Synergistic Effects of Nutritional Formula on Joint Inflammation Through Modulation of Bone Metabolism in Rats

TL;DR

A nutritional formula combining turmeric, glucosamine, bone powder, and collagen reduces joint inflammation and improves joint health in rats by modulating bone metabolism and inflammation.

Contribution

The study demonstrates a synergistic effect of a multi-component nutritional formula on joint inflammation through modulation of bone metabolism.

Findings

The formula reduced inflammation-related cytokines and oxidative stress in rats.

Micro-CT showed joint architecture restoration and improved physiological status.

In vitro analysis confirmed reduced MMP13 and CTX-1 levels, indicating slowed joint deterioration.

Abstract

Background: Joint inflammation is significantly connected with progressive joint deterioration, potentially increasing the incidence of persistent major clinical challenges and global disability. Nutrient-based preventive strategies have been explored to investigate the interventive efficacy of the proposed prescribed formula for joint inflammation. However, the synergistic ameliorative effects of the nutritional formula should be evaluated to investigate its impact on joint inflammation. Methods: A prescribed formula including turmeric (T), N-acetylglucosamine (G), enzymatically hydrolyzed bone powder (E), and undenatured type II collagen (U) was comprehensively evaluated for its synergistic effects on joint inflammation and the underlying mechanisms. A rat model established using the Hulth method was used to evaluate the interventive effects in vivo. Moreover, in vitro analysis using the murine chondrogenic cell line ATDC5 was performed to validate the intervention and its mechanism of action. Results: The prescribed formula was shown to synergistically reduce levels of inflammation-related cytokines, reduce oxidative stress, and enhance bone metabolism to promote joint regeneration. Micro-Computed Tomography (Micro-CT) analysis revealed restoration of joint architecture and ameliorated physiological status upon formula intervention. In vitro analysis further validated the synergistic alleviation of inflammation and oxidation, as well as reductions in MMP13 and CTX-1 levels, which implies that modulating bone metabolism alleviates the deterioration and inflammation of joint architecture. Conclusions: The synergistic formula in this study achieves synchronous modulation of several core pathological pathways, yielding synergistic modulation of joint inflammation. Nutrient-based interventions or preventive strategies show promising effects against joint inflammation and progressive mechanistic deterioration.

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Full text

1. Introduction

Joint inflammation refers to local inflammatory manifestations induced by infection, metabolic abnormalities, or autoimmune reaction [1]. With the increasing prevalence of exercise, knee swelling emerges as a common manifestation of widespread joint deterioration, accompanied by significant inflammatory responses that may increase the incidence of osteoarthritis (OA), a major clinical challenge and a leading cause of global disability [2,3,4]. This pathological triad results in motion constraint, functional impairment, and a substantially diminished quality of life for millions worldwide [5]. Escalating participation in recreational and competitive sports, coupled with an aging demographic, has significantly amplified the prevalence of joint injury and post-traumatic OA, thereby expanding the at-risk population [6]. Current pharmacologic mainstays, including non-steroidal anti-inflammatory drugs and intra-articular corticosteroid injections, offer predominantly symptomatic and transient relief. Their long-term utility is critically limited by a well-documented profile of systemic adverse effects and a fundamental inability to halt or reverse the underlying structural deterioration of joint tissues [7,8,9,10]. Thus, it is drastically necessary to investigate innovative strategies that not only alleviate symptoms but also fundamentally provide early prevention of joint inflammation.

The pathophysiological landscape of joint inflammation or OA is recognized as a complex, multifactorial interplay among inflammatory, metabolic, mechanical, and immunological processes [11,12]. Central to this network is a self-perpetuating cycle of low-grade inflammation, driven by pro-inflammatory cytokines such as IL-1β and TNF-α, which promotes overexpression of matrix-degrading enzymes and suppresses the synthesis of essential cartilage components, like type II collagen and aggrecan [13]. Concurrently, oxidative stress and dysregulated immune responses further exacerbate tissue damage and impair repair mechanisms. This intricate network of interacting pathways reveals a critical limitation of mono-therapeutic approaches: targeting a single node within this web is inherently insufficient to arrest the disease process. This insight has catalyzed a paradigm shift in therapeutic development towards multi-targeted strategies capable of concurrently addressing several pathological axes.

Within this conceptual framework, evidence-based nutritional interventions offer a promising avenue for multi-targeted intervention. Several natural compounds have demonstrated targeted activity against specific joint inflammation. Curcumin, the primary bioactive polyphenol in turmeric (T), is a potent inhibitor of the NF-κB signaling pathway, a master regulator of inflammation and matrix catabolism [14]. N-Acetylglucosamine (G) serves as an indispensable biosynthetic precursor for glycosaminoglycans, directly supporting chondrocyte anabolic activity and the restoration of the cartilage matrix architecture [15]. Enzymatically Hydrolyzed Bone Meal (E) provides a highly bioavailable source of microcrystalline hydroxyapatite and collagen peptides, which are crucial for maintaining subchondral bone mineral density and mechanical competence, thereby indirectly stabilizing the overlying cartilage [16]. In a distinct immunomodulatory approach, undenatured type II collagen (UC-II, U) has been shown to promote oral immune tolerance, potentially reducing the autoimmune component of OA pathogenesis by modulating dendritic and T-cell responses to native type II collagen [17,18,19,20,21]. While human trials and meta-analyses have substantiated the safety and modest efficacy of these individual agents, their isolated use likely represents a suboptimal approach to addressing OA’s complexity. The strategic integration of such compounds, possessing complementary and non-overlapping mechanisms, is a logical step forward, mirroring successful combination therapies in oncology and cardiology and holding the potential to unlock synergistic efficacy. However, key questions regarding their prescribed formula, cellular biocompatibility, and integrated effects on chondrocyte viability, inflammation, and matrix homeostasis remain entirely uninterpreted.

Herein, a comprehensive study was conducted using a prescribed formula including T, G, E, and U to evaluate their synergistic effects on joint inflammation and its underlying mechanisms. The enhanced interventive effect of the formula on joint inflammation was demonstrated in a rat model established using the Hulth method. The prescribed formula synergistically decreased the level of inflammation-related cytokines, reduced oxidative stress, and enhanced bone metabolism to promote joint regeneration. Micro-Computed Tomography (Micro-CT) analysis revealed the restoration of joint architecture and the ameliorated physiological status upon formula intervention. In vitro analysis further validated the synergistic alleviation of inflammation and identified the detailed mechanism, showing that decreased MMP13 expression helps prevent the deterioration of joint architecture. Thus, we demonstrate the synergistic effects of the investigated formula on joint inflammation, potentially providing preventive strategies for progressive joint deterioration or OA.

2. Materials and Methods

2.1. Reagents

The individual components of the synergistic formula were sourced as follows: T was purchased from Indena SpA (Milan, Italy), G was purchased from Shandong Runde Biotechnology (Taian, China), E was purchased from Ovita Biotech (Wuhan, China), and U was purchased from Bioiberica, S.A.U (Esplugues de Llobregat, Spain). RIPA lysate and TRIzol reagent were purchased from Solarbio (Beijing, China). Anti-rabbit aggrecan, the anti-rabbit MMP13, anti-rabbit anabolic markers collagen type II (COL2A1), and anti-rabbit GAPDH were purchased from Proteintech (Wuhan, China). Nitrocellulose blotting membrane was purchased from Millipore (Billerica, MA, USA). HRP-conjugated secondary antibody and ECL Western blotting substrate were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Recombinant mouse IL-1β was purchased from UA Bioscience (Nanjing, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Waltham, MA, USA). ATDC5 murine chondrogenic cells were obtained from Shangcheng Beinachuanglian Biotechnology (Xinyang, China).

2.2. Animals and Treatment

Male Sprague–Dawley (SD) rats (8 weeks old, 250 ± 5 g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animal study protocol was approved by the Institutional Animal Committee of Nankai University (protocol code SYXK-2019-0001). All animal procedures were conducted in accordance with the National Institutes of Health (NIH) guidelines and the ARRIVE reporting standards. A total of 64 SPF Sprague Dawley rats were randomly divided into 8 groups (n = 8 per group) using a random number table: Sham group, Model group, Turmeric group (T), Enzymatically Hydrolyzed Bone Meal group (E), N-Acetylglucosamine group (G), Undenatured Type II Collagen group (U), TEU combination group (TEU), and UEG combination group (UEG) (Table S1). After a seven-day acclimation period, post-traumatic osteoarthritis (PTOA) was induced in the right knee joint via the Hulth method. Then, the rats were anesthetized with an intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg). Under aseptic conditions, a medial parapatellar incision was made to expose the knee joint cavity. The anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) were transected, followed by transection of the medial collateral ligament (MCL). The medial meniscus was then completely excised, with meticulous care taken to preserve the integrity of the articular cartilage surfaces. The joint capsule, muscle, and subcutaneous tissue were sutured in layers with absorbable sutures, and the skin was closed. Sham-operated control rats underwent an identical surgical exposure of the knee joint, including capsulotomy, but without any ligament transection or meniscectomy. All animals received a single subcutaneous injection of buprenorphine (0.05 mg/kg) for postoperative analgesia and erythromycin ointment was applied to the sutured skin. Daily observation for signs of infection or discomfort was conducted.

On the first postoperative day, Hulth rats were randomly assigned to 6 treatment groups, a Sham surgery group, and a Model group for control (n = 8). The intervention group received daily gavage of the corresponding formula for 28 consecutive days, while the Sham and Model groups received the vehicle (0.5% CMC-Na, 5 mL/kg) during treatment. Body weight and general health state were monitored regularly.

2.3. Sample Collection and Processing

Rats were deeply anesthetized twenty-four hours after the final administration. Blood was collected via retro-orbital sinus bleeding, allowed to clot at room temperature for 30 min, and then centrifuged at 3000 rpm for 15 min. Serum aliquots were immediately frozen and stored at −80 °C for subsequent biochemical analyses. Following euthanasia by anesthetic overdose, the right knee joints were carefully dissected, stripped of excessive musculature while preserving the joint capsule, and fixed in 4% paraformaldehyde solution for 48 h at 4 °C for subsequent imaging and histological processing.

2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

Serum and cell culture supernatant samples were collected for analysis of inflammatory cytokines, oxidative stress, etc. IL-6, IL-18, TNF-α, and CTX-1 levels were determined by ELISA using commercial kits (Jiangsu Meibiao Biological Technology, Yancheng, China), according to the manufacturer’s protocol. Oxidative stress was evaluated by determining the levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) using assay kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Standard curves were generated to calculate the concentrations of corresponding targets based on absorbance values at 450 nm (OD_450nm_) measured with a microplate reader (TECAN Infinite M200 Pro NanoQuant, Männedorf, Switzerland).

2.5. Micro-Computed Tomography (Micro-CT)

The intact right femur was fixed in formalin for at least 2 days and scanned using micro-CT. High-resolution micro-CT was performed using the NMC-200 system (Quantum FX; PerkinElmer, Waltham, MA, USA) at a voltage of 90 kV, a current of 180 μA, and a resolution of 20 μm/pixel to evaluate microstructural changes in the subchondral bone of the rat knee joint. Data was acquired using the cruiser software (V2.5.0) with an axial field of view (FOV) of 16.014 mm, reconstructed voxel size of 0.051 × 0.051 × 0.051 mm^3^, and a spatial resolution better than 7.5 μm. Quantitative parameters, including bone mineral density (BMD, g/cm^3^), bone volume fraction (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm), were calculated from the reconstructed 3D images using the manufacturer’s dedicated analysis software of Recon (V2.3).

2.6. Histopathological Examination

Knee joints were decalcified in 10% ethylenediaminetetraacetic acid (pH 7.4) for 28 days with constant agitation. Tissues were then dehydrated, paraffin-embedded, and sectioned coronally at a thickness of 5 μm. For general morphological assessment, sections were stained with Hematoxylin and Eosin (H&E). To specifically identify osteoclasts and assess bone resorption activity, adjacent sections were stained using a commercial Tartrate-Resistant Acid Phosphatase (TRAP) kit (Sigma-Aldrich, St. Louis, MO, USA).

2.7. Cell Culture and Treatment

The widely used ATDC5 murine chondrogenic cell line was cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO_2_ incubator. Inflammatory chondrocyte injury was established by stimulating the cells with 10 ng/mL recombinant mouse IL-1β for 24 h. Cells were treated with different concentrations of the corresponding formula. The potential cytotoxicity of the test agents was evaluated using Cell Counting Kit-8 (CCK-8) (Yeasen Biotechnology, Shanghai, China) following the steps provided by the manufacturer. The non-cytotoxic concentration range for each reagent was defined as the concentration causing a reduction in cell viability of less than 10%. Each experiment was repeated three times using independent cell cultures from separate batches to ensure biological reproducibility.

2.8. Flow Cytometry

To assess the cytocompatibility and confirm the safety profile of the different compound combinations, cell apoptosis was analyzed using flow cytometry. Following the orthogonal experimental design, chondrocytes were treated with various concentration ratios of the synergistic combination as determined in the preliminary screening. After 24 h of incubation, cells were collected and washed with cold PBS, and then resuspended in 1× binding buffer at a density of 1 × 10^6^ cells/mL. Subsequently, the cell suspension was stained with 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) for 15 min. The cell samples were then analyzed using a flow cytometer (FACSymphony A1, BD Biosciences, San Jose, CA, USA), and the acquired data were processed using FlowJo software (v10.10, FlowJo LLC, Ashland, OR, USA). Each experimental group was analyzed in triplicate, and the data were statistically compared to the untreated control group to evaluate the impact of the different combinatorial ratios on chondrocyte survival.

2.9. Western Blot and Fluorescent Analysis

The total protein extract was prepared by treating ATDC5 cells of different groups with RIPA lysis buffer. The protein was separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose (NC) membrane for subsequent Western blot analysis. Three independent biological replicates were run simultaneously for each group, and the corresponding membranes of the various target proteins were incubated with the corresponding primary antibodies against COL2A1 (1:1000), MMP13 (1:500), and GAPDH (1:1000) at 4 °C overnight. The membranes were washed with PBST three times, followed by incubation with HRP-conjugated goat anti-rabbit IgGs (1:5000) for 1 h at room temperature. After washing, the target proteins were detected using an ECL Western blotting substrate and visualized using chemiluminescence with a ChemiDoc MP imaging system (BIO-RAD, Hercules, CA, USA) after optimized exposure. The protein levels were quantified by determining the gray value of the bands using Image J (v1.54).

Immunofluorescence staining for type II collagen was conducted to visualize the synthesis and distribution of a key cartilage matrix component. Cells were fixed, permeabilized, blocked, and incubated overnight with a primary antibody against type II collagen (1:200). After washing, the cells were incubated with an appropriate fluorophore-conjugated secondary antibody, and the nuclei were counterstained with DAPI. Fluorescence images were captured using a confocal laser scanning microscope (Leica TCS SP8).

2.10. Statistical Analysis

All quantitative data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (v8.4.3, San Diego, CA, USA). The normality of data distribution for each group was verified using the Shapiro–Wilk test. For comparisons of parameters among the nine in vivo experimental groups or multiple in vitro treatment groups, one-way analysis of variance (ANOVA) was applied. When ANOVA indicated a statistically significant difference (p < 0.05), Tukey’s honestly significant difference (HSD) post hoc test was used for all pairwise comparisons between group means. A two-tailed p-value of less than 0.05 was considered statistically significant for all tests.

3. Results

3.1. Effects of SF on Joint Inflammation in Rats

As illustrated in Figure 1A, the beneficial effects of the prescribed formula on joint inflammation were analyzed in rats aged 8 weeks after establishing the Hulth model. The doses of the corresponding supplements were determined based on the recommended daily nutrient intake (40 mg for U, 300 mg for G, and 1000 mg for T and E) and the previous experiences to allow the appropriate determination of administration in rats. Daily observations of rat weight and behavioral abnormalities were recorded to evaluate the potential side effects of the SF. The results indicate that neither the Hulth method nor the intervention with the SF had a significant impact on body weight (Figure 1B). Similar variations in body weight were observed across groups at the end of the experiment compared to baseline measurements (Figure 1C). After sacrifice, organ indices for the liver, lung, heart, kidney, and spleen were analyzed and indicated no statistically significant variations (Table S2).

Serological analysis indicated the presence of systemic inflammation and oxidative stress in Hulth rats. Compared with the Sham group, the Model group showed significantly elevated serum levels of pro-inflammatory cytokines, including IL-6, IL-18, and TNF-α. Increased oxidative stress was demonstrated by decreased levels of SOD and GSH, along with increased ROS and MDA. After intervention, restoration of inflammation-related cytokines was observed with a significant synergistic effect of the SF (Figure 1D–F). Moreover, increased levels of SOD and GSH confirmed the synergistic effects of the SF on reducing oxidative stress (Figure 1G,H), along with decreases in MDA and ROS (Figure 1I,J). These results systematically demonstrate the alleviating effect of the interventive supplements on joint inflammation, with a significant synergistic effect. The mean values of each group and the significance statistics are shown in Table S3.

3.2. Effect of SF on Joint Microstructure Degeneration

Micro-CT and 3D reconstruction analyses revealed pronounced structural deterioration in the knee joints of the Model group, characterized by increased bone porosity, apparent bone loss, and erosion on articular surfaces. These damages were ameliorated in interventive groups, as evidenced by reduced porosity and smoother articular surfaces. Notably, the TEU group achieved the most significant restoration of joint architecture and inhibition of abnormal osteophyte formation, where the structure recovered to a state comparable to that of the Sham group (Figure S1). As indicated in Figure 2A, pathological osteophyte formation was successfully induced by the modeling procedure. The presence of smaller osteophytes in interventive groups indicates that the treatments effectively inhibited osteophyte growth and mitigated associated pathological alterations. Two-dimensional image analyses further demonstrated that the Model group exhibited decreased BMD, BMC, BV, BV/TV, Tb.N, and Tb.Th compared with the Control group, accompanied by cartilage thinning and increased bone surface area (Figure 2B–G). Although no statistically significant differences were observed in subchondral bone area, BV, or BMD among the groups, an increase in BV/TV was noted in the interventive groups, suggesting mild cortical thickening. While the Model group displayed evident bone loss and trabecular sparsity, these structural abnormalities were improved following intervention (Figure 2H,I). Generally, the SF exhibited only mild effects on bone elemental characteristics during the experimental period, with a more significant impact identified on joint microstructure and mechanistic restoration.

3.3. Modulation of SF on Bone Metabolism and Regeneration

Articular cartilage morphology was assessed using H&E staining. Compared with the Sham group, the Model group exhibited severe cartilage degeneration, characterized by surface fibrillation, fissures, erosion, disorganized chondrocytes, and marked inflammatory infiltration (Figure 3A). These alterations indicate disrupted cartilage matrix homeostasis. Histological evaluation revealed improved cartilage structure in the therapeutic intervention groups. Treated groups, particularly the TEU combination group, displayed smoother surfaces, orderly chondrocyte alignment, and reduced infiltration. Mankin scoring (Table S3) confirmed successful modeling and therapeutic efficacy. The total score was significantly higher in the Model group than in the Sham group. Intervention significantly reduced the total score, with the most pronounced effects on surface integrity and chondrocyte preservation (Figure S2).

Joint inflammation progression involves subchondral bone remodeling. TRAP staining revealed dense clusters of TRAP-positive multinucleated osteoclasts in the subchondral bone of the Model group (Figure 3B, arrow), indicating elevated bone resorptive activity associated with sclerosis, micro-fractures, and cyst formation. SF intervention markedly reduced the number of TRAP-positive cells in this region, with the UEG synergistic formula group showing significant effects. This suggests that the treatments not only protected cartilage but also inhibited abnormal subchondral bone resorption, likely contributing to osteochondral unit homeostasis.

3.4. Effects of SF on Cell Inflammation and Oxidative Stress

The beneficial effects of the SF were assessed in vitro after establishing inflammatory ATDC5 cells with IL-1β. Cell viability was defined after incubation with different concentrations of individual supplements (0.1–100 μg/mL). The results indicate that the investigated nutrients showed no significant cytotoxicity, with U, T, and G enhancing cell viability (Figure 4A). These non-cytotoxic ranges guided the sequential design for identifying synergistic ratios and selecting the high and low concentrations for subsequent experiments.

Analysis of inflammation-related cytokines revealed that the Model group exhibited significantly elevated levels of pro-inflammatory factors, including IL-6, IL-18, and TNF-α. These factors were reduced to varying degrees after treatment (Figure 4B–G), with both high- and low-dose UEG groups significantly lowering the level of TNF-α. The pronounced effects of TEU on related cytokines demonstrated its synergistic activity against inflammation. The Model group exhibited enhanced oxidative stress, as reflected by decreased SOD and GSH levels and elevated MDA. This increased oxidative stress was ameliorated after SF intervention, confirming its synergistic effects on redox balance (Figure 4H–M). Notably, while all treatments reduced oxidative stress markers, the low-ratio formulation demonstrated a significantly stronger effect in elevating SOD levels compared to the high-ratio formulation. The significant improvements in oxidative stress-related indices by TEU or UEG demonstrate a clear synergistic effect.

Flow cytometric analysis confirmed increased cell apoptosis in the Model group. The intervention groups demonstrated significantly reduced apoptosis, with the enhanced alleviative effect demonstrating the superior efficacy of the SF (Figure 4N,O). These results confirm that the bioactive compounds, at different concentrations, exert anti-inflammatory and anti-oxidative effects, collectively establishing the cytocompatibility and functional efficacy of the optimized formulations. The mean values of each group and the significance statistics are shown in Table S4.

3.5. Regulation of SF on Joint Inflammation Through Modulation of Bone Metabolism

Immunofluorescence staining in ATDC5 cells visually demonstrated that the fluorescence signal of type II collagen was weakened and its distribution was sparse in the cells and matrix of the Model group. After intervention, both the signal intensity and the deposition range of type II collagen were markedly enhanced and improved, especially in the groups with supplemented TEU and UEG (Figure 5A). Western blot results provided further protein-level evidence related to the regulatory effects of SF, as well as the supporting information of uncropped graphs in Figure S3. Though individual bioactive compounds led to decreased levels of COL2A1, it was markedly restored to near-normal levels in the UEG group, demonstrating its synergistic effect (Figure 5B,C). The results indicate that the expression of the catabolic enzyme MMP13 was significantly downregulated after intervention with the SF (Figure 5B,D). The decreased expression of MMP13 after SF intervention indicates the underlying mechanism of matrix degradation, which is closely correlated with bone formation. Serological analysis indicated that the elevated serum level of the bone resorption marker CTX-I in the Model rats was effectively reduced after intervention (Figure 5E). Collectively, these multi-level results demonstrate that the SF comprehensively ameliorated joint inflammation by restoring anabolic–catabolic balance in cartilage and concurrently modulating abnormal subchondral bone resorption and formation.

4. Discussion

Joint deterioration is a complex degenerative disease affecting the entire joint architecture. Its effective management requires simultaneous intervention across multiple interconnected pathological processes, including structural destruction, metabolic imbalance, and a pro-inflammatory microenvironment [22,23]. This study systematically evaluated the comprehensive interventive effects of a multi-component synergistic formulation on joint inflammation through combined in vivo and in vitro experiments. The results indicate that the formulation attenuated macrostructural and histopathological joint damage through multi-target, disease-modifying effects at the systemic, tissue, and molecular levels. This was achieved by inhibiting the inflammation–oxidative stress axis and coordinately regulating the metabolism of the cartilage matrix alongside the remodeling of subchondral bone [24]. The synergistic effects of the SF were assessed using Bliss independence analysis to identify interactions between treatments and the combination groups (TEU and UEG). As summarized in Tables S5 and S6, the results indicate that the UEG group consistently exhibited synergistic effects on inflammatory markers and antioxidant markers in both in vivo and in vitro settings, particularly under high-concentration conditions. TEU combination results suggest that the absence of G may compromise formulation efficacy. MDA was the only indicator where both combinations exhibited antagonism, suggesting that lipid peroxidation may involve different regulatory mechanisms. The consistent synergistic performance of UEG across multiple inflammatory and oxidative stress markers highlights the critical role of G in enabling beneficial component interactions.

The structural integrity of the joint is fundamental to its normal biomechanical function. Micro-CT analysis in this study revealed that Hulth rats developed significant subchondral bone loss, trabecular bone thinning, and erosion of the articular surface—alterations that directly compromise joint mechanical stability [25,26,27]. Intervention with the SF, particularly in the most effective treatment group (TEU), effectively reversed this pathological structural damage, promoted the repair of bone microstructure, and inhibited the formation of abnormal osteophytes. Notably, although joint characteristics such as bone area, bone volume, and bone mineral density of the subchondral bone did not show statistically significant changes, the consistent improvement in the bone volume fraction (BV/TV), coupled with evident restoration of three-dimensional morphology, suggests that the intervention may have primarily enhanced the quality and spatial architecture of the bone microstructure rather than merely increasing its mineralization. This beneficial effect is crucial for reconstructing a mechanically robust subchondral bone microstructure.

The SF intervention exhibited multi-dimensional protective effects on joint inflammation. Importantly, tartrate-resistant acid phosphatase staining further revealed the formulation’s capacity to effectively suppress abnormally activated osteoclasts in the subchondral bone region, thereby directly intervening in a core cellular event driving bone remodeling imbalance [28]. Corroborating systemic evidence was provided by serum biomarker analysis, which demonstrated that the formulation concurrently reduced levels of multiple pro-inflammatory cytokines and mitigated key indicators of oxidative stress. A significant increase in bone resorption marker CTX-1 was observed upon inflammation, and SF intervention drastically restored its level, indicating that the formulation likely acts via a common upstream regulatory mechanism related to bone adsorption and metabolism. It is noteworthy that although trends toward improved bone microarchitecture were observed, some quantitative parameters, such as BMD and BV, did not reach statistical significance. This discrepancy indicates that the formulation preferentially affects cellular activity and microstructural organization rather than gross mineral accumulation, particularly within the relatively short intervention period employed in this study.

Moreover, in vitro experiments on ATDC5 chondrocytes indicated that the formulation effectively reversed the metabolic imbalance characteristic of joint inflammation. This was manifested by significantly downregulated expression of the pivotal catabolic enzyme MMP13, alongside upregulated expression of essential anabolic components of type II collagen [29]. This bidirectional regulatory effect was consistently validated at protein translation levels. It is well-established that pro-inflammatory cytokines such as IL-1β and TNF-α can activate canonical signaling pathways like NF-κB, which synergistically drives the expression of matrix-degrading enzymes while inhibiting cartilage matrix biosynthesis and exacerbating oxidative stress [30,31,32,33]. The individual components within this SF are posited to work in concert to disrupt this cascade. For instance, curcumin, a known inhibitor of the NF-κB pathway, likely contributes to the primary anti-inflammatory and antioxidant effects. N-acetylglucosamine serves as a crucial biosynthetic precursor for matrix components, and undenatured type II collagen may participate in modulating local immune responses. Together, these constituents target multiple pathways to disrupt the vicious cycle whereby inflammation precipitates matrix degradation, thereby restoring the anabolic capacity of chondrocytes. Furthermore, the formulation’s pronounced inhibition of osteoclast activity strongly suggests an additional intervention in the core signaling axis governing bone resorption [34,35,36,37]. This constitutes a key mechanism for the protection of subchondral bone and maintenance of functional homeostasis within the osteochondral unit.

Although consistent trends observed across multiple analyses and significant differences detected in key outcomes (with effect sizes and 95% confidence intervals reported in the Section 3) support the reliability of our main findings, this study has limited statistical power to detect small effect sizes. A post hoc consideration of statistical power indicates that the current design is primarily sensitive to moderate-to-large effects, and subtle differences may remain undetected. Therefore, the results should be interpreted in conjunction with variability and confidence interval widths, and non-significant findings should not be regarded as definitive evidence of absence. Larger sample sizes and independent validation will be required in future studies to strengthen statistical robustness. The Hulth model represents an aggressive post-traumatic osteoarthritis phenotype, which may exaggerate inflammatory and degenerative responses compared to the more heterogeneous and slowly progressing joint disease observed in the general population. Therefore, the magnitude of the effects observed may not directly reflect clinical outcomes. In addition, despite consistent trends observed in animal and cell models, the relatively short duration of intervention limits conclusions regarding long-term joint protection or regeneration. Structural remodeling processes typically require a longer time frame; thus, longer-term studies are needed. Additionally, translating multi-component nutritional formulations into clinical practice poses further challenges, including variability in absorption, metabolism, and individual optimal dosing. Therefore, before making clear clinical recommendations, it is essential to conduct long-term safety and efficacy assessments in well-designed randomized clinical trials.

In conclusion, the SF achieves synchronous modulation of several core pathological pathways in this study. This multi-target, multi-pathway interventive strategy aligns closely with the complex etiology of joint inflammation and the contemporary need for preventive management. Nutrient-based interventions or preventive strategies show promise for addressing joint inflammation and progressive deterioration. While the findings are encouraging, they should be interpreted within the context of the study’s limitations. Future studies integrating mechanistic investigations and clinical validation will be critical to further elucidate the therapeutic potential of the formulation.

5. Conclusions

We have investigated the effects of a synergistic formula comprising turmeric (T), N-acetylglucosamine (G), enzymatically hydrolyzed bone powder (E), and undenatured type II collagen (U) on joint inflammation and its underlying mechanisms. In vivo analysis in a rat model established with the Hulth method demonstrated the synergistic effects of the prescribed formula, validating the benefits of complexed nutrients for joint inflammation. Micro-CT analysis revealed restoration of joint architecture and physiological status rather than changes in BMC or similar measures. In vitro analysis confirmed synergistic alleviation of inflammation and oxidation, as well as the underlying mechanism involving modulation of bone metabolism. Therefore, it is possible that nutrient complexes may have enhanced, synergistic beneficial effects on health or diseases. However, these findings should be interpreted with caution due to several limitations, including the inherent constraints of the animal model, the relatively small sample size, and the short observation period. These factors highlight the need for further investigation with improved study designs before any clinical recommendations can be made. In conclusion, nutrient-based interventions or preventive strategies show promise for addressing joint inflammation and progressive mechanistic deterioration.