Electron-injected Pd@CeO2 nanozymes for multifaceted ROS scavenging an…

Morphological and structural analysis of Pd@CeO₂

Pd@CeO₂ nanozymes were synthesized via a multi-step process depicted in Fig. 1a. Uniform Pd nanocubes (~ 18 nm) were initially formed by reducing Na₂PdCl₄ using ascorbic acid, polyvinylpyrrolidone, and potassium bromide, ensuring monodispersed and precisely sized nanoparticles (Fig. 1b–c). Subsequently, cerium nitrate was introduced in the presence of L-Arginine as a capping agent, facilitating CeO₂ nanoparticles’ self-assembly onto the Pd nanocube surfaces. This process resulted in a distinct core-shell Pd@CeO₂ structure, confirmed by TEM imaging showing complete and uniform encapsulation of Pd cores by CeO₂ shells (Fig. 1d–e). Energy-dispersive X-ray spectroscopy (EDS) elemental mapping (Fig. 1f) demonstrated the spatial distribution of Pd, Ce, and O, further confirming successful core-shell formation. The crystallinity of Pd and Pd@CeO₂ phases was validated through X-ray diffraction (XRD), which exhibited characteristic peaks corresponding to both materials (Figure S1). Dynamic light scattering (DLS) measurements confirmed that Pd@CeO₂ nanozymes exhibited a mean particle diameter of 103.9 ± 0.56 nm (Fig. 1g), whereas the zeta potential measurement indicated a surface charge of −19.5 ± 0.47 mV (Fig. 1h).

To elucidate the electron-injection mechanism, X-ray photoelectron spectroscopy (XPS) was employed. Analysis of the Ce 3 d spectra revealed a marked increase in the Ce³⁺/Ce⁴⁺ ratio in Pd@CeO₂ (34.23%/65.77%) compared with pristine CeO₂ (21.29%/78.71%). Furthermore, peak fitting of the Ce 3d spectra revealed that Pd@CeO₂ exhibits overall lower binding energies than CeO₂ (Fig. 1i). In particular, the enhanced intensity of Ce³⁺ peaks at these shifted positions indicates electron enrichment at the CeO₂ interface, arising from interfacial electron transfer from Pd to CeO₂, which provides robust ROS-scavenging performance. Stability assessments over 7 days revealed minimal particle aggregation or charge alterations, underscoring the nanoparticle’s suitability for physiological applications (Figure S2).

Synthesis and characterization of Pd@CeO₂ nanozymes. (a) Schematic illustration of the Pd@CeO₂ nanozymes preparation process. (b) TEM images of Pd nanocubes. (c) Electron diffraction (SAED) pattern of Pd nanocubes. (d) TEM images of Pd@CeO₂ nanozymes. (e) SAED pattern of Pd@CeO₂ nanozymes. (f) Element mapping of Pd, Ce, O, and the merged image of these three elements. (g–h) Particle size distribution and zeta potential of Pd@CeO₂ nanozymes. (i) XPS of Pd@CeO₂ nanozymes

Enzyme-mimicking activities of Pd@CeO₂ and mechanistic insights from DFT calculations

ROS including hydroxyl radicals (·OH), superoxide anions (·O₂⁻), and hydrogen peroxide (H₂O₂), are pivotal in numerous biological and pathological processes [31, 32]. We first evaluated the ·OH scavenging capability of Pd@CeO₂ using electron spin resonance (ESR) spectroscopy. ESR spectra (Fig. 2a) displayed a distinct 1:2:2:1 quartet signal characteristic of DMPO-·OH adducts generated through the Fenton reaction. Compared to controls, Pd@CeO₂ exhibited significantly reduced ESR signal intensity, demonstrating superior ·OH scavenging due to the enhanced catalytic effect of Pd@CeO₂. Subsequently, the SOD-like activity of Pd@CeO₂ was investigated. ESR spectra of ·O₂⁻ (Fig. 2c) revealed a pronounced reduction in the typical 1:1:1:1 quartet upon treatment with Pd@CeO₂, confirming efficient superoxide neutralization. Quantitative analysis using the nitro blue tetrazolium (NBT) assay (Fig. 2d–e) further validated this enhanced performance, as Pd@CeO₂ exhibited the lowest absorbance at 560 nm, reflecting the highest inhibition rate (48.72%) compared with CeO₂ (24.81%) and Pd + CeO₂ mixtures (37.18%). CAT-like activity, essential for H₂O₂ detoxification, was evaluated using a colorimetric assay (Fig. 2g–h). Pd@CeO₂ showed the lowest absorbance at 405 nm and a markedly higher H₂O₂ inhibition rate (77.1%) compared to CeO₂ (40.8%) and Pd + CeO₂ (44.7%). Additionally, dissolved oxygen measurements revealed rapid catalytic decomposition of H₂O₂ to O₂ upon Pd@CeO₂ treatment, increasing O₂ concentrations from 7.5 µg/mL to 28.3 µg/mL within 20 min—significantly outperforming CeO₂ nanoparticles (21.2 µg/mL) (Fig. 2i). These results demonstrate that Pd@CeO₂ exhibits comprehensive ROS scavenging capacity, integrating ·OH, ·O₂⁻, and H₂O₂ elimination through synergistic multi-enzyme–like activities.

To elucidate the mechanism underlying the exceptional catalytic performance of the CeO₂ (111) slab decorated with Pd clusters, density functional theory (DFT) calculations were performed. A Pd@CeO₂ model was constructed and compared with the pristine CeO₂ slab (Fig. 3a). Owing to the pronounced empty-band distribution in CeO₂, the position of the d-band center relative to the Fermi level was carefully considered. For the CeO₂ (111) slab, the d-band center is located at 3.13 eV (Fig. 3b). Upon adsorption of a Pd cluster, the d-band center shifts downward by 0.38 eV to 2.75 eV (Fig. 3c), indicating a weakened adsorption strength of oxygen intermediates at the surface Ce sites. These results demonstrate that the incorporation of Pd clusters induces pronounced electronic interactions within the catalyst, resulting in a downward shift of the d-band center. This adjustment optimizes the adsorption of essential intermediates and thereby enhances catalytic efficiency, as illustrated in Fig. 3d. The charge density difference analysis further confirms the electronic modulation induced by the Pd clusters. As shown in Fig. 3e, adsorption of a Pd cluster results in a charge transfer of approximately 1.15 electrons from the cluster to the CeO₂ substrate, as determined by Bader charge analysis. This electron donation increases the electron density on the substrate, strengthening electronic interactions on CeO₂. This observation is consistent with the projected density of states (PDOS) analysis, which reveals the downward shift of the d-band center. Furthermore, the adsorption energies of oxygen intermediates on the Pd@CeO₂ surface were systematically evaluated. The reaction energy barriers of each elementary step were also calculated, revealing a clear relationship between the overlap of transition-metal d orbitals and the adsorption of oxygen intermediates on the catalyst surface (Fig. 3f). For both CeO₂ and Pd@CeO₂ systems, we found that the rate-determining step (RDS) for H₂O₂ decomposition producing ·OH radicals (i.e., the peroxidase-like reaction) is associated with higher energy barriers compared to H₂O₂ decomposition producing O₂ (i.e., the CAT-like reaction), with values of 1.93 eV vs. 1.14 eV and 1.42 eV vs. 1.08 eV, respectively. This result suggests that the CAT-like pathway predominates in the overall reaction. Notably, the adsorption of Pd clusters significantly reduces the RDS. Specifically, the formation of *O is identified as the RDS for Pd@CeO₂, with a calculated energy barrier of 1.08 eV, which is lower than the 1.14 eV barrier observed in the pristine CeO₂ system. This reduction can be attributed to the enhanced electron density at the Ce sites induced by Pd cluster adsorption, which is consistent with the Bader charge analysis, and facilitates H₂O₂ reduction and consequently promotes O₂ evolution, thereby conferring superior CAT-like activity. These findings provide fundamental insights into how Pd clusters regulate the electronic structure of CeO₂, thereby improving catalytic activity.

ROS elimination by Pd@CeO₂ nanozymes. (a) ESR spectra of ·OH radicals trapped by DMPO in different samples (positive group, CeO₂, Pd + CeO₂ and Pd@CeO₂). (b) Schematic representation of the SOD-like catalytic mechanism of Pd@CeO₂. (c) ESR spectra of ·O₂⁻ scavenging activity for CeO₂, Pd + CeO₂, and Pd@CeO₂. (d) NBT assay showing the reduction of ·O₂⁻ by Pd@CeO₂ through decreased absorbance at 560 nm. (e) Inhibition rates of ·O₂⁻ by different formulations. (f) Schematic of the CAT-like catalytic mechanism of Pd@CeO₂. (g) CAT-like activity of Pd@CeO₂ was evaluated using a catalase assay kit, where the decrease in absorbance at 405 nm reflects H₂O₂ decomposition. (h) Inhibition rates of H₂O₂ decomposition by CeO₂, Pd + CeO₂, and Pd@CeO₂. (i) Oxygen generation during H₂O₂ decomposition by CeO₂ NPs, Pd + CeO₂ NPs, and Pd@CeO₂

DFT-based mechanistic understanding of Pd@CeO₂ catalysis. (a) Models of the CeO₂ (111) slab (denoted as CeO₂) and the CeO₂ (111) slab with an adsorbed Pd cluster (denoted as Pd@CeO₂). Spheres in teal, red and grey represent cerium, oxygen and palladium, respectively. (b–c) PDOS plots of the (b) CeO₂ and (c) Pd@CeO₂. (d) Schematic illustration showing that the d-band center shift of CeO₂ induced by Pd₄ modulates the binding ability of O-containing intermediates. (e) Charge density difference distribution of the Pd@CeO₂, and insert is Bader charge analysis of Pd₄ on CeO₂ (111) slab. The pink and cyan volumes represent electron depletion and accumulation, respectively. Spheres in teal, red and grey represent cerium, oxygen and palladium, respectively. (f) Energy profiles and the corresponding configurations for the formation of surface peroxo species on the CeO₂ (111) slab, either pristine (denoted as CeO₂) or decorated with a Pd cluster (denoted as Pd@CeO₂). The rate-determining step (RDS) is highlighted in the plots. Spheres in blue, white, pink and red represent cerium, hydrogen, oxygen and palladium, respectively

In vitro ROS-scavenging capability of Pd@CeO₂ nanozymes

Building on the robust enzyme-mimetic catalytic activities of Pd@CeO₂, we evaluated their cellular uptake, biocompatibility, and cytoprotective performance against oxidative stress. The internalization of Pd@CeO₂ nanozymes was monitored in HUVEC and RAW 264.7 cells using FITC labeling. Fluorescence microscopy and flow cytometry consistently demonstrated efficient, time-dependent cellular uptake of Pd@CeO₂, with uptake efficiency reaching over 95% within 12 h and approaching complete internalization by 24 h (Fig. 4a–b). Biocompatibility assessment using CCK-8 assays revealed negligible cytotoxicity of Pd@CeO₂ at concentrations ≤ 50 µg/mL in both HUVEC and RAW 264.7 cells (Fig. 4c–d). Longitudinal analysis over a three-day period confirmed the absence of adverse effects on cell proliferation, highlighting the suitability of Pd@CeO₂ for biomedical applications (Figure S3). Next, we evaluated the protective role of Pd@CeO₂ against H₂O₂-induced oxidative injury. Pretreatment with Pd@CeO₂ markedly enhanced HUVEC viability (91.66%), outperforming CeO₂ (72.72%) and Pd + CeO₂ mixtures (79.79%) (Fig. 4e). Consistently, intracellular ROS levels, visualized by DCFH-DA fluorescence, were substantially diminished in the Pd@CeO₂ group, underscoring its superior scavenging efficacy relative to the controls (Fig. 4f and i and Figure S4–S5). Calcein-AM/PI staining further confirmed enhanced cell survival and reduced cell death following Pd@CeO₂ treatment under oxidative stress conditions (Fig. 4g and j). Flow cytometry analyses using Annexin V-FITC/PI staining demonstrated a substantially lower apoptosis rate in Pd@CeO₂-treated cells, highlighting robust anti-apoptotic effects (Fig. 4h and k). Collectively, the findings establish Pd@CeO₂ as a safe and efficient nanozymes with superior antioxidant and anti-apoptotic properties, highlighting its promise for biomedical applications in oxidative stress–driven pathologies.

Biocompatibility and protective effects of Pd@CeO₂ against oxidative damage. (a) Flow cytometry analysis of FITC-labeled Pd@CeO₂ uptake by HUVECs and RAW 264.7 macrophages at different time points (0, 2, 12, and 24 h). (b) quantification of flow cytometry results. (c) Cytotoxicity of Pd@CeO₂ on HUVECs at various concentrations (0, 10, 20, 50, 80, 160, 200, and 320 µg/mL). (d) Cytotoxicity of Pd@CeO₂ on RAW 264.7 macrophages at various concentrations (0, 10, 20, 50, 80, 160, 200, and 320 µg/mL). (e) Cell viability of CeO₂, Pd + CeO₂, and Pd@CeO₂ (50 µg/mL) against H₂O₂-induced oxidative damage. (f) Flow cytometric analysis of intracellular ROS levels in oxidatively stressed HUVEC cells. (g) Statistical analysis of cell mortality in injured cells. (h) Statistical analysis of apoptosis rate in each group. (i) DCFH-DA fluorescence microscopy images showing ROS levels in each group. Scale bars: 20 μm. (j) Calcein-AM/PI fluorescence staining of HUVECs treated with H₂O₂ and or different formulations. Scale bars: 50 μm. (k) Flow cytometry analysis of apoptosis rates after different treatments

Molecular mechanisms of Pd@CeO₂ in suppressing apoptosis and inflammation

To further elucidate the protective mechanisms conferred by Pd@CeO₂ nanozymes against oxidative stress-induced injury, we evaluated their influence on apoptosis-related pathways. Apoptosis, prominently regulated by proteins including Bcl-2 (anti-apoptotic), Bax (pro-apoptotic), and cleaved Caspase-3 (executioner of apoptosis), is central to I/R injury pathology [33, 34]. Western blot analysis (Fig. 5a–d) demonstrated that Pd@CeO₂ significantly elevated Bcl-2 expression while concurrently suppressing Bax and cleaved Caspase-3 levels compared to CeO₂ and Pd + CeO₂. These findings indicate a robust inhibition of apoptosis via modulation of the Bax/Bcl-2/caspase-3 pathway, underscoring the potent cytoprotective capabilities of Pd@CeO₂.

Inflammation represents another pivotal aspect of I/R injury, which is further aggravated by excessive ROS generation [35, 36]. Employing an LPS-stimulated RAW 264.7 macrophage inflammation model, ELISA assays revealed that Pd@CeO₂ markedly attenuated secretion of key pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 compared to the LPS-treated control groups (Fig. 5e–g). This highlights the substantial anti-inflammatory efficacy of Pd@CeO₂ in cellular models. To uncover the molecular basis underlying these anti-inflammatory effects, quantitative PCR and Western blot analyses assessed the expression of inflammation-related factors NF-κB and PPAR-γ. The LPS-driven increase in NF-κB mRNA expression was substantially suppressed by Pd@CeO₂, highlighting its robust capacity to modulate this pivotal pro-inflammatory transcription factor (Fig. 5h). Conversely, Pd@CeO₂ significantly upregulated PPAR-γ mRNA, supporting its role in resolving inflammation (Fig. 5i). Protein-level analyses further confirmed these results, demonstrating reduced phosphorylation of NF-κB p65 and increased PPAR-γ protein expression in Pd@CeO₂-treated groups compared to controls (Fig. 5j–l). These results suggest that Pd@CeO₂ modulate inflammatory responses by downregulating the NF-κB pathway and upregulating the anti-inflammatory PPAR-γ pathway.

Anti-apoptotic and anti-inflammatory mechanisms of Pd@CeO₂. (a) Western blot analysis of Bcl-2, Bax, and cleaved caspase-3 protein expression levels. Quantification of (b) Bcl-2, (c) Bax, and (d) caspase-3 protein expression levels. ELISA results showing (e) TNF-α, (f) IL-1β, and (g) IL-6 secretion levels after treated with CeO₂, Pd + CeO₂, or Pd@CeO₂. Quantification of (h) NF-κB and (i) PPAR-γ mRNA expression levels by qPCR. (j) Western blot analysis of p-NF-κB p65, NF-κB p65, and PPAR-γ protein expression. Quantification of (k) p-p65 and (l) PPAR-γ protein expression ratio

Restoration of angiogenic capacity by Pd@CeO₂ in oxidative environments

Building upon the demonstrated anti-apoptotic and anti-inflammatory properties of Pd@CeO₂, we further investigated its angiogenic capacity under oxidative stress, a critical factor in tissue repair and flap survival. Angiogenesis is notably impaired by I/R injury, thus emphasizing its importance in recovery [37, 38]. qPCR analysis revealed that Pd@CeO₂ treatment markedly upregulated VEGFA mRNA, a key driver of angiogenesis, in H₂O₂-stimulated HUVECs compared with controls, CeO₂, and Pd + CeO₂ (Fig. 6a). Western blot analyses corroborated this, revealing substantially higher VEGFA protein levels in Pd@CeO₂-treated cells relative to H₂O₂-exposed cells (Fig. 6b–c). These results highlight the strong capacity of Pd@CeO₂ to reestablish angiogenic signaling pathways disrupted by oxidative stress. We next evaluated endothelial cell migration using scratch assays. H₂O₂ exposure markedly impaired migratory capacity, whereas treatment with CeO₂, Pd + CeO₂, and especially Pd@CeO₂ significantly promoted wound closure (Fig. 6d). Experiments further confirmed that Pd@CeO₂ achieved the most pronounced enhancement of migration, surpassing all other treatments (Fig. 6f). Finally, we assessed angiogenic functionality via endothelial tube formation assays. H₂O₂-induced oxidative stress severely disrupted tube formation, evident by reduced branching and network complexity. Notably, pretreatment with Pd@CeO₂ effectively restored endothelial tube formation, significantly enhancing total branching length compared to CeO₂ and Pd + CeO₂ treatments ((Fig. 6e and g). These collective results position Pd@CeO₂ as a highly effective nanozyme, capable of significantly promoting angiogenesis under oxidative stress, thereby highlighting its substantial therapeutic potential in ischemic tissue repair.

In vitro investigations revealed that Pd@CeO₂ orchestrates multifaceted cytoprotective mechanisms by reducing oxidative stress, inhibiting apoptosis through the Bcl-2/Bax/caspase axis, and dampening inflammatory cascades mediated by NF-κB and PPAR-γ (Fig. 6h), thereby establishing a coherent mechanistic basis for its therapeutic potential.

Enhancement of angiogenesis in oxidative stress-damaged HUVECs by Pd@CeO₂. (a) qPCR analysis of VEGFA mRNA expression following treatment with CeO₂, Pd + CeO₂, or Pd@CeO₂. (b) Western blot assessment of VEGFA protein expression. (c) Quantification of VEGFA protein expression level. (d) Statistical analysis of wound repair rate of each group. (e) Statistical evaluation of the total dendritic branching length across experimental groups. (f) Representative images of the scratch assay in HUVECs under H₂O₂-induced oxidative stress. Scale bar: 200 μm. (g) Representative images of tube formation assays in HUVECs exposed to H₂O₂. Scale bar: 200 μm. (h) Diagrammatic representation of Pd@CeO₂-mediated mechanisms in attenuating oxidative stress, inhibiting apoptosis, and suppressing inflammation

Therapeutic efficacy of Pd@CeO₂ in a rat skin flap ischemia–reperfusion injury model

To preliminarily assess the in vivo biosafety of Pd@CeO₂ nanozymes, we examined their biocompatibility through hemocompatibility and histological studies. Hemolysis assays revealed an exceptionally low hemolysis rate (S1-S2). Histological examination of major organs (heart, liver, spleen, lung, and kidney) by H&E staining at 7 and 14 days revealed no detectable pathological alterations, thereby reinforcing the excellent biosafety profile of Pd@CeO₂ (Figure S7).

Subsequently, therapeutic efficacy was evaluated using a rat dorsal skin flap I/R injury model. After 6 h of ischemia followed by reperfusion, extensive flap necrosis was observed in the untreated controls, whereas Pd@CeO₂ treatment markedly preserved tissue integrity and minimized necrotic damage (Fig. 7a-b). Real-time microcirculation assessed via laser speckle imaging revealed significantly enhanced blood perfusion in the Pd@CeO₂ group, which achieved the highest flap survival rate (85.42%) compared to CeO₂ (60.10%), Pd + CeO₂ (77.14%), and untreated groups (55.76%) (Fig. 7b). H&E staining further demonstrated significant preservation of tissue architecture in Pd@CeO₂-treated flaps, characterized by intact epidermal layers, well-maintained hair follicles, and normal dermal collagen deposition, contrasting sharply with severe epidermal thinning in control groups (Fig. 7c). Given the promising in vitro angiogenic effects of Pd@CeO₂, we subsequently evaluated its pro-angiogenic capabilities within an in vivo I/R skin flap model. Immunohistochemical staining for the endothelial marker CD31 was performed to assess neovascularization within flap tissues. Results indicated compromised angiogenesis in the I/R group, exhibiting microvascular densities of approximately 4.0 vessels per field. In contrast, groups treated with Pd + CeO₂ and particularly Pd@CeO₂ displayed considerably enhanced neovascularization, with microvessel densities of 13.5 and 16.0, respectively (Fig. 7d). These findings underscore the superior efficacy of Pd@CeO₂ in promoting angiogenesis under I/R injury conditions.

Evaluation of the therapeutic effects of Pd@CeO₂ in a rat skin flap I/R injury model. (a) Schematic illustration of the therapeutic process in the I/R injury model. (b) Representative images of skin flaps on day 7 post-surgery for the I/R, CeO₂, Pd + CeO₂, and Pd@CeO₂. (c) HE staining of distal skin flap tissue sections. Scale bars: 200 μm. (d) Representative images of immunohistochemical staining of CD31 in I/R, CeO₂, Pd + CeO₂, and Pd@CeO₂ groups

Mitigation of inflammation and apoptosis by Pd@CeO₂ in I/R skin flaps

To further validate the anti-inflammatory potential of Pd@CeO₂, immunohistochemical staining of CD68 was conducted to quantify macrophage infiltration, indicative of inflammation within skin flaps. As shown in Fig. 8a, markedly elevated macrophage densities were detected in both the I/R and saline groups (≈ 11.17 each), reflecting pronounced inflammatory activation. In contrast, macrophage infiltration was substantially reduced in the CeO₂ (≈ 6.0) and Pd + CeO₂ groups (≈ 4.17), with the most pronounced attenuation observed in the Pd@CeO₂-treated group (≈ 3.5), thereby underscoring the potent in vivo anti-inflammatory efficacy of Pd@CeO₂. Additionally, TUNEL assays were employed to evaluate apoptosis within the flap tissues. Distinctly higher densities of TUNEL-positive cells were detected in the I/R and saline control groups, reflecting significant apoptotic activity (apoptosis rates of 92.67% and 95.67%, respectively). Treatment groups showed substantially reduced apoptosis rates, with CeO₂ at 45.68%, Pd + CeO₂ at 44.49%, and notably, Pd@CeO₂ demonstrating the lowest apoptosis at 43.08% (Fig. 8b). Finally, DHE staining indicated that Pd@CeO₂ treatment effectively suppressed ROS accumulation in flap tissues, reinforcing the correlation between robust ROS scavenging and improved flap viability and structural integrity (Fig. 8c). These results collectively confirm that Pd@CeO₂ effectively mitigates apoptosis, potentially via robust ROS scavenging and reduced inflammatory signaling, thus underscoring its substantial therapeutic potential in addressing oxidative and inflammatory damage during I/R injury.

Anti-inflammatory and anti-apoptotic effects of Pd@CeO₂ in an I/R skin-flap model. (a) Immunohistochemical staining and semi-quantitative analysis of CD68 to evaluate macrophage densities in the skin flap tissues, with red arrows indicating macrophages, bar = 100 μm. (b) TUNEL staining and quantitative assessment to detect apoptotic cells in skin flap tissues, with red arrows highlighting apoptotic cells. Scale bars: 50 μm. (c) ROS levels and semi-quantitative analysis in skin flap tissues assessed via DHE staining. Scale bars: 100 μm

Transcriptomic analysis of Pd@CeO₂-mediated protection against I/R injury

To gain comprehensive insights into the molecular mechanisms underlying the therapeutic actions of Pd@CeO₂ in I/R injury, transcriptomic profiling was conducted. Hierarchical clustering of differentially expressed genes (DEGs) revealed distinct transcriptional signatures among the Control, I/R, and Pd@CeO₂-treated groups (Fig. 9a). Volcano plots further delineated significantly altered gene expressions, highlighting 2004 upregulated and 2167 downregulated genes when comparing the control and I/R groups. In contrast, Pd@CeO₂ treatment induced significant expression changes, with 269 genes upregulated and 138 genes downregulated compared to the I/R group (Fig. 9b). Gene Ontology (GO) enrichment analysis indicated that Pd@CeO₂ treatment notably influenced biological processes related to inflammation and immune responses, cellular components associated with membrane and extracellular matrix, and molecular functions involving receptor activity and signaling transduction (Fig. 9c). Notably, enriched biological processes such as “inflammatory response”, “immune response”, and “cellular response to cytokine stimulus” were significantly modulated upon Pd@CeO₂ treatment, indicating its robust anti-inflammatory potential at the transcriptomic level. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further highlighted key signaling pathways significantly regulated by Pd@CeO₂ treatment, including TNF, Chemokine, and PPAR signaling pathways (Fig. 9d). These results corroborate earlier findings regarding the involvement of these pathways in inflammatory modulation and tissue repair, providing robust transcriptomic validation for the anti-inflammatory and regenerative effects observed in previous cellular and histological assessments.

In vivo transcriptomic analysis of rat with I/R injury. (a) Hierarchical clustering heatmap depicting DEG in different groups. (b) Volcano plots. (c) GO enrichment analysis. (d) KEGG pathway enrichment scatterplot