Abstract Osteoarthritis (OA), the fourth leading cause of global disability, is marked by progressive cartilage loss, synovial inflammation, and dysregulated bone remodeling. Current therapeutic strategies are hindered by systemic adverse effects and insufficient intra-articular drug bioavailability, underscoring the demand for enhanced delivery mechanisms. In this study, we developed a CeO[2] nanozyme-driven gel system synergistically integrated with the herbal monomer rhein (RH). The coordinatively unsaturated sites on the CeO[2] nanozymes triggered RH self-assembly into a gel through surface coordination interactions. Comprehensive characterization demonstrated that the CeO[2]/RH gels exhibit synergistic reactive oxygen/nitrogen species (RONS)-scavenging capability, leveraging both the antioxidant enzyme-mimetic activity of CeO[2] and the intrinsic properties of RH. The redox-modulatory activity of CeO[2]/RH gels promoted macrophage polarization from M1 pro-inflammatory to M2 anti-inflammatory phenotypes through phenotypic reprogramming. This immunomodulatory shift substantially reduced inflammatory mediator secretion in macrophages, thereby suppressing chondrocyte apoptosis and senescence through downregulation of the IL-6/JAK2/STAT1 signaling axis via macrophage-chondrocyte crosstalk. In vivo validation using a modified Hulth-induced rat OA model demonstrated the CeO[2]/RH gels’ therapeutic superiority. Collectively, our findings establish the CeO[2]/RH gels as a synergistic therapeutic platform for OA intervention, demonstrating dual competency in RONS-scavenging precision and immunomodulatory microenvironment remodeling through redox-biology-mediated cellular cross-talk regulation. Keywords: Osteoarthritis, Synovial macrophages, Gel, Enzyme-mimetic activity, Anti-inflammatory Graphical abstract Image 1 [37]Open in a new tab 1. Introduction Osteoarthritis (OA), a prevalent degenerative joint disease affecting approximately 250 million people worldwide, constitutes the fourth leading cause of disability [[38]1]. This condition manifests distinct pathological characteristics including articular cartilage degradation, aberrant angiogenesis, subchondral bone remodeling, osteophyte development, and synovial membrane inflammation [[39]2]. Although current therapeutic strategies such as nonsteroidal anti-inflammatory drugs (NSAIDs) and hyaluronic acid (HA) provide symptomatic relief, their efficacy is transient. Moreover, their clinical utility is substantially constrained by dose-dependent adverse effects, particularly in chronic disease management [[40]3,[41]4]. This biological constraint underscores the critical need to develop novel drug delivery systems capable of implementing multi-targeted therapeutic strategies. Emerging evidence has delineated macrophages as central regulators in osteoarthritis pathogenesis, with recent studies establishing their dual role in both initiating and amplifying inflammatory cascades [[42]5,[43]6]. Mechanistic investigations reveal that microenvironmental oxidative stress, quantified by elevated reactive oxygen/nitrogen species (RONS) levels, drives phenotypic reprogramming through redox-sensitive signaling pathways [[44]7]. Crucially, this polarization establishes a self-perpetuating inflammatory loop wherein RONS-generated oxidative stress and cytokine secretion reciprocally reinforce macrophage activation [[45]8]. This sustained inflammatory response accelerates OA progression by triggering cellular apoptosis and irreversible joint tissue degeneration [[46]9]. Consequently, modulating RONS homeostasis within the synovial microenvironment alongside macrophage polarization regulation represents a promising strategy for managing and intervening in OA-associated inflammatory processes. Nanozymes, engineered nanomaterials exhibiting intrinsic enzyme-mimetic activities, present distinct advantages over natural enzymes through their enhanced stability and cost-effectiveness [[47]10,[48]11]. These artificial enzymes demonstrate particular efficacy in scavenging reactive oxygen species (ROS) by emulating antioxidant defense systems such as catalase (CAT) and superoxide dismutase (SOD) [[49]12,[50]13]. Among diverse nanozymes, cerium oxide (CeO[2]) nanozymes stand out as excellent candidates for enzyme-mimicking applications due to their unique chemical properties. However, the clinical translation of CeO[2] nanozymes for OA treatment remains challenging due to their tendency to aggregate under physiological conditions and their suboptimal biodistribution [[51]14]. To overcome these limitations, improving intra-articular retention of CeO[2] nanozymes is crucial, as it directly impacts their sustained therapeutic efficacy. In this context, hydrogel-based delivery systems have emerged as a promising strategy, leveraging their injectability, defect-conforming adaptability, and structural similarity to the native extracellular matrix [[52]15]. Their three-dimensional porous networks provide a hydrated microenvironment that promotes cellular infiltration while simultaneously acting as a versatile platform for sustained and localized drug releasee [[53]16]. Thus, integrating CeO[2] nanozymes into hydrogel matrices offers a rational strategy to synergize ROS-neutralizing capabilities with cartilage-protective scaffolding functions, forming a multifunctional therapeutic paradigm for OA intervention. Recent advances in supramolecular chemistry have enabled the direct self-assembly of natural herbal small molecules into functional hydrogels while preserving their native chemical structures [[54]17]. These herb-derived hydrogels demonstrate intrinsic self-delivery properties, conferring enhanced solubility, improved therapeutic precision, and reduced cytotoxicity compared to conventional synthetic hydrogel systems. Pioneering studies have validated the feasibility of this strategy by demonstrating the supramolecular assembly of diverse herbal bioactive constituents, including polysaccharides, flavonoids, and terpenoids [[55][18], [56][19], [57][20]]. Of particular interest is rhein (RH), a bioactive anthraquinone primarily derived from Rheum palmatum L. (Dahuang in traditional Chinese medicine), which exhibits broad-spectrum bioactivities encompassing anti-inflammatory, antioxidant, and tissue-protective effects [[58]21,[59]22]. Notably, Yang et al. demonstrated RH's capacity for spontaneous hydrogel formation via non-covalent molecular interactions [[60]23]. Building upon this foundation, we propose a synergistic gel system integrating RH's inherent therapeutic properties with CeO[2] nanozyme functionality, designed to address the multifactorial pathogenesis of OA through combined pharmacological and bioengineering strategies. Here, we developed a catalytic gel system combining CeO[2] nanozymes with RH that demonstrated enhanced therapeutic outcomes for OA. As established in prior research [[61]23], RH demonstrates self-assembly capability into a gel state at concentrations exceeding the critical gelation threshold of 5 mg/mL. In contrast, our CeO[2]-integrated system achieved gelation at a significantly lower RH concentration (2 mg/mL), with surface coordination interactions identified as the dominant mechanistic driver of enhanced assembly efficiency. This assembly strategy effectively mitigated the dose-dependent toxicity associated with conventional high-concentration RH gel formulations. Subsequently, the resultant CeO[2]/RH composite gels underwent comprehensive physicochemical characterization. Crucially, the synergistic integration of CeO[2] and RH conferred exceptional RONS-scavenging capacity, validated across both in vitro assays and in vivo models. The redox-modulatory activity of the system drove phenotypic reprogramming, effectively promoting macrophage polarization from M1 pro-inflammatory to M2 anti-inflammatory phenotypes. This immunomodulatory shift substantially reduced inflammatory mediator secretion in macrophages, thereby suppressing chondrocyte apoptosis and senescence through downregulation of the IL-6/JAK2/STAT1 signaling axis via macrophage-chondrocyte cross-communication. In vivo validation using a modified Hulth-induced rat osteoarthritis model demonstrated the CeO[2]/RH gels’ therapeutic superiority, with histopathological analyses revealing marked improvements in articular cartilage integrity. Collectively, our findings establish the CeO[2]/RH gels as a synergistic therapeutic platform for OA intervention ([62]Scheme 1), demonstrating dual competency in RONS-scavenging precision and immunomodulatory microenvironment remodeling through redox-biology-mediated cellular cross-talk regulation. Scheme 1. [63]Scheme 1 [64]Open in a new tab Schematic diagram delineating the therapeutic mechanism of CeO[2]/RH gels in OA management, created with MedPeer (medpeer.cn). 2. Materials and methods 2.1. Preparation of CeO[2]/RH gels CeO[2] nanoparticles (CeO[2] NPs) (8 mg) and RH (2 mg) were mixed in a 1.5 mL EP tube, followed by the addition of 1 mL of 0.2 M NaHCO[3] solution. The mixture was ultrasonicated (100 W, 5 min) to achieve uniform dispersion, resulting in the formation of the CeO[2]/RH self-assembled gels. 2.2. Intracellular RONS detection Intracellular ROS and RNS levels were monitored using fluorescent probes. DCFH-DA and DAF-FM DA were employed to detect ROS and RNS, respectively, based on the oxidation of DCFH to fluorescent DCF and the fluorescence response of DAF-FM. RAW264.7 cells were pretreated with lipopolysaccharide (LPS, 1 μg/mL) for 24 h, incubated with the probes at 37 °C for 30 min, and washed three times with base medium to remove residual dye. Fluorescence imaging was performed using laser scanning confocal microscopy (excitation: 488 nm, emission: 525 nm). 2.3. EdU analysis for cell proliferation RAW264.7 cells were seeded in confocal dishes and cultured overnight. After pretreatment with 300 μM H[2]O[2] for 4 h under designated conditions, the supernatant was removed, and the cells were incubated with 10 μM EdU at 37 °C for 2 h. Subsequently, cells were fixed with 4 % paraformaldehyde (15 min, room temperature), permeabilized with 0.5 % Triton X-100, and stained with click reaction solution and DAPI according to the manufacturer's protocol (30 min, room temperature). After three washes with PBS, EdU-labeled DNA was visualized by laser scanning confocal microscopy. 2.4. Senescence-associated β-galactosidase staining Cellular senescence was assessed using the SA-β-galactosidase (SA-β-gal) staining kit. Briefly, treated cells were seeded in 6-well plates, washed twice with PBS, and fixed with β-galactosidase fixative (1 mL per well, 15 min, room temperature). After three PBS washes, cells were incubated with β-Gal staining working solution (1 mL per well) at 37 °C for 16 h. SA-β-gal activity was quantified by bright-field microscopy imaging. 2.5. Measurements of cytokines Cytokine levels were quantified by ELISA using commercially available kits. Briefly, RAW264.7 cells were pretreated with LPS for 24 h, and cell culture supernatants were collected. A standard curve was generated according to the manufacturer's protocol. Absorbance was measured at 450 nm using a microplate reader, and cytokine concentrations were calculated based on the standard curve. 2.6. Western blot analysis Protein expression levels of p-p65, p-JAK2, and p-STAT1 were analyzed by western blotting. Treated cells were lysed with RIPA buffer containing protease/phosphatase inhibitors. After 30 min of ice-cold lysis, lysates were centrifuged (12,000×g, 15 min, 4 °C), and supernatants were collected. Protein concentrations were quantified using a BCA assay kit. Equal amounts of protein (20–50 μg) were denatured in 6 × Laemmli buffer by boiling at 100 °C for 5 min, separated via 10 % SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5 % (w/v) non-fat milk in TBST (1 × ) for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against p-p65 (1:1000), p-JAK2 (1:1000), p-STAT1 (1:1000), and β-actin (1:1000). After TBST washes, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 2.5 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents and quantified with ImageLab software. 2.7. Phenotypic analysis of macrophages The expressions of CD86 and CD206 in treated RAW264.7 cells were detected by flow cytometry. A single-cell suspension was prepared following LPS and IL-4 treatment. The cells were then incubated with APC-F4/80 antibody, FITC-CD86 antibody, and PE-CD206 antibody at room temperature for 30 min, protected from light. Finally, the samples were analyzed by flow cytometry. 2.8. Macrophage phenotypic characterization Macrophage polarization was assessed by flow cytometry through detection of CD86 (M1 marker) and CD206 (M2 marker) expression in RAW264.7 cells. Briefly, cells were stimulated with LPS and IL-4 for 24 h to induce polarization, followed by preparation of single-cell suspensions. Cells were stained with APC-conjugated anti-F4/80 (1:200), FITC-conjugated anti-CD86 (1:100), and PE-conjugated anti-CD206 (1:100) antibodies in the dark at 25 °C for 30 min. Flow cytometric analysis was performed using a BD FACSCelesta system, with data processed by FlowJo v10.9 software. 2.9. Therapeutic evaluation of CeO[2]/RH gels in OA rat model All animal procedures were approved by the Animal Ethics Committee of Yangzhou University Medical School (Approval No. 202402928). Female Sprague-Dawley (SD) rats (8-week-old, 180–220 g) underwent OA induction via modified Hulth surgery under Zoletil anesthesia (50 mg/kg, intraperitoneal). The right knee joint was shaved, disinfected with iodophor, and incised longitudinally (2 cm) to transect the medial collateral ligament and anterior cruciate ligament, followed by medial meniscectomy. Postoperative infections were prevented by intramuscular penicillin (80,000 U/day, 3 days). Rats were randomized into five groups (n = 3 per group): (1) Sham (skin incision only); (2) OA (surgery only); (3) OA + CeO[2] NPs (4 mg/kg); (4) OA + RH (1 mg/kg); (5) OA + CeO[2]/RH gels (4 mg/kg CeO[2] + 1 mg/kg RH). Treatments were administered intra-articularly every 14 days from postoperative day 7. At 8 weeks post-treatment, rats were euthanized for knee joint collection. Specimens were fixed in 4 % paraformaldehyde (48 h), decalcified in 10 % EDTA (6 weeks), paraffin-embedded, and sectioned (5 μm). Morphological changes were analyzed through: Micro-CT scanning (SkyScan 1176, Bruker) with 3D reconstruction; Histopathological evaluation via H&E, toluidine blue, and Safranin O/Fast Green staining; Immunohistochemical analysis of cartilage degradation markers. 2.10. Statistical analysis Data were shown as mean ± standard deviation and the statistically significant differences between control and experimental groups were evaluated using Student's t-test for column analyses and Two-way ANOVA for grouped analyses in Prism 9.0 software (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). 3. Results and discussion 3.1. Synthesis and characterization of CeO[2]/RH gels In this study, CeO[2] NPs with antioxidant enzyme-mimetic activity were synthesized using a hydrothermal method according to the previous work ([65]Fig. 1a) [[66]24]. Transmission electron microscopy (TEM) revealed that the CeO[2] NPs exhibited a uniform morphology with an average size of approximately 4 nm ([67]Fig. 1b). To investigate the phase structure of the synthesized CeO[2] NPs, X-ray diffraction (XRD) spectroscopy was employed to analyze the crystal purity and structural information. All diffraction peaks matched the cubic fluorite structure of CeO[2] (PDF#65–2975), specifically at 2θ values of 28.55°, 33.08°, 47.48°, 56.34°, 59.09°, 69.42°, 76.70°, and 79.08°, corresponding to the (111), (200), (220), (311), (222), (400), (331), and (420) crystal planes, respectively ([68]Fig. 1c). The absence of any impurity peaks confirmed the successful synthesis of pure CeO[2] NPs. To further investigate the surface properties of the CeO[2] NPs, Fourier transform infrared spectroscopy (FTIR) was conducted. The FTIR spectrum in [69]Fig. 1d revealed characteristic surface hydration features of CeO[2], with a broad absorption band spanning 3100-3700 cm^−1 indicative of overlapping O-H stretching vibrations from surface hydroxyl groups (Ce-OH) [[70]25]. A distinct peak centered at 1562 cm^−1 further confirmed hydroxyl adsorption through its characteristic bending vibration mode (δ-OH). Next, we attempted to introduce CeO[2] NPs into RH solution. Surprisingly, the gelation was observed in the above mixed system. As shown in [71]Fig. 1e, CeO[2] NPs (8 mg/mL) were added to an RH solution (2 mg/mL) and subjected to ultrasonic treatment, resulting in a fluid-like behavior in about 3 min. After 5 min of ultrasound, the solution quickly transformed into a gel. Notably, the CeO[2]/RH gel exhibited syringe deliverability ([72]Fig. 1f), enabling precise intra-articular injection for targeted therapeutic delivery. Subsequently, scanning electron microscopy (SEM) characterization revealed an interconnected porous microstructure of CeO[2]/RH gels ([73]Fig. 1g). Energy-dispersive spectroscopy (EDS) results demonstrated that C, N, O, and Ce were evenly distributed throughout the gels ([74]Fig. S1). As previous report [[75]23], RH demonstrates direct self-assembly into hydrogels at concentrations exceeding the critical threshold of 5 mg/mL. However, a lower RH concentration (2 mg/mL) was applied in [76]Fig. 1e. This difference in concentration implied that CeO[2] NPs played important role in triggering the gelation of RH. Then, we carefully investigated the gelation condition in the mixed system. As shown in [77]Fig. S2, CeO[2] NPs and RH were mixed at varying concentrations, subjected to 5-min sonication, and monitored for macroscopic phase behavior evolution. Below a CeO[2] concentration of 6 mg/mL, the CeO[2]/RH composite system exhibited Newtonian fluid behavior. Conversely, at CeO[2] concentrations ≥6 mg/mL combined with 2 mg/mL RH, the system underwent spontaneous gelation. These findings revealed a direct correlation between CeO[2] concentration and gel formation. Fig. 1. [78]Fig. 1 [79]Open in a new tab Synthesis and characterization of CeO[2]/RH gels. (a) Scheme illustrating the synthesis of CeO[2] NPs. (b) TEM image of CeO[2] NPs. (c) XRD spectrum of CeO[2] NPs. (d) FTIR spectrum of CeO[2] NPs. (e) Photographs showing the formation of gels. (f) Photograph of injecting CeO[2]/RH gels fluid into PBS. (g) SEM image of CeO[2]/RH gels. (h) Strain-dependent (frequency: 1.0 Hz) and (i) Stress sweep measurements of CeO[2]/RH gels at a frequency of 1.0 Hz. (j) Time sweep at 0.5 % strain and 1Hz frequency. (k) Step-strain detection for three cycles at high strain (30 %) and low strain (0.5 %). (l) Frequency sweep at 0.5 % strain. (m) Viscosity switch in a continuous step rate test with stepwise measurements. (n) Adhesion test of CeO[2]/RH gels to AISI 316L and PTFE. To investigate the pivotal interactions governing the stability of the CeO[2]/RH gels, we systematically evaluated four dispersing agents: urea (hydrogen bond disruptor), Tween 20 (hydrophobic interaction modulator), NaCl (ionic interaction inhibitor), and ethylenediaminetetraacetic acid (EDTA, a metal-chelating agent) [[80]26]. As shown in [81]Fig. S3, even at an elevated concentration of 50 mM Tween 20, the CeO[2]/RH gels retained structural integrity, excluding hydrophobic interactions as the main assembly mechanism. Similarly, no significant sol transition occurred after treatment with 8 M urea, which competitively disrupt weak hydrogen bonds through stronger hydrogen bond formation, or with 1 M NaCl, which screens electrostatic interactions. In stark contrast, incubation with 100 mM EDTA induced complete gel-to-sol transition, which is might attributed to the reduced cross-linking density of the gel after breaking the surface coordination effects between RH and CeO[2]. Previous crystallographic analyses reveal that Ce^4+ ions on the (111) facets of CeO[2] NPs exhibit six coordinatively unsaturated sites [[82]27], which would facilitate the coordination bonding with oxygen-rich functional groups (e.g., -COOH, -OH) in RH. These results conclusively demonstrate that surface coordination interactions, rather than conventional non-covalent interactions, dominates the assembly and stabilization of the CeO[2]/RH gels. Next, the viscoelastic property of the CeO[2]/RH gels was investigated through rheological experiments. As shown in [83]Fig. 1h, at a constant frequency of 1.0 Hz, the storage modulus (G′) was greater than the loss modulus (G″) when the strain was below 10 %, suggesting a gel-like state. However, as the strain exceeded 10 %, G′ decreased below G″, indicating a transition from a gel state to a sol state. The stress sweep curves of CeO[2]/RH gels at a frequency of 1.0 Hz were also measured ([84]Fig. 1i). The mechanical strength of the gels was assessed by the yield stress (τ), defined as the critical shear stress at which the gel structure collapses (intersection of G′ and G″). Below 26 Pa, G′ remains larger than G″, indicating that CeO[2]/RH gels maintain structural integrity under frictional pressure as injectable gels. Additionally, the G′ consistently exceeded the G" ([85]Fig. 1j and k), indicating the structural stability of the CeO[2]/RH gels. Next, to evaluate the strain-induced damage and self-healing properties of CeO[2]/RH gels, a continuous stepwise oscillatory strain sweep was applied between 0.5 % and 30 % (frequency: 1.0 Hz). As shown in [86]Fig. 1l, exposure to a high dynamic strain (30 %) resulted in a significant reduction in the G′ value, indicating partial structural breakdown of the gel network. Remarkably, when the strain was reduced to 0.5 %, the G′ value rapidly recovered to its original level, demonstrating the gel's intrinsic self-healing capability. The viscosity variations observed in CeO[2]/RH gels also supported the aforementioned findings. As shown in [87]Fig. 1m, under high-speed shearing, the gel structure was disrupted, leading to a sharp viscosity reduction. Upon cessation of shearing, the gels exhibited rapid creep recovery, with viscosity returning to near-initial values. This reversible behavior persisted over multiple cycles, demonstrating the CeO[2]/RH gel's inherent stability and self-healing properties. Furthermore, CeO[2]/RH gels demonstrated robust substrate adhesion to diverse materials, including AISI 326L stainless steel and polytetrafluoroethylene (PTFE), highlighting their versatility in interfacial bonding applications ([88]Fig. 1n). Together, CeO[2]/RH gels exhibit key injectable gel characteristics, making them suitable for intra-articular applications. 3.2. RONS scavenging capacity of CeO[2]/RH gels RONS are identified as key factors in the pathophysiology of OA. They contribute to tissue damage through oxidation and nitrosation processes, which, in turn, exacerbate inflammation and accelerate degenerative changes in joint tissues [[89]28]. CeO[2], as a wide-used antioxidant nanozyme, is expected to confer antioxidant properties on the resulting gels. Consequently, we initially assessed the capacity of CeO[2]/RH gels to scavenge RONS in vitro. As depicted in [90]Fig. S4, at a constant concentration of RH at 2 mg/mL, the hydroxyl radical (•OH) scavenging efficiency of CeO[2]/RH gels exhibited a dependence on the concentration of CeO[2]. In comparison to CeO[2] alone at a concentration of 64 mg/mL, the CeO[2]/RH gels demonstrated a superior antioxidant activity, enhancing the •OH clearance rate of CeO[2] from 9.7 % to 22.3 %. Subsequently, we evaluated the reactive nitrogen species (RNS) scavenging activity of CeO[2]/RH gels. [91]Fig. S5 illustrated that among the CeO[2]/RH gels containing varying concentrations of CeO[2] NPs, the nitric oxide (NO) concentration was approximately 16 μM following treatment with 64 μg/mL of CeO[2] in the gels. However, the application of this CeO[2]/RH gels further mitigated the release of NO, achieving a concentration of 12 μM. Notably, the pronounced spectral absorption interference from RH severely impeded accurate quantification of SOD activity within the composite systems. Consequently, this investigation focused solely on evaluating the SOD-mimetic activity of CeO[2] NPs. As demonstrated in [92]Fig. S6, CeO[2] NPs exhibited robust, concentration-dependent SOD-mimetic behavior, and this catalytic functionality remained intact following their incorporation into the gel network. To systematically assess the antioxidant cytoprotective effects of CeO[2]/RH gels, we formulated a systematic experimental strategy comprising the following models: (1) LPS-induced inflammatory oxidative stress, (2) H[2]O[2]-mediated direct oxidation, and (3) UV irradiation-triggered oxidative damage. First, we treated RAW264.7 macrophage cells with LPS, followed by systematic evaluation of the RONS-scavenging therapeutic potential of CeO[2]/RH gels. For detection of RONS, fluorescent probes were employed: 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for ROS, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) for RNS, and Hoechst for nucleus staining. [93]Fig. 2a illustrated distinct fluorescence responses across experimental groups. While LPS-treated samples exhibited intense green fluorescence compared to the control group, this LPS-induced fluorescence signal was significantly attenuated in the CeO[2]-, RH-, and CeO[2]/RH gel-treated groups. Notably, the CeO[2]/RH gels displayed the weakest fluorescence intensity, indicating superior inhibitory efficacy ([94]Fig. 2b). Quantitative analysis of fluorescence intensities corroborated these observations, as statistically significant differences were confirmed in [95]Fig. 2c. Given the pivotal role of mitochondria in RONS generation, we employed the mitochondrial-targeted fluorescent probe MitoSOX to quantify mitochondrial superoxide levels in LPS-stimulated RAW264.7 cells. This well-validated method enables specific detection of mitochondrial superoxide (O[2]^•-), distinguishing it from cytosolic ROS species, thereby providing precise spatial resolution for oxidative stress analysis under inflammatory conditions [[96]29]. As demonstrated in [97]Fig. 2d and e, MitoSOX fluorescence imaging revealed a marked elevation in mitochondrial O[2]^•- production following LPS stimulation. Notably, these LPS-induced oxidative bursts were substantially attenuated upon treatment with CeO[2]/RH gels, with fluorescence intensity decreasing by 41 % compared to the LPS-only group. This ROS modulation aligns with the CeO[2]/RH gels' ability to stabilize mitochondrial membrane potential (JC-1 assay: as evidenced by the increased JC-1 aggregate/monomer ratio vs. LPS group) ([98]Fig. S7). Next, we developed a parallel oxidative stress model by challenging RAW264.7 cells with H[2]O[2]. This H[2]O[2] exposure protocol was optimized to induce distinct yet comparable oxidative damage patterns, enabling systematic evaluation of therapeutic interventions across multiple stress paradigms. As illustrated in [99]Fig. 2f, the flow cytometric quantification demonstrated that the CeO[2]/RH gels suppressed H[2]O[2]-induced intracellular ROS levels by 62.3 %. Corroboratively, in the fluorescence imaging experiment ([100]Fig. 2g), H[2]O[2]-treated RAW264.7 cells exhibited intense green fluorescence compared to untreated controls, indicative of elevated oxidative stress. This H[2]O[2]-induced fluorescence signal was significantly attenuated in the group of CeO[2]/RH gels. Quantitative analysis confirmed the superior ROS-scavenging efficacy of the composite gels ([101]Fig. S8), aligning with its enhanced antioxidant capacity observed in previous assays. Building upon the CeO[2]/RH gels' demonstrated superior antioxidant performance, we conducted systematic investigations into its cytoprotective potential against oxidative damage. Quantitative CCK-8 analysis ([102]Fig. 2h) revealed that pre-treatment with the CeO[2]/RH gels significantly restored H[2]O[2]-impaired RAW264.7 viability from 56 % (H[2]O[2]-only group) to 86 %. Notably, the CeO[2]/RH gels exhibited synergistic cytoprotection, outperforming both CeO[2] NPs (1.3-fold improvement) and RH alone (1.2-fold improvement) in rescuing H[2]O[2]-induced viability loss. Quantitative apoptosis analysis via Propidium iodide (PI)/Annexin V-FITC dual staining ([103]Fig. 2i) corroborated the CCK-8 viability trends, demonstrating that CeO[2]/RH gels co-treatment reduced late apoptotic cells (Q2) by 96 % (57.6 %→2.0 %, gels group vs. H[2]O[2] group) and total apoptotic cells (Q1 + Q2 + Q3) by 88 %. This dramatic suppression of cell death aligns with the CeO[2]/RH gels' ability to restore proliferative capacity (EdU assay, as indicated by higher red fluorescence intensity in newly divided cells) ([104]Fig. 2j and k), mechanistically explaining its superior cytoprotection over monotherapies. Furthermore, the robust cytoprotective efficacy of CeO[2]/RH gels was validated in UV-induced cellular injury models, with CCK-8 assay and flow cytometry analysis ([105]Fig. S9). Together, CeO[2]/RH gels, with significant antioxidant capacity, exhibits a superior cytoprotective effect against oxidative stress. This antioxidant-preservation synergy, driven by CeO[2]'s antioxidant enzyme-mimic activity and RH's inherent performance, positions it as a next-generation countermeasure against oxidative stress. Fig. 2. [106]Fig. 2 [107]Open in a new tab Antioxidant performance evaluation of CeO[2]/RH gels. (a) Fluorescence imaging of RONS in LPS-stimulated RAW264.7 cells using DCFH-DA (green, ROS) and DAF-FM DA (green, RNS) probes. The nuclei were stained with Hoechst (blue). (b,c) Normalized fluorescence intensity quantification confirming ROS and RNS suppression in different groups (n = 3). (d) Mitochondrial O[2]^•^- detection and (e) quantitative analysis (n = 3) in LPS-stimulated RAW264.7 cells via MitoSOX red probe. (f) Flow cytometric analysis of intracellular ROS using DCFH-DA in H[2]O[2]-treated RAW264.7 cells. (g) H[2]O[2]-induced ROS visualization by DCFH-DA fluorescence. (h) CCK-8 assay comparing cell viability across H[2]O[2] treatment groups. (i) Apoptosis profiling by flow cytometry post-H[2]O[2] exposure. (j) Representative confocal microscopy images and (k) quantitative analysis of EdU-positive RAW264.7 cells, demonstrating proliferative activity following different treatments (n = 3). (For interpretation of the references to