). This study tested fucoidan from the Persian Gulf algae \u003cem\u003eSargassum tenerrimum\u003c/em\u003e on adjuvant-induced RA in Wistar rats. The isolated fucoidan demonstrated excellent scavenging capability (83.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35%) on 2,2-diphenyl-1-picrylhydrazyl radicals at a dosage of 6 mg/mL. RA was induced in rats via Freund's adjuvant injection. From days 5 to 23, rats (n\u0026thinsp;=\u0026thinsp;10/group) received daily placebo, fucoidan (50, 100, 150 mg/kg), or prednisolone (10 mg/kg). Fucoidan effectively reduced inflammation and alleviated symptoms associated with adjuvant-induced RA in a dose-dependent manner. Hematoxylin and eosin staining strongly supported the previous results regarding fucoidan's effectiveness, especially at 150 mg/kg, in reducing inflammation and repairing joint structure. Safranin O staining confirmed that better joint and cartilage health was linked to higher fucoidan doses. Immunohistochemical analysis showed that fucoidan treatment significantly lowered levels of the inflammatory cytokines IL-1β and TNF-α in the joint tissue of RA rats. Moreover, fucoidan treatment led to a dose-dependent reduction in inflammatory biochemical parameters such as myeloperoxidase, nitric oxide, malondialdehyde, and C-reactive protein. In the joint,tissue. Fucoidan reduced the activity of inflammatory genes (T-bet, RORγt), leading to decreased differentiation of Th1 and Th17 cells, while enhancing the activity of GATA3 and FOXP3 genes, which promoted Th2 and Treg cell polarization. In contrast to prednisolone, fucoidan elevated the levels of Nrf2 and HO-1 in the plantar joint.Fucoidan from \u003cem\u003eS. tenerrimum\u003c/em\u003e has strong antioxidant and immunomodulatory benefits, making it a promising option for managing complex inflammation like RA.\u003c/p\u003e","manuscriptTitle":"Therapeutic Potential of Fucoidan from a Persian Gulf Alga (Sargassum tenerrimum) in Rheumatoid Arthritis: In Vivo Evaluation in a Rat Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 16:16:05","doi":"10.21203/rs.3.rs-7981987/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-26T16:11:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T16:58:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119939162593502622759761293048617047614","date":"2025-12-13T21:37:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T10:43:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326673589390617028857327337077616956095","date":"2025-10-31T16:15:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258252562546529468424784458844189373485","date":"2025-10-31T14:55:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-31T14:51:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-31T05:29:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-31T05:27:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammopharmacology","date":"2025-10-29T16:18:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e8e6d1e5-25f3-4b8a-a7cc-8a3d213a09cc","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:01:30+00:00","versionOfRecord":{"articleIdentity":"rs-7981987","link":"https://doi.org/10.1007/s10787-026-02165-x","journal":{"identity":"inflammopharmacology","isVorOnly":false,"title":"Inflammopharmacology"},"publishedOn":"2026-02-28 15:58:18","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-11-12 16:16:05","video":"","vorDoi":"10.1007/s10787-026-02165-x","vorDoiUrl":"https://doi.org/10.1007/s10787-026-02165-x","workflowStages":[]},"version":"v1","identity":"rs-7981987","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7981987","identity":"rs-7981987","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}