A Complete Freund’s Adjuvant-Induced Murine Model as an Experimental Platform to Explore Systemic Inflammation in Rheumatoid Arthritis

A Complete Freund’s Adjuvant-Induced Murine Model as an Experimental Platform to Explore Systemic Inflammation in Rheumatoid Arthritis | 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 A Complete Freund’s Adjuvant-Induced Murine Model as an Experimental Platform to Explore Systemic Inflammation in Rheumatoid Arthritis Flávia Heloísa da Silva, Gabriella Lopes Cappellaro, Matheus Felipe Zazula, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8523680/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that compromises joint integrity and triggers extra-articular manifestations associated with oxidative distress and systemic inflammation. This study analysed the onset of inflammation in the CFA-induced arthritis model by characterising peripheral molecular and tissue changes to elucidate the systemic inflammatory mechanisms. Methods: For this purpose, 16 male Wistar rats (12 weeks) were divided into two groups; the ART received intradermal and intra-articular CFA, while the CTL received saline solution; the oedema/arthritic index was monitored during the experiment. Synovial fluid, plasma, lymph nodes, rectus femoris muscle, and peritoneal lavage were analysed. Results: Principal component analysis revealed a difference between CTL and ART in multiple compartments. In the lymph node, Dimension 1 (89.38%) associated the ART group with greater oxidative damage (LPO, H₂O₂, O₂⁻, NO, PCO) and increased activity of SOD, CAT, and NP-SH, with lower levels of GPx. In muscle, Dimension 1 (78.11%) linked the ART group to increased TNF-α, IL-1β, Mfn1, Hsp70, and Nrf2, while Dimension 2 reflected an atrophic axis influenced by increased myostatin. In plasma, Dimension 1 (88.97%) associated the ART with increased activity of CAT, SOD, LPO, and RS, indicating oxidative distress with an adaptive antioxidant response. In peritoneal macrophages, Dimension 1 (86.92%) indicated increased activity (number, adhesion, phagocytosis, lysosomal volume, RS). Conclusion: It is concluded that CFA induces a multifactorial systemic inflammatory state, integrated with redox imbalance and mitochondrial dysregulation, approaching the extra-articular phenotypes of RA found in human patients, providing a basis for pathophysiological studies and targeted interventions. Joint Pathology CFA-model Chronical Inflammation Oxidative Distress Macrophage Activation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Rheumatoid arthritis (RA) is a chronic, symmetrical, autoimmune inflammatory disease characterized by thickening of the synovial membrane, progressive destructive arthropathy, joint deformities, and functional limitation ( 1 ). Pain, edema, and morning stiffness are some of the common symptoms reported, usually manifested symmetrically, mainly in small and medium-sized joints ( 2 ). As the disease progresses, comorbidities such as dyslipidemia, hypertension, and insulin resistance may arise ( 3 ), as well as pulmonary and renal dysfunction cardiovascular changes ( 4 ) among other comorbidities that aggravate the clinical picture and reduce quality of life and life expectancy ( 5 ) The initial phase of RA is marked by a preclinical period of autoimmunity, evidenced by the presence of autoantibodies such as anti-citrullinated peptides (anti-CCP) and rheumatoid factor (RF), which precede clinical manifestations ( 6 ). The transition between the preclinical phases of RA and the phase of joint inflammation is not fully understood, but it is marked by heterogeneous, non-standardized, and non-mandatory intermediate stages that favor the chronicity of the disease ( 7 ). In the joint microenvironment, macrophages, neutrophils, and T and B lymphocytes are recruited, which secrete proinflammatory cytokines, activate osteoclasts, and promote tissue damage ( 8 ) Activated neutrophils, through NADPH oxidase, produce reactive oxygen species (RS), which contribute to cartilage degradation and synovial damage ( 9 ). Type B synoviocytes (fibroblast-like synoviocytes, or activated synoviocytes) amplify the inflammatory process by secreting cytokines, recruiting Th17 cells, and degrading the extracellular matrix, while also promoting dysfunctional angiogenesis and chronic hypoxia ( 8 ). Experimental models of RA induction have been widely used to study the pathophysiology of RA and test therapeutic approaches, as they allow control over the onset of the disease, facilitate monitoring of its progression, and help clarify the genetic, endocrine, and biomechanical pathways involved in joint disease ( 10 ). The CFA-induced model stands out for inducing an intense inflammatory response, with evident joint manifestations such as edema, joint deformity, cartilage destruction, and cellular infiltrate ( 11 ). However, it is often pointed out as limited because it does not completely capture the pathogenesis of human RA, mimicking an acute initial inflammatory response, differing from what occurs at the onset of human RA ( 12 ). Despite these limitations, recent evidence indicates that CFA also promotes systemic inflammatory changes, such as increased anti-CCP, oxidative stress, activation of the kallikrein-kinin system (KKS), and elevated RANKL, suggesting involvement beyond the joint compartment ( 13 ). Nonetheless, there is no complete understanding of the extent of systemic inflammation triggered by this model. In this context, the present study aims to investigate the onset of systemic inflammation in the CFA-induced arthritis model by characterizing molecular and tissue alterations, the mediators involved, and the possible impacts on peripheral physiological systems. Thus, we seek to validate the CFA model not only as a tool for studying local arthritis but also as an experimental platform for understanding the systemic mechanisms of inflammation, which are often present in patients with RA. MATERIALS AND METHODS Ethical Procedures All experimental procedures were approved by the institution's Animal Ethics Committee (CEUA 13/2022) and conducted in accordance with international animal welfare guidelines and the recommendations of the ARRIVE 2.0 guidelines. The sample size was calculated based on data from previous studies with similar protocols ( 14 )and statistical analysis with 80% power and a significance level (α) of 5%. Animals The present study was conducted with 16 male Wistar rats ( Rattus norvegicus , Rodentia: Muridae ), with an average age of 12 weeks at the start of the experiment. The animals were housed in polypropylene cages with a maximum capacity of three individuals per unit, kept under controlled environmental conditions (temperature of 24 ± 1°C, 12-hour light/dark cycle), with ad libitum access to water and standard commercial feed. The animals were obtained from the Central Animal Facility of the UNIOESTE and were acclimatized for 14 days in the Sector Animal Facility, to minimize adaptive stress before the start of the experimental interventions. After the acclimatization period, the animals were randomly distributed into two experimental groups: control group (CTL, n = 8) and arthritis group (ART, n = 8), in two independent experiments. Arthritis Induction Arthritis induction was performed using Complete Freund's Adjuvant (CFA) containing Mycobacterium butyricum (0.5 mg/mL; Difco®), according to the protocol described by Gomes et al. (2014). The ART group received an intradermal injection of 50 µL of CFA near the base of the tail. After seven days, a second injection was administered, this time intra-articularly, with 50 µL of CFA introduced into the synovial cavity of the right tibiofemoral joint, anatomically guided by the infrapatellar tendon. The CTL group underwent the same manipulation protocol and received equivalent volumes of sterile 0.9% saline solution, both administered subcutaneously and intra-articularly. Evaluation of Joint Parameters The progression of the joint inflammatory process was monitored by an evaluator blinded to group allocation by measuring the diameter of the tibiofemoral joint in the medial-lateral axis using a manual caliper with automatic return. Measurements were taken at six time points: the day of the intradermal injection (baseline assessment – BA), the day of the intra-articular injection (AV1), and on alternate subsequent days (AV2, AV3, AV4, and AV5). For each time point, three consecutive measurements were taken, and the mean value (in centimeters) was considered. The severity of joint edema was assessed based on the arthritic index, calculated as the mean difference between the diameter values obtained in the AV1 to AV5 assessments in relation to the baseline value (BA), expressed in millimeters ( 16 ). The animals were euthanized nine days after intra-articular injection, by exposure to carbon dioxide followed by guillotine decapitation ( 17 ). All analyses were performed by experienced researchers blinded to the experimental conditions. Sample Collection and Preparation The joint capsule was exposed and washed with 100 µL of 0.9% isotonic saline solution containing 4 µL of 5% EDTA as an anticoagulant ( 15 ) The collected synovial fluid was used for cellular analysis. Blood was collected via decapitation in tubes containing EDTA and centrifuged at 4,000 g for 10 minutes to obtain plasma, which was stored at − 80°C until analysis. The popliteal lymph nodes were removed, frozen in liquid nitrogen, and stored at − 80°C for further processing. To collect peritoneal macrophages, 10 mL of ice-cold PBS buffer pH 7.4 was injected into the peritoneal cavity. After a gentle massage to detach macrophages, collection was performed through a small incision in the abdominal wall. These cells were kept on ice until centrifugation, counting, and dilution. Cell Counting and Characterization in Synovial Wash Twenty microliters of synovial fluid were diluted in Türk's solution (glacial acetic acid, 1% methylene blue, and distilled water) for total leukocyte counting in a Neubauer chamber, considering an approppriate dilution factor (80 to 380 µL). The count was performed under a light microscope with a 40x objective, counting cells in four quadrants, and the results were expressed in cells/mm³. Smears with 5 µL of the sample were stained by May-Grünwald and Giemsa for differential leukocyte counting under a light microscope at 100x magnification ( 18 ) Macrophage activity For all macrophage assays, 100 µL of solution containing 2 x 10⁶ cells/mL was plated in 96-well plates and incubated for 45 min at 37°C for macrophage adhesion. The adhesion capacity of macrophages was evaluated after cell incubation and fixation with 50% methanol. After that, cells were stained with Giemsa solution (0.1%) for 10 minutes. The plate was washed with PBS, and the dye was solubilized in 200 µL of 50% methanol. Absorbance was read at 550 nm ( 19 ). Phagocytic capacity was assessed by incubating the cells for 30 min with zymosan and neutral red solution. After this period, the cells were fixed with Baker's calcium formalin solution and washed with PBS. The neutral red inside the phagosomes was solubilized using an extraction solution, and the absorbance was read at 550 nm ( 20 ). For the analysis of lysosomal retention volume, the cells were incubated with a 2% neutral red solution. After 30 minutes, the supernatant was discarded, and the wells were washed with PBS to remove the neutral red that had not been internalized into the cells. An extraction solution was added to solubilize the neutral red retained within the lysosomes, and the absorbance was read at 550 nm ( 21 ). Evaluation of Reactive Species and Oxidative Stress RS were measured by the oxidation of 2',7'-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF). For this purpose, 10 µL of each sample type was incubated with 10 mM Tris-HCl buffer (pH 7.4) and 1 mM DCFH-DA for 1 hour, with fluorescence reading (excitation 488 nm, emission 520 nm) in a spectrophotometer ( 22 ). Lipid peroxidation was assessed by the thiobarbituric acid (TBARS) reaction method, which quantifies malondialdehyde (MDA), the final product of lipid peroxidation. In plasma, 200 µL of the sample reacted with TBA, SDS, and acetic acid, incubated at 95°C for 2 hours, with a reading at 532 nm. In the lymph node, peroxidation was quantified by the formation of the Fe²⁺-xylenol orange complex, measured at 560 nm ( 23 ). Oxidative damage to proteins was determined by quantifying carbonyl groups, derived from protein oxidation, through reaction with 2,4-dinitrophenylhydrazine (DNPH) and reading at 360 nm. ( 24 ). Superoxide production was estimated by the formation of formazan blue from the reduction of nitroblue tetrazolium (NBT). The homogenates were incubated for 1 hour in a medium containing 0.2% NBT in PBS at 37°C. Then, 120 µL of 2M KOH and 140 µL of DMSO (dimethyl sulfoxide) were added to solubilize the NBT. After 30 minutes, the absorbance was read at 550 nm ( 25 ). Hydrogen peroxide production was measured based on the oxidation of phenol red by peroxidase. Each well received 100 µL of the phenol red solution containing peroxidase and zymosan, and they were incubated for 30 minutes. Then, 10 µL of 1 M NaOH was added, and after 30 min, the absorbance was read at 550 nm ( 25 ). Nitric oxide production was estimated indirectly by quantifying nitrite (NO₂⁻), the main stable metabolite of NO, using the Griess reagent. For this purpose, 100 µL of the sample was incubated with 100 µL of Griess reagent (equimolar solution of 1% sulfanilic acid and 0.1% N-(1-naphthyl) ethylenediamine in 2.5% phosphoric acid) for 10 minutes at room temperature, protected from light. The formation of the azo complex was determined by reading the absorbance at 540 nm. Nitrite concentrations were calculated based on a standard curve of sodium nitrite (NaNO₂) ( 26 ). Analysis of Antioxidant Enzymes SOD activity was assessed by two complementary methods. In lymph node homogenates, SOD was quantified based on its ability to inhibit the reduction of nitroblue tetrazolium (NBT) to formazan, measured by absorbance at 560 nm ( 27 ). In plasma, activity was determined by the inhibition of epinephrine autooxidation in an alkaline medium, monitored spectrophotometrically at 480 nm ( 28 ). CAT activity was measured based on the decomposition of hydrogen peroxide (H₂O₂), determined by the decrease in absorbance at 240 nm in phosphate buffer (pH 7.0). The reaction was conducted in cuvettes containing 20 µL of the sample and 105 µL of 0.3 mM H₂O₂, with immediate measurement of substrate decomposition ( 29 ). The GPx activity was evaluated indirectly by the rate of oxidation of reduced glutathione (GSH), coupled with the decomposition of NADPH in a system containing glutathione reductase. The reaction was monitored by the decrease in absorbance at 340 nm ( 30 ). Quantification of Non-Enzymatic Antioxidants The concentrations of GSH and non-protein thiols (NP-SH) were determined by reaction with 5,5'-dithiobis(2-nitrobenzoate) (DTNB), which forms a yellow chromophore measured at 415 nm. For this purpose, proteins were precipitated with 30% trichloroacetic acid (TCA) and centrifuged at 7,000 g for 10 minutes at 4°C, using the supernatant for quantification ( 31 ). Western Blotting Muscle tissue (proximal lateral region) was dissected, placed in cryotubes, frozen in liquid nitrogen, and stored at − 80°C. Protein extraction was performed by homogenizing approximately 30 mg of muscle in 300 µL of ice-cold buffer A (10 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 1 mM NaF, 10 µg/mL aprotinin, 10 mM β-glycerophosphate, 1 mM PMSF, 1 mM DTT, 2 mM sodium orthovanadate, in 10 mM HEPES, pH 7.9). After incubation on ice for 15 minutes, the samples were centrifuged at 16,000 × g for 45 minutes at 4°C, and the supernatant was collected. Protein concentrations were determined by the Bradford (1976) method. The samples (80 µg of protein) were mixed with the loading buffer (200 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 2.75 mM β-mercaptoethanol, 0.04% bromophenol blue), heated at 100°C for 5 minutes, and subjected to 12% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes using the HEP-1 Transfer System (ThermoFischer®, USA) at 400 mA for 3 hours. After verifying the transfer with 0.5% Ponceau S, the membranes were blocked with 1% BSA in TBS-T for 2 hours. The membranes were incubated overnight (4°C) with primary antibodies (Table 1 ), followed by washing with TBS-T and incubation with peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology, USA) for 2 hours. Detection was performed with 3,3',5,5'-tetramethylbenzidine (TMB). Densitometric analysis was performed using ImageJ, normalized to β-actin. Table 1 Antibodies used for Western Blotting Target Primary Antibody Species Dilution Source Nrf2 anti-Nrf2 Mouse 1:1000 Santa Cruz, sc-365949 Hsp70 anti-Hsp70 Rabbit 1:1000 Sigma-Aldrich, SAB4200797 Bax anti-Bax Mouse 1:1000 Santa Cruz, sc-7480 ATP5A1 anti-ATP synthase subunit α Rabbit 1:1000 Sigma-Aldrich, SAB5700821 Mfn1 anti-Mitofusina-1 Rabbit 1:1000 Sigma-Aldrich, SAB2106161 TNF-α anti-TNF-alpha Mouse 1:1000 Santa Cruz, sc-52746 IL-1β anti-Interleucina-1 beta Mouse 1:1000 Santa Cruz, sc-52012 Miostatina anti-Myostatin Rabbit 1:1000 Sigma-Aldrich, SAB1305392 β-actina anti-β-actina Mouse 1:5000 Sigma-Aldrich, A5316 Statistical Analysis Data were expressed as mean ± standard deviation and analyzed using descriptive and inferential statistics in the R program (version 4.4.0). To choose the appropriate statistical test, data were evaluated for normality (Shapiro-Wilk test). When the assumptions were satisfied, the data were submitted to the t-test for independent samples and the Mann-Whitney test for nonparametric data. For serum variables that were evaluated at different times, repeated-measures ANOVA was performed, followed by Tukey-HSD post-hoc testing, when appropriate. In all cases, the level of significance adopted was 5%. Matrices A, B, and C were standardized and analyzed using principal component analysis (PCA). In principal component analysis, factor loadings were defined as the correlations of each variable with the composition of the component, the factor being a new statistical variable defined by the set of factor loadings. The factor loadings resulting from the principal components were evaluated for significance using Single Factor Analysis of Variance, assuming the experimental groups as a fixed factor with Tukey-HSD post-test. RESULTS Functional Characteristics Analysis of the joint edema variable (Fig. 1A) revealed highly significant effects of both the experimental group (χ² = 226.1; p < 0.0001) and the experimental time (χ² = 60.3; p < 0.0001), indicating that both the onset and progression of joint inflammation were strongly modulated by the arthritic condition and the post-induction period. The intercept was also highly significant (χ² = 1793.6; p < 0.0001), reinforcing the robustness of the model. From the initial time point (AV1), ART animals exhibited significantly higher joint edema than CTL animals (p < 0.001), reflecting the rapid onset of synovial inflammation. Notably, this difference remained stable and highly significant throughout AV3, AV4, and AV5, suggesting the maintenance of an active and persistent inflammatory state. Analysis of the joint index (Fig. 1B) revealed changes consistent with a pronounced joint response, with significant effects for the group factor (χ² = 226.31; p < 0.0001) and time (χ² = 54.71; p < 0.0001), with significance also for the intercept (χ² = 40.64; p < 0.0001). At AV2, the ART group had systematically higher values compared to the CTL group, reflecting early immune activation in peripheral compartments. This difference was sustained at all subsequent times (t03, t04, t05; p < 0.001), suggesting chronic inflammation. Within the ART group, a gradual decline in the inflammatory index was observed after AV2 (p < 0.001, in all). This pattern reaffirms an intense systemic inflammatory response with an acute onset, followed by progressive modulation, while remaining at a high level. Body Characteristics and Markers of Arthritis Regarding body characteristics and markers of arthritis onset (Table 2 ), ART animals show no significant difference in final body weight compared to CTL (p = 0.1811). However, a significant reduction in the relative weight of the rectus femoris muscle was observed in ART animals compared to CTL (p < 0.0001), indicating a characteristic inflammation-associated reduction in muscle mass. The arthritic index showed a marked increase in the ART group compared to CTL (p = 0.0001), confirming the development of systemic and joint inflammation in the experimental model. Similarly, analysis of the area under the joint edema curve showed a significant increase in ART animals compared to CTL (p = 0.0001), reflecting persistent joint inflammation. In the synovial lavage, the total leukocyte count, expressed as a logarithm, was significantly higher in the ART group than in the CTL group (p = 0.0009), accompanied by an intense change in the cell profile. ART animals showed a marked reduction in the percentage of mononuclear cells compared to CTL (p = 0.0009), along with a significant increase in the polymorphonuclear cell population (p = 0.0009), characterizing an acute inflammatory infiltrate. Table 2 Morphophysiological and inflammatory parameters in the CTL and ART groups. Values expressed as mean ± standard error. Different letters in the same row indicate significant differences (p < 0.05). Variable CTL ART p-Value Body Weight (g) 376.12 ± 42.20 347.87 ± 37.93 0.1811 Rectus Femoris (g/100 g B.W.) 0.33 ± 0.01 a 0.28 ± 0.01 b < 0.0001 Arthritic Index (A.U.)* 0.28 ± 2.08 b 30.38 ± 4.46 a 0.0001 Articular Edema (% x days )* 909.48 ± 26.96 b 1208.71 ± 43.38 a 0.0001 Synovial Leukocytes (log)* 4.99 ± 0.32 b 7.42 ± 0.54 a 0.0009 Mononuclear Cells (%)* 96.06 ± 2.42 a 27.06 ± 4.60 b 0.0009 Polymorphonuclear Cells (%)* 3.93 ± 2.42 b 72.93 ± 4.60 a 0.0009 Markers of Oxidative Distress in the Popliteal Lymph Node PCA performed with parameters related to oxidative stress in the popliteal lymph node (Fig. 2) showed a clear separation between the CTL and ART groups. The model revealed that axis 1 explained 89.38% of the total variance (AV = 8.04), while axis 2 contributed 4.77% (AV = 0.43), totaling 94.15% of the explained variance. This separation demonstrates systematic changes in the local redox profile in response to the induced inflammatory process. The individuals in the ART group were mostly distributed in the positive quadrant of axis 1 and were positively associated with vectors related to oxidative damage, such as increased LPO, H₂O₂, O₂⁻, NO, and PCO, in addition to hyperactivation of antioxidant enzymes such as SOD, CAT, and NP-SH, and reduced GPx activity. This pattern suggests increased oxidative stress and compensatory activation of antioxidant systems in the lymphatic microenvironment. The 99% confidence ellipses indicate homogeneous internal distributions within each group and confirm the robustness of the multivariate separation between the experimental conditions. The direction and magnitude of the vectors demonstrate the high correlation between the oxidative damage variables and the local inflammatory condition represented by the ART group. Muscle Markers PCA performed with inflammatory, mitochondrial, and regulatory parameters in the rectus femoris muscle (Fig. 3) showed a clear separation between CTL and ART groups. The model revealed that axis 1 explained 78.11% of the total variance (AV = 4.69), while axis 2 contributed 18.31% (AV = 1.10), totaling 96.42% of explained variance. This separation demonstrates systematic changes in the inflammatory and mitochondrial muscle environment in response to the arthritic process. Individuals in the ART group were predominantly distributed in the positive quadrant of axis 1 and were positively associated with the vectors TNF-α, IL-1β, Mfn1, Hsp70, Nrf2, Bax, and ATP5A1, markers related to inflammatory stress, mitochondrial dysregulation, and cellular stress response. This pattern indicates a functionally compromised muscle environment, with activation of pro-inflammatory and stress-adaptive signaling pathways. Axis 2, in turn, was influenced exclusively by MYO, a negative regulator of muscle mass, which allowed its interpretation as an atrophic axis. Although it did not contribute significantly to the separation between the groups, its vector position shows an independent dimension of the inflammatory response, possibly associated with the trophic regulation of muscle tissue. The 99% confidence ellipses indicate cohesive internal distributions within each group, confirming the robustness of the multivariate separation. The direction and magnitude of the vectors reveal a high correlation between inflammatory-mitochondrial markers and the systemic inflammatory condition in the ART group, reflecting the repercussion of the arthritis model on skeletal muscle. Plasma Oxidative Distress Markers PCA performed with the parameters of the antioxidant system and oxidative damage in plasma (Fig. 4) showed clear discrimination between the CTL and ART groups. The model revealed that axis 1 explained 88.97% of the total variance (AV = 3.56), while axis 2 contributed 6.55% (AV = 0.26), totaling 95.52% of the explained variance. This separation demonstrates systematic changes in plasma redox balance in response to the inflammatory process. Animals in the ART group were predominantly distributed in the positive quadrant of axis 1 and were also positively associated with the CAT, SOD, LPO, and RS vectors, indicating a profile characterized by a simultaneous increase in oxidative damage and the activity of classic antioxidant enzymes. This pattern reflects an adaptive response to exacerbated systemic oxidative stress, with compensatory activation of plasma defense mechanisms. Axis 2 was not strongly influenced by any variable, functioning as a secondary and poorly discriminative dimension in the model. The 99% confidence ellipses indicate cohesive internal distributions within each group, confirming the robustness of the multivariate separation. The direction and magnitude of the vectors highlight the strong correlation between systemic oxidative stress and the inflammatory condition of the ART group, reflecting the impact of the arthritis model on plasma redox parameters. Peritoneal Macrophage Activity PCA analysis applied to functional variables of peritoneal macrophages showed clear separation between the ART and CTL groups, reflecting marked changes in phagocytic activity and reactive species production in response to the inflammatory condition (Fig. 5). Axis 1, called Phagocytic Activity, accounted for 86.92% of the explained variance (AV = 5.22) and clearly segregated the experimental groups along this axis. The animals in the ART group were positioned in the positive quadrant and were strongly associated with the variables Number of Macrophages, Adhesion, Phagocytosis, Lysosomal Volume, and production of reactive species, classic indicators of phagocytic activation and increased microbicidal capacity. This pattern suggests exacerbated functional activation of macrophages in the context of systemic inflammation induced by arthritis. Axis 2, with lower discriminatory power (VR = 9.11%; AV = 0.55), was named Reactive Species Production. It represented a secondary functional dimension, with a greater contribution from the O₂⁻ variable, although without significant change between groups. The confidence ellipses (99%) indicated well-defined distributions with low overlap between groups, reinforcing the statistical robustness of the observed segmentation. The vector pattern indicates that the exacerbated phagocytic response of peritoneal macrophages is directly related to the arthritic condition, demonstrating their involvement in maintaining systemic inflammation. DISCUSSION In this study, it was demonstrated that the CFA-induced AR mouse model causes intense and sustained joint inflammation, as well as a robust systemic inflammatory response, evidenced by changes in multiple tissue compartments. The rapid onset and prolonged maintenance of joint edema observed in arthritic animals reinforce the effectiveness of the experimental model used to study chronic inflammation using CFA ( 33 ). Local inflammation was characterized by joint edema and polymorphonuclear infiltrate in the synovium, with differentiation of CD4⁺ T lymphocytes into Th1 and Th17, resulting in the release of IFN-γ, TNF-α, and IL-17. These mediators activate macrophages, stimulate hyperproliferation of type B synoviocytes, and promote additional production of proinflammatory cytokines, including TNF-α, IL-1, and IL-6 ( 34 , 35 ) TNF-α and IL-1 directly contribute to joint edema, as they promote swelling and the influx of inflammatory cells into the joint, while the production of inducible nitric oxide synthase (iNOS) by activated macrophages inhibits vascular contractility, impairing fluid drainage ( 36 ). These mechanisms characterize initial acute inflammation, confirming CFA as a potent synovial inducer ( 18 , 37 ) In muscle, inflammatory cytokines such as IL-1β and TNF-α induce atrophy via TAK1 kinase activation, triggering the NF-κB, p38 MAPK, and ERK pathways, raising myostatin levels, which in turn inhibit myoblastic differentiation and increase protein degradation Myostatin also stimulates TNF-α in synovial fibroblasts via PI3K-Akt-AP-1, reinforcing its role as a negative regulator of muscle growth and regeneration ( 38 ). Hsp70 generally exerts anti-inflammatory effects. showed that its extracellular form inhibits mediators such as IL-6, IL-8, and MCP-1 in synoviocytes from RA patients via suppression of the MAPK and NF-κB pathways. However, this protein has a dual function and can also act in a pro-inflammatory manner, promoting IL-6 secretion and Th17/Treg imbalance( 39 ) Thus, its increase, as observed in this study, may reflect both a compensatory response and the intensification of inflammation, supporting its clinical relevance as an indirect marker of inflammatory activity, even in seronegative patients ( 40 ). Nrf2 activation induces antioxidant and anti-inflammatory genes, acting as a compensatory mechanism to limit cellular damage, modulate immunity, and restore redox balance, establishing a link between oxidative stress and immuno-inflammatory regulation ( 41 ). Under basal conditions, Nrf2 remains at low levels due to Keap1-mediated degradation, but under oxidative stress, modifications in this protein promote stabilization, accumulation, and nuclear translocation of Nrf2, which activates antioxidant genes and defense enzymes ( 42 ) The absence of this regulation compromises the detoxification of reactive species and redox homeostasis, as observed in the ART group. In addition, Nrf2 can inhibit the NF-κB pathway, reduce the expression of inflammatory cytokines, and repress genes such as IL-6 and IL-1β, evidencing anti-inflammatory action in addition to redox regulation ( 42 ). Mfn1 is crucial for mitochondrial bioenergetics and osteogenic differentiation, especially under oxidative stress. Its reduction, observed in this study, indicates a decrease in mitochondrial fusion, fragmentation, lower ATP production, and greater oxidative stress. Gu et al., (2022) point out that the imbalance between fusion and fission can increase Bax both through interaction with Mfn2, compensating for the reduction in Mfn1, and through its pro-apoptotic action in the face of mitochondrial dysfunction. The increase in Bax promotes cristae remodeling, fragmentation, and loss of membrane potential possibly related to the reduction of ATP5A1, which is essential for mitochondrial function ( 8 ). As seen, the activation of inflammatory pathways and mitochondrial dysfunction compromise muscle tissue, resulting in loss of RF mass and apoptotic activation, a pattern typical of sarcopenia in RA ( 44 ). The oxidative damage observed in the RF muscle also extended to the popliteal lymph node, plasma, and peritoneal lavage, evidencing an increase in LPO, H₂O₂, O₂⁻, NO, PCO, NP-SH, TBARS, and RS, hyperactivation of SOD and CAT, and reduction of GPx ( 45 ), confirming the systemic dissemination of redox balance. The intensification of oxidative stress, with damage to lipids, proteins, and DNA, aggravates inflammation and cellular dysfunction ( 46 ). Excess NO can react with O₂⁻ to form highly reactive and deleterious peroxynitrite, while high PCO levels indicate intense protein carbonylation, reducing structural and enzymatic functionality and further contributing to tissue impairment ( 47 ). Increased SOD and CAT activity may represent a compensatory response to Nrf2-mediated RS accumulation, as these enzymes convert O₂⁻ to H₂O₂ and promote its degradation into water and oxygen. In contrast, the reduction in GPx observed in this study compromises the neutralization of peroxides, favoring the accumulation of highly reactive species, ferroptosis, and greater cellular vulnerability, possibly due to excessive consumption or failure in the regeneration of reduced glutathione (GSH) ( 46 ). This imbalance, with increased SOD and CAT but GPx deficiency, indicates insufficient neutralization of peroxides and maintenance of oxidative stress, since the compensatory response does not compensate for antioxidant dysfunction ( 46 ) Studies in patients with RA show a similar profile, with increased MDA and reduced GSH, reinforcing the relationship between redox imbalance, inflammation, and tissue damage ( 48 ). Similarly, our findings indicate that increased activation of oxidative pathways, coupled with partial inefficiency of antioxidant mechanisms, aggravated systemic inflammation. The systemic inflammatory response also involved an increase in the number of peritoneal macrophages, whose increased adhesion, phagocytosis, and lysosomal retention reflect the recruitment of circulating monocytes and, to a lesser extent, the local proliferation of resident macrophages. The increased cell adhesion observed is consistent with recruited macrophages, which are more activated and phagocytic than resident macrophages ( 49 ) Phagocytosis eliminates apoptotic particles and cells, preventing the release of danger signals (DAMPs) and modulating inflammation. The increase in cytoplasmic vacuoles in macrophages may explain the increase in the lysosomal retention rate, indicative of intense intracellular digestion, stimulating the secretion of IL-1, TNF-α, and reactive species, amplifying inflammation ( 50 ). The processes involving innate immunity are regulated by inflammatory cytokines and other mediators that favor the migration, differentiation, and survival of macrophages in the peritoneal cavity. In summary, the CFA-induced AR mouse model reproduces key aspects of the disease, including joint inflammation, systemic response, mitochondrial dysfunction, and redox imbalance, reflecting its complexity. The limitations of this study include analysis at a single time point and the absence of functional approaches to confirm molecular mechanisms. Thus, future studies should adopt longitudinal designs, complementary models, and pharmacological interventions, in addition to clinical validation, to deepen the understanding of pathological mechanisms and support therapeutic strategies aimed at redox balance and immune modulation. Declarations Funding: No specific funding was received for this work. Author Contribution All authors made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; drafted the work or revised it critically for important intellectual content; approved the version to be published; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Acknowledgements: The authors acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the postgraduate scholarships awarded to the graduate students involved in this study. Data Availability All data supporting the findings of this study are available from the corresponding author upon reasonable request. References Machaj D, Płaczek A, Cyboran K, Siedlak A, Białas F. 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12:39:16","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":214733,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8523680/v1/73760c3004b90d12874768c9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eA Complete Freund’s Adjuvant-Induced Murine Model as an Experimental Platform to Explore Systemic Inflammation in Rheumatoid Arthritis\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRheumatoid arthritis (RA) is a chronic, symmetrical, autoimmune inflammatory disease characterized by thickening of the synovial membrane, progressive destructive arthropathy, joint deformities, and functional limitation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Pain, edema, and morning stiffness are some of the common symptoms reported, usually manifested symmetrically, mainly in small and medium-sized joints (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). As the disease progresses, comorbidities such as dyslipidemia, hypertension, and insulin resistance may arise (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), as well as pulmonary and renal dysfunction cardiovascular changes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) among other comorbidities that aggravate the clinical picture and reduce quality of life and life expectancy (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe initial phase of RA is marked by a preclinical period of autoimmunity, evidenced by the presence of autoantibodies such as anti-citrullinated peptides (anti-CCP) and rheumatoid factor (RF), which precede clinical manifestations (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The transition between the preclinical phases of RA and the phase of joint inflammation is not fully understood, but it is marked by heterogeneous, non-standardized, and non-mandatory intermediate stages that favor the chronicity of the disease (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In the joint microenvironment, macrophages, neutrophils, and T and B lymphocytes are recruited, which secrete proinflammatory cytokines, activate osteoclasts, and promote tissue damage (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) Activated neutrophils, through NADPH oxidase, produce reactive oxygen species (RS), which contribute to cartilage degradation and synovial damage (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Type B synoviocytes (fibroblast-like synoviocytes, or activated synoviocytes) amplify the inflammatory process by secreting cytokines, recruiting Th17 cells, and degrading the extracellular matrix, while also promoting dysfunctional angiogenesis and chronic hypoxia (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExperimental models of RA induction have been widely used to study the pathophysiology of RA and test therapeutic approaches, as they allow control over the onset of the disease, facilitate monitoring of its progression, and help clarify the genetic, endocrine, and biomechanical pathways involved in joint disease (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The CFA-induced model stands out for inducing an intense inflammatory response, with evident joint manifestations such as edema, joint deformity, cartilage destruction, and cellular infiltrate (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, it is often pointed out as limited because it does not completely capture the pathogenesis of human RA, mimicking an acute initial inflammatory response, differing from what occurs at the onset of human RA (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Despite these limitations, recent evidence indicates that CFA also promotes systemic inflammatory changes, such as increased anti-CCP, oxidative stress, activation of the kallikrein-kinin system (KKS), and elevated RANKL, suggesting involvement beyond the joint compartment (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Nonetheless, there is no complete understanding of the extent of systemic inflammation triggered by this model.\u003c/p\u003e \u003cp\u003eIn this context, the present study aims to investigate the onset of systemic inflammation in the CFA-induced arthritis model by characterizing molecular and tissue alterations, the mediators involved, and the possible impacts on peripheral physiological systems. Thus, we seek to validate the CFA model not only as a tool for studying local arthritis but also as an experimental platform for understanding the systemic mechanisms of inflammation, which are often present in patients with RA.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthical Procedures\u003c/h2\u003e \u003cp\u003e All experimental procedures were approved by the institution's Animal Ethics Committee (CEUA 13/2022) and conducted in accordance with international animal welfare guidelines and the recommendations of the ARRIVE 2.0 guidelines. The sample size was calculated based on data from previous studies with similar protocols (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)and statistical analysis with 80% power and a significance level (α) of 5%.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eThe present study was conducted with 16 male \u003cem\u003eWistar\u003c/em\u003e rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e, Rodentia: \u003cem\u003eMuridae\u003c/em\u003e), with an average age of 12 weeks at the start of the experiment. The animals were housed in polypropylene cages with a maximum capacity of three individuals per unit, kept under controlled environmental conditions (temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 12-hour light/dark cycle), with ad libitum access to water and standard commercial feed. The animals were obtained from the Central Animal Facility of the UNIOESTE and were acclimatized for 14 days in the Sector Animal Facility, to minimize adaptive stress before the start of the experimental interventions. After the acclimatization period, the animals were randomly distributed into two experimental groups: control group (CTL, n\u0026thinsp;=\u0026thinsp;8) and arthritis group (ART, n\u0026thinsp;=\u0026thinsp;8), in two independent experiments.\u003c/p\u003e\n\u003ch3\u003eArthritis Induction\u003c/h3\u003e\n\u003cp\u003eArthritis induction was performed using Complete Freund's Adjuvant (CFA) containing Mycobacterium butyricum (0.5 mg/mL; Difco\u0026reg;), according to the protocol described by Gomes et al. (2014). The ART group received an intradermal injection of 50 \u0026micro;L of CFA near the base of the tail. After seven days, a second injection was administered, this time intra-articularly, with 50 \u0026micro;L of CFA introduced into the synovial cavity of the right tibiofemoral joint, anatomically guided by the infrapatellar tendon. The CTL group underwent the same manipulation protocol and received equivalent volumes of sterile 0.9% saline solution, both administered subcutaneously and intra-articularly.\u003c/p\u003e\n\u003ch3\u003eEvaluation of Joint Parameters\u003c/h3\u003e\n\u003cp\u003eThe progression of the joint inflammatory process was monitored by an evaluator blinded to group allocation by measuring the diameter of the tibiofemoral joint in the medial-lateral axis using a manual caliper with automatic return. Measurements were taken at six time points: the day of the intradermal injection (baseline assessment \u0026ndash; BA), the day of the intra-articular injection (AV1), and on alternate subsequent days (AV2, AV3, AV4, and AV5). For each time point, three consecutive measurements were taken, and the mean value (in centimeters) was considered.\u003c/p\u003e \u003cp\u003eThe severity of joint edema was assessed based on the arthritic index, calculated as the mean difference between the diameter values obtained in the AV1 to AV5 assessments in relation to the baseline value (BA), expressed in millimeters (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe animals were euthanized nine days after intra-articular injection, by exposure to carbon dioxide followed by guillotine decapitation (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). All analyses were performed by experienced researchers blinded to the experimental conditions.\u003c/p\u003e\n\u003ch3\u003eSample Collection and Preparation\u003c/h3\u003e\n\u003cp\u003eThe joint capsule was exposed and washed with 100 \u0026micro;L of 0.9% isotonic saline solution containing 4 \u0026micro;L of 5% EDTA as an anticoagulant (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) The collected synovial fluid was used for cellular analysis. Blood was collected via decapitation in tubes containing EDTA and centrifuged at 4,000 g for 10 minutes to obtain plasma, which was stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. The popliteal lymph nodes were removed, frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further processing.\u003c/p\u003e \u003cp\u003eTo collect peritoneal macrophages, 10 mL of ice-cold PBS buffer pH 7.4 was injected into the peritoneal cavity. After a gentle massage to detach macrophages, collection was performed through a small incision in the abdominal wall. These cells were kept on ice until centrifugation, counting, and dilution.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting and Characterization in Synovial Wash\u003c/h2\u003e \u003cp\u003eTwenty microliters of synovial fluid were diluted in T\u0026uuml;rk's solution (glacial acetic acid, 1% methylene blue, and distilled water) for total leukocyte counting in a Neubauer chamber, considering an approppriate dilution factor (80 to 380 \u0026micro;L). The count was performed under a light microscope with a 40x objective, counting cells in four quadrants, and the results were expressed in cells/mm\u0026sup3;. Smears with 5 \u0026micro;L of the sample were stained by May-Gr\u0026uuml;nwald and Giemsa for differential leukocyte counting under a light microscope at 100x magnification (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMacrophage activity\u003c/h3\u003e\n\u003cp\u003eFor all macrophage assays, 100 \u0026micro;L of solution containing 2 x 10⁶ cells/mL was plated in 96-well plates and incubated for 45 min at 37\u0026deg;C for macrophage adhesion. The adhesion capacity of macrophages was evaluated after cell incubation and fixation with 50% methanol. After that, cells were stained with Giemsa solution (0.1%) for 10 minutes. The plate was washed with PBS, and the dye was solubilized in 200 \u0026micro;L of 50% methanol. Absorbance was read at 550 nm (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhagocytic capacity was assessed by incubating the cells for 30 min with zymosan and neutral red solution. After this period, the cells were fixed with Baker's calcium formalin solution and washed with PBS. The neutral red inside the phagosomes was solubilized using an extraction solution, and the absorbance was read at 550 nm (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the analysis of lysosomal retention volume, the cells were incubated with a 2% neutral red solution. After 30 minutes, the supernatant was discarded, and the wells were washed with PBS to remove the neutral red that had not been internalized into the cells. An extraction solution was added to solubilize the neutral red retained within the lysosomes, and the absorbance was read at 550 nm (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eEvaluation of Reactive Species and Oxidative Stress\u003c/h3\u003e\n\u003cp\u003eRS were measured by the oxidation of 2',7'-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF). For this purpose, 10 \u0026micro;L of each sample type was incubated with 10 mM Tris-HCl buffer (pH 7.4) and 1 mM DCFH-DA for 1 hour, with fluorescence reading (excitation 488 nm, emission 520 nm) in a spectrophotometer (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLipid peroxidation was assessed by the thiobarbituric acid (TBARS) reaction method, which quantifies malondialdehyde (MDA), the final product of lipid peroxidation. In plasma, 200 \u0026micro;L of the sample reacted with TBA, SDS, and acetic acid, incubated at 95\u0026deg;C for 2 hours, with a reading at 532 nm. In the lymph node, peroxidation was quantified by the formation of the Fe\u0026sup2;⁺-xylenol orange complex, measured at 560 nm (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxidative damage to proteins was determined by quantifying carbonyl groups, derived from protein oxidation, through reaction with 2,4-dinitrophenylhydrazine (DNPH) and reading at 360 nm. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuperoxide production was estimated by the formation of formazan blue from the reduction of nitroblue tetrazolium (NBT). The homogenates were incubated for 1 hour in a medium containing 0.2% NBT in PBS at 37\u0026deg;C. Then, 120 \u0026micro;L of 2M KOH and 140 \u0026micro;L of DMSO (dimethyl sulfoxide) were added to solubilize the NBT. After 30 minutes, the absorbance was read at 550 nm (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHydrogen peroxide production was measured based on the oxidation of phenol red by peroxidase. Each well received 100 \u0026micro;L of the phenol red solution containing peroxidase and zymosan, and they were incubated for 30 minutes. Then, 10 \u0026micro;L of 1 M NaOH was added, and after 30 min, the absorbance was read at 550 nm (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNitric oxide production was estimated indirectly by quantifying nitrite (NO₂⁻), the main stable metabolite of NO, using the Griess reagent. For this purpose, 100 \u0026micro;L of the sample was incubated with 100 \u0026micro;L of Griess reagent (equimolar solution of 1% sulfanilic acid and 0.1% N-(1-naphthyl) ethylenediamine in 2.5% phosphoric acid) for 10 minutes at room temperature, protected from light. The formation of the azo complex was determined by reading the absorbance at 540 nm. Nitrite concentrations were calculated based on a standard curve of sodium nitrite (NaNO₂) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Antioxidant Enzymes\u003c/h2\u003e \u003cp\u003eSOD activity was assessed by two complementary methods. In lymph node homogenates, SOD was quantified based on its ability to inhibit the reduction of nitroblue tetrazolium (NBT) to formazan, measured by absorbance at 560 nm (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In plasma, activity was determined by the inhibition of epinephrine autooxidation in an alkaline medium, monitored spectrophotometrically at 480 nm (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCAT activity was measured based on the decomposition of hydrogen peroxide (H₂O₂), determined by the decrease in absorbance at 240 nm in phosphate buffer (pH 7.0). The reaction was conducted in cuvettes containing 20 \u0026micro;L of the sample and 105 \u0026micro;L of 0.3 mM H₂O₂, with immediate measurement of substrate decomposition (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe GPx activity was evaluated indirectly by the rate of oxidation of reduced glutathione (GSH), coupled with the decomposition of NADPH in a system containing glutathione reductase. The reaction was monitored by the decrease in absorbance at 340 nm (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of Non-Enzymatic Antioxidants\u003c/h2\u003e \u003cp\u003eThe concentrations of GSH and non-protein thiols (NP-SH) were determined by reaction with 5,5'-dithiobis(2-nitrobenzoate) (DTNB), which forms a yellow chromophore measured at 415 nm. For this purpose, proteins were precipitated with 30% trichloroacetic acid (TCA) and centrifuged at 7,000 g for 10 minutes at 4\u0026deg;C, using the supernatant for quantification (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting\u003c/h2\u003e \u003cp\u003eMuscle tissue (proximal lateral region) was dissected, placed in cryotubes, frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Protein extraction was performed by homogenizing approximately 30 mg of muscle in 300 \u0026micro;L of ice-cold buffer A (10 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 1 mM NaF, 10 \u0026micro;g/mL aprotinin, 10 mM β-glycerophosphate, 1 mM PMSF, 1 mM DTT, 2 mM sodium orthovanadate, in 10 mM HEPES, pH 7.9). After incubation on ice for 15 minutes, the samples were centrifuged at 16,000 \u0026times; g for 45 minutes at 4\u0026deg;C, and the supernatant was collected. Protein concentrations were determined by the Bradford (1976) method.\u003c/p\u003e \u003cp\u003eThe samples (80 \u0026micro;g of protein) were mixed with the loading buffer (200 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 2.75 mM β-mercaptoethanol, 0.04% bromophenol blue), heated at 100\u0026deg;C for 5 minutes, and subjected to 12% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes using the HEP-1 Transfer System (ThermoFischer\u0026reg;, USA) at 400 mA for 3 hours. After verifying the transfer with 0.5% Ponceau S, the membranes were blocked with 1% BSA in TBS-T for 2 hours.\u003c/p\u003e \u003cp\u003eThe membranes were incubated overnight (4\u0026deg;C) with primary antibodies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), followed by washing with TBS-T and incubation with peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology, USA) for 2 hours. Detection was performed with 3,3',5,5'-tetramethylbenzidine (TMB). Densitometric analysis was performed using ImageJ, normalized to β-actin.\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\u003eAntibodies used for Western Blotting\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary Antibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNrf2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-Nrf2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta Cruz, sc-365949\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHsp70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-Hsp70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich, SAB4200797\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-Bax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta Cruz, sc-7480\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATP5A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-ATP synthase subunit α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich, SAB5700821\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMfn1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-Mitofusina-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich, SAB2106161\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-TNF-alpha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta Cruz, sc-52746\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-Interleucina-1 beta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSanta Cruz, sc-52012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiostatina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-Myostatin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich, SAB1305392\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eanti-β-actina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:5000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSigma-Aldrich, A5316\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=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and analyzed using descriptive and inferential statistics in the R program (version 4.4.0). To choose the appropriate statistical test, data were evaluated for normality (Shapiro-Wilk test). When the assumptions were satisfied, the data were submitted to the t-test for independent samples and the Mann-Whitney test for nonparametric data. For serum variables that were evaluated at different times, repeated-measures ANOVA was performed, followed by Tukey-HSD post-hoc testing, when appropriate. In all cases, the level of significance adopted was 5%.\u003c/p\u003e \u003cp\u003eMatrices A, B, and C were standardized and analyzed using principal component analysis (PCA). In principal component analysis, factor loadings were defined as the correlations of each variable with the composition of the component, the factor being a new statistical variable defined by the set of factor loadings. The factor loadings resulting from the principal components were evaluated for significance using Single Factor Analysis of Variance, assuming the experimental groups as a fixed factor with Tukey-HSD post-test.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Characteristics\u003c/h2\u003e \u003cp\u003eAnalysis of the joint edema variable (Fig.\u0026nbsp;1A) revealed highly significant effects of both the experimental group (χ\u0026sup2; = 226.1; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and the experimental time (χ\u0026sup2; = 60.3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating that both the onset and progression of joint inflammation were strongly modulated by the arthritic condition and the post-induction period. The intercept was also highly significant (χ\u0026sup2; = 1793.6; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), reinforcing the robustness of the model. From the initial time point (AV1), ART animals exhibited significantly higher joint edema than CTL animals (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), reflecting the rapid onset of synovial inflammation. Notably, this difference remained stable and highly significant throughout AV3, AV4, and AV5, suggesting the maintenance of an active and persistent inflammatory state.\u003c/p\u003e \u003cp\u003eAnalysis of the joint index (Fig.\u0026nbsp;1B) revealed changes consistent with a pronounced joint response, with significant effects for the group factor (χ\u0026sup2; = 226.31; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and time (χ\u0026sup2; = 54.71; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with significance also for the intercept (χ\u0026sup2; = 40.64; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). At AV2, the ART group had systematically higher values compared to the CTL group, reflecting early immune activation in peripheral compartments. This difference was sustained at all subsequent times (t03, t04, t05; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting chronic inflammation. Within the ART group, a gradual decline in the inflammatory index was observed after AV2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, in all). This pattern reaffirms an intense systemic inflammatory response with an acute onset, followed by progressive modulation, while remaining at a high level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBody Characteristics and Markers of Arthritis\u003c/h2\u003e \u003cp\u003eRegarding body characteristics and markers of arthritis onset (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), ART animals show no significant difference in final body weight compared to CTL (p\u0026thinsp;=\u0026thinsp;0.1811). However, a significant reduction in the relative weight of the rectus femoris muscle was observed in ART animals compared to CTL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating a characteristic inflammation-associated reduction in muscle mass.\u003c/p\u003e \u003cp\u003eThe arthritic index showed a marked increase in the ART group compared to CTL (p\u0026thinsp;=\u0026thinsp;0.0001), confirming the development of systemic and joint inflammation in the experimental model. Similarly, analysis of the area under the joint edema curve showed a significant increase in ART animals compared to CTL (p\u0026thinsp;=\u0026thinsp;0.0001), reflecting persistent joint inflammation.\u003c/p\u003e \u003cp\u003eIn the synovial lavage, the total leukocyte count, expressed as a logarithm, was significantly higher in the ART group than in the CTL group (p\u0026thinsp;=\u0026thinsp;0.0009), accompanied by an intense change in the cell profile. ART animals showed a marked reduction in the percentage of mononuclear cells compared to CTL (p\u0026thinsp;=\u0026thinsp;0.0009), along with a significant increase in the polymorphonuclear cell population (p\u0026thinsp;=\u0026thinsp;0.0009), characterizing an acute inflammatory infiltrate.\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\u003eMorphophysiological and inflammatory parameters in the CTL and ART groups. Values expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Different letters in the same row indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCTL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eART\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody Weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e376.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;42.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e347.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;37.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.1811\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRectus Femoris (g/100 g B.W.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArthritic Index (A.U.)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.08 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.46 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArticular Edema (% x days )*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e909.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;26.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1208.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;43.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSynovial Leukocytes (log)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMononuclear Cells (%)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e96.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymorphonuclear Cells (%)*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;2.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u0026thinsp;4.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0009\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=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMarkers of Oxidative Distress in the Popliteal Lymph Node\u003c/h2\u003e \u003cp\u003ePCA performed with parameters related to oxidative stress in the popliteal lymph node (Fig.\u0026nbsp;2) showed a clear separation between the CTL and ART groups. The model revealed that axis 1 explained 89.38% of the total variance (AV\u0026thinsp;=\u0026thinsp;8.04), while axis 2 contributed 4.77% (AV\u0026thinsp;=\u0026thinsp;0.43), totaling 94.15% of the explained variance. This separation demonstrates systematic changes in the local redox profile in response to the induced inflammatory process. The individuals in the ART group were mostly distributed in the positive quadrant of axis 1 and were positively associated with vectors related to oxidative damage, such as increased LPO, H₂O₂, O₂⁻, NO, and PCO, in addition to hyperactivation of antioxidant enzymes such as SOD, CAT, and NP-SH, and reduced GPx activity. This pattern suggests increased oxidative stress and compensatory activation of antioxidant systems in the lymphatic microenvironment. The 99% confidence ellipses indicate homogeneous internal distributions within each group and confirm the robustness of the multivariate separation between the experimental conditions. The direction and magnitude of the vectors demonstrate the high correlation between the oxidative damage variables and the local inflammatory condition represented by the ART group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMuscle Markers\u003c/h2\u003e \u003cp\u003ePCA performed with inflammatory, mitochondrial, and regulatory parameters in the rectus femoris muscle (Fig.\u0026nbsp;3) showed a clear separation between CTL and ART groups. The model revealed that axis 1 explained 78.11% of the total variance (AV\u0026thinsp;=\u0026thinsp;4.69), while axis 2 contributed 18.31% (AV\u0026thinsp;=\u0026thinsp;1.10), totaling 96.42% of explained variance. This separation demonstrates systematic changes in the inflammatory and mitochondrial muscle environment in response to the arthritic process. Individuals in the ART group were predominantly distributed in the positive quadrant of axis 1 and were positively associated with the vectors TNF-α, IL-1β, Mfn1, Hsp70, Nrf2, Bax, and ATP5A1, markers related to inflammatory stress, mitochondrial dysregulation, and cellular stress response. This pattern indicates a functionally compromised muscle environment, with activation of pro-inflammatory and stress-adaptive signaling pathways. Axis 2, in turn, was influenced exclusively by MYO, a negative regulator of muscle mass, which allowed its interpretation as an atrophic axis. Although it did not contribute significantly to the separation between the groups, its vector position shows an independent dimension of the inflammatory response, possibly associated with the trophic regulation of muscle tissue. The 99% confidence ellipses indicate cohesive internal distributions within each group, confirming the robustness of the multivariate separation. The direction and magnitude of the vectors reveal a high correlation between inflammatory-mitochondrial markers and the systemic inflammatory condition in the ART group, reflecting the repercussion of the arthritis model on skeletal muscle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePlasma Oxidative Distress Markers\u003c/h2\u003e \u003cp\u003ePCA performed with the parameters of the antioxidant system and oxidative damage in plasma (Fig.\u0026nbsp;4) showed clear discrimination between the CTL and ART groups. The model revealed that axis 1 explained 88.97% of the total variance (AV\u0026thinsp;=\u0026thinsp;3.56), while axis 2 contributed 6.55% (AV\u0026thinsp;=\u0026thinsp;0.26), totaling 95.52% of the explained variance. This separation demonstrates systematic changes in plasma redox balance in response to the inflammatory process. Animals in the ART group were predominantly distributed in the positive quadrant of axis 1 and were also positively associated with the CAT, SOD, LPO, and RS vectors, indicating a profile characterized by a simultaneous increase in oxidative damage and the activity of classic antioxidant enzymes. This pattern reflects an adaptive response to exacerbated systemic oxidative stress, with compensatory activation of plasma defense mechanisms. Axis 2 was not strongly influenced by any variable, functioning as a secondary and poorly discriminative dimension in the model.\u003c/p\u003e \u003cp\u003eThe 99% confidence ellipses indicate cohesive internal distributions within each group, confirming the robustness of the multivariate separation. The direction and magnitude of the vectors highlight the strong correlation between systemic oxidative stress and the inflammatory condition of the ART group, reflecting the impact of the arthritis model on plasma redox parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePeritoneal Macrophage Activity\u003c/h2\u003e \u003cp\u003ePCA analysis applied to functional variables of peritoneal macrophages showed clear separation between the ART and CTL groups, reflecting marked changes in phagocytic activity and reactive species production in response to the inflammatory condition (Fig.\u0026nbsp;5). Axis 1, called Phagocytic Activity, accounted for 86.92% of the explained variance (AV\u0026thinsp;=\u0026thinsp;5.22) and clearly segregated the experimental groups along this axis. The animals in the ART group were positioned in the positive quadrant and were strongly associated with the variables Number of Macrophages, Adhesion, Phagocytosis, Lysosomal Volume, and production of reactive species, classic indicators of phagocytic activation and increased microbicidal capacity. This pattern suggests exacerbated functional activation of macrophages in the context of systemic inflammation induced by arthritis. Axis 2, with lower discriminatory power (VR\u0026thinsp;=\u0026thinsp;9.11%; AV\u0026thinsp;=\u0026thinsp;0.55), was named Reactive Species Production. It represented a secondary functional dimension, with a greater contribution from the O₂⁻ variable, although without significant change between groups. The confidence ellipses (99%) indicated well-defined distributions with low overlap between groups, reinforcing the statistical robustness of the observed segmentation. The vector pattern indicates that the exacerbated phagocytic response of peritoneal macrophages is directly related to the arthritic condition, demonstrating their involvement in maintaining systemic inflammation.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, it was demonstrated that the CFA-induced AR mouse model causes intense and sustained joint inflammation, as well as a robust systemic inflammatory response, evidenced by changes in multiple tissue compartments. The rapid onset and prolonged maintenance of joint edema observed in arthritic animals reinforce the effectiveness of the experimental model used to study chronic inflammation using CFA (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLocal inflammation was characterized by joint edema and polymorphonuclear infiltrate in the synovium, with differentiation of CD4⁺ T lymphocytes into Th1 and Th17, resulting in the release of IFN-γ, TNF-α, and IL-17. These mediators activate macrophages, stimulate hyperproliferation of type B synoviocytes, and promote additional production of proinflammatory cytokines, including TNF-α, IL-1, and IL-6 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) TNF-α and IL-1 directly contribute to joint edema, as they promote swelling and the influx of inflammatory cells into the joint, while the production of inducible nitric oxide synthase (iNOS) by activated macrophages inhibits vascular contractility, impairing fluid drainage (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). These mechanisms characterize initial acute inflammation, confirming CFA as a potent synovial inducer (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn muscle, inflammatory cytokines such as IL-1β and TNF-α induce atrophy via TAK1 kinase activation, triggering the NF-κB, p38 MAPK, and ERK pathways, raising myostatin levels, which in turn inhibit myoblastic differentiation and increase protein degradation Myostatin also stimulates TNF-α in synovial fibroblasts via PI3K-Akt-AP-1, reinforcing its role as a negative regulator of muscle growth and regeneration (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHsp70 generally exerts anti-inflammatory effects. showed that its extracellular form inhibits mediators such as IL-6, IL-8, and MCP-1 in synoviocytes from RA patients via suppression of the MAPK and NF-κB pathways. However, this protein has a dual function and can also act in a pro-inflammatory manner, promoting IL-6 secretion and Th17/Treg imbalance(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) Thus, its increase, as observed in this study, may reflect both a compensatory response and the intensification of inflammation, supporting its clinical relevance as an indirect marker of inflammatory activity, even in seronegative patients (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNrf2 activation induces antioxidant and anti-inflammatory genes, acting as a compensatory mechanism to limit cellular damage, modulate immunity, and restore redox balance, establishing a link between oxidative stress and immuno-inflammatory regulation (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Under basal conditions, Nrf2 remains at low levels due to Keap1-mediated degradation, but under oxidative stress, modifications in this protein promote stabilization, accumulation, and nuclear translocation of Nrf2, which activates antioxidant genes and defense enzymes (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) The absence of this regulation compromises the detoxification of reactive species and redox homeostasis, as observed in the ART group. In addition, Nrf2 can inhibit the NF-κB pathway, reduce the expression of inflammatory cytokines, and repress genes such as IL-6 and IL-1β, evidencing anti-inflammatory action in addition to redox regulation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMfn1 is crucial for mitochondrial bioenergetics and osteogenic differentiation, especially under oxidative stress. Its reduction, observed in this study, indicates a decrease in mitochondrial fusion, fragmentation, lower ATP production, and greater oxidative stress. Gu et al., (2022) point out that the imbalance between fusion and fission can increase Bax both through interaction with Mfn2, compensating for the reduction in Mfn1, and through its pro-apoptotic action in the face of mitochondrial dysfunction. The increase in Bax promotes cristae remodeling, fragmentation, and loss of membrane potential possibly related to the reduction of ATP5A1, which is essential for mitochondrial function (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs seen, the activation of inflammatory pathways and mitochondrial dysfunction compromise muscle tissue, resulting in loss of RF mass and apoptotic activation, a pattern typical of sarcopenia in RA (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe oxidative damage observed in the RF muscle also extended to the popliteal lymph node, plasma, and peritoneal lavage, evidencing an increase in LPO, H₂O₂, O₂⁻, NO, PCO, NP-SH, TBARS, and RS, hyperactivation of SOD and CAT, and reduction of GPx (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), confirming the systemic dissemination of redox balance. The intensification of oxidative stress, with damage to lipids, proteins, and DNA, aggravates inflammation and cellular dysfunction (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Excess NO can react with O₂⁻ to form highly reactive and deleterious peroxynitrite, while high PCO levels indicate intense protein carbonylation, reducing structural and enzymatic functionality and further contributing to tissue impairment (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreased SOD and CAT activity may represent a compensatory response to Nrf2-mediated RS accumulation, as these enzymes convert O₂⁻ to H₂O₂ and promote its degradation into water and oxygen. In contrast, the reduction in GPx observed in this study compromises the neutralization of peroxides, favoring the accumulation of highly reactive species, ferroptosis, and greater cellular vulnerability, possibly due to excessive consumption or failure in the regeneration of reduced glutathione (GSH) (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis imbalance, with increased SOD and CAT but GPx deficiency, indicates insufficient neutralization of peroxides and maintenance of oxidative stress, since the compensatory response does not compensate for antioxidant dysfunction (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) Studies in patients with RA show a similar profile, with increased MDA and reduced GSH, reinforcing the relationship between redox imbalance, inflammation, and tissue damage (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Similarly, our findings indicate that increased activation of oxidative pathways, coupled with partial inefficiency of antioxidant mechanisms, aggravated systemic inflammation.\u003c/p\u003e \u003cp\u003eThe systemic inflammatory response also involved an increase in the number of peritoneal macrophages, whose increased adhesion, phagocytosis, and lysosomal retention reflect the recruitment of circulating monocytes and, to a lesser extent, the local proliferation of resident macrophages. The increased cell adhesion observed is consistent with recruited macrophages, which are more activated and phagocytic than resident macrophages (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/p\u003e \u003cp\u003ePhagocytosis eliminates apoptotic particles and cells, preventing the release of danger signals (DAMPs) and modulating inflammation. The increase in cytoplasmic vacuoles in macrophages may explain the increase in the lysosomal retention rate, indicative of intense intracellular digestion, stimulating the secretion of IL-1, TNF-α, and reactive species, amplifying inflammation (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). The processes involving innate immunity are regulated by inflammatory cytokines and other mediators that favor the migration, differentiation, and survival of macrophages in the peritoneal cavity.\u003c/p\u003e \u003cp\u003eIn summary, the CFA-induced AR mouse model reproduces key aspects of the disease, including joint inflammation, systemic response, mitochondrial dysfunction, and redox imbalance, reflecting its complexity. The limitations of this study include analysis at a single time point and the absence of functional approaches to confirm molecular mechanisms. Thus, future studies should adopt longitudinal designs, complementary models, and pharmacological interventions, in addition to clinical validation, to deepen the understanding of pathological mechanisms and support therapeutic strategies aimed at redox balance and immune modulation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eNo specific funding was received for this work.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; drafted the work or revised it critically for important intellectual content; approved the version to be published; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThe authors acknowledge the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) and the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) for the postgraduate scholarships awarded to the graduate students involved in this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMachaj D, Płaczek A, Cyboran K, Siedlak A, Białas F. 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Dispon\u0026iacute;vel em: https://creativecommons.org/licenses/by/4.0/\u003c/li\u003e\n\u003cli\u003eLouwe PA, Badiola Gomez L, Webster H, Perona-Wright G, Bain CC, Forbes SJ, et al. Recruited macrophages that colonize the post-inflammatory peritoneal niche convert into functionally divergent resident cells. Nature Communications. 2021;12(1):1770. \u003c/li\u003e\n\u003cli\u003eBergman M, Salman H, Bessler H, Omanski M, Punsky I, Djaldetti M. Interaction between phagocytosis and IL-1 production by rat peritoneal macrophages. Biomedicine \u0026amp; Pharmacotherapy. 2002;56:159\u0026ndash;62. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Joint Pathology, CFA-model, Chronical Inflammation, Oxidative Distress, Macrophage Activation","lastPublishedDoi":"10.21203/rs.3.rs-8523680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8523680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction: \u003c/strong\u003eRheumatoid arthritis (RA) is a chronic systemic inflammatory disease that compromises joint integrity and triggers extra-articular manifestations associated with oxidative distress and systemic inflammation. This study analysed the onset of inflammation in the CFA-induced arthritis model by characterising peripheral molecular and tissue changes to elucidate the systemic inflammatory mechanisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eFor this purpose, 16 male \u003cem\u003eWistar\u003c/em\u003e rats (12 weeks) were divided into two groups; the ART received intradermal and intra-articular CFA, while the CTL received saline solution; the oedema/arthritic index was monitored during the experiment. Synovial fluid, plasma, lymph nodes, rectus femoris muscle, and peritoneal lavage were analysed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePrincipal component analysis revealed a difference between CTL and ART in multiple compartments. In the lymph node, Dimension 1 (89.38%) associated the ART group with greater oxidative damage (LPO, H₂O₂, O₂⁻, NO, PCO) and increased activity of SOD, CAT, and NP-SH, with lower levels of GPx. In muscle, Dimension 1 (78.11%) linked the ART group to increased TNF-α, IL-1β, Mfn1, Hsp70, and Nrf2, while Dimension 2 reflected an atrophic axis influenced by increased myostatin. In plasma, Dimension 1 (88.97%) associated the ART with increased activity of CAT, SOD, LPO, and RS, indicating oxidative distress with an adaptive antioxidant response. In peritoneal macrophages, Dimension 1 (86.92%) indicated increased activity (number, adhesion, phagocytosis, lysosomal volume, RS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eIt is concluded that CFA induces a multifactorial systemic inflammatory state, integrated with redox imbalance and mitochondrial dysregulation, approaching the extra-articular phenotypes of RA found in human patients, providing a basis for pathophysiological studies and targeted interventions.\u003c/p\u003e","manuscriptTitle":"A Complete Freund’s Adjuvant-Induced Murine Model as an Experimental Platform to Explore Systemic Inflammation in Rheumatoid Arthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 10:39:56","doi":"10.21203/rs.3.rs-8523680/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6ec779f-6453-4aab-a8fc-7c3d32f75689","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T12:11:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 10:39:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8523680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8523680","identity":"rs-8523680","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}