A dual-pronged strategy: multi-bioactive nanoplatform synergizes ROS antidote and iron stabilization to protect against liver ischemia-reperfusion injury

Abstract

Liver ischemia-reperfusion injury (LIRI) is a severe and nearly unavoidable complication in hepatectomy and liver transplantation, accompanied by intense oxidative stress and ferroptosis. Traditional clinical therapies often face challenges such as poor bioavailability, short circulation time, and low stability, which restrict their efficacy in regulating complex pathological microenvironments. Here, we developed a biocompatible and multi-bioactive nanoplatform, Se@MelP, comprising a selenium nanoparticle (SeNP) core encapsulated within a melanin-like shell. Specifically, selenium (Se) was incorporated into selenoproteins in the form of selenocysteine to regulate redox reactions, while the melanin-like shell simultaneously scavenged reactive oxygen species (ROS) and chelated Fe²⁺, together synergistically alleviating LIRI. Se@MelP possessed excellent ROS scavenging activity, strong iron chelating capability, and favorable biosafety. In vitro experiments confirmed that Se@MelP effectively eliminated excessive ROS, alleviated Fe²⁺ overload, and mitigated mitochondrial damage, protecting cells from oxidative stress and ferroptosis. Upon intravenous administration in the LIRI model, Se@MelP markedly improved liver function, attenuated tissue injury, and mitigated oxidative stress, inflammatory responses, and ferroptosis. Notably, Se@MelP activated the AMPK/Nrf2 pathway, enhancing the endogenous antioxidant defenses and anti-ferroptotic capacity, thereby conferring protection against liver injury. This multi-bioactive nanoplatform offers a promising therapeutic strategy for LIRI and other oxidative stress‑related liver diseases.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04600-1.

Introduction

Liver ischemia-reperfusion injury (LIRI) refers to the paradoxical aggravation of liver damage resulting from the reperfusion phase after a transient ischemic insult [1, 2]. It remains a major and nearly unavoidable complication in clinical procedures such as hepatectomy and transplantation [3–6]. The occurrence of LIRI can trigger liver inflammation, fibrosis, and even hepatic failure, directly affecting the prognosis of patients undergoing liver-related surgeries [7, 8]. Therefore, timely and effective intervention is crucial to mitigate or even reverse LIRI. Recent studies have shown that oxidative stress, inflammation, and ferroptosis are central contributors to LIRI development [9–11]. Ferroptosis, in particular, is a new form of regulated cell death characterized by iron-driven accumulation of toxic lipid peroxides [12, 13]. During LIRI, a synergistic ferroptosis network involving iron overload, lipid peroxidation, and inactivation of the key antioxidant enzyme glutathione peroxidase 4 (GPX4), collectively exacerbates liver damage [14–16]. Studies have shown that clinical antioxidant N-acetylcysteine [17] and iron chelator deferoxamine [18] could effectively alleviate the damage caused by ischemia-reperfusion (IR), yet their therapeutic applicability is hampered by poor bioavailability, short circulation time, low stability, and severe adverse effects [6, 19, 20]. Therefore, developing safe and effective therapeutic strategies is urgently needed for LIRI.

Given these challenges, biocompatible nanomaterials bypass the limitations of molecular drugs by integrating enhanced stability, controllable pharmacokinetics, and robust activity at the injury site, representing a promising alternative direction [21–23]. Selenium (Se), an essential human trace element, plays key roles in antioxidant defense, immune modulation, and anticancer activity [24–26]. It functions as selenocysteine in the active centers of crucial antioxidant enzymes, including glutathione peroxidases (GPXs) and thioredoxin reductase (TXNRD), serving as an essential redox-active component [27, 28]. Notably, Se also participates in the ferroptosis process through pathways such as regulating the activity of GPX4 [29–31]. However, the narrow therapeutic window of Se poses a major challenge to its clinical application, as excessive intake readily induces toxicity [32]. To mitigate these limitations, Se has been engineered into nanoparticle forms. Compared to conventional inorganic and organic selenocompounds, selenium nanoparticles (SeNPs) exhibit better bioavailability and safety, attracting extensive attention from the biomedical research in recent years [33, 34]. Nevertheless, like other single-component nanomaterials, SeNPs alone exhibit limited efficacy due to their inability to concurrently address the multiple pathogenic pathways involved in the complex pathological microenvironment. To overcome this, integrating multi-functional and synergistic activities into one nanoplatform is expected to offer enhanced therapeutic efficacy.

Melanin is a natural pigment widely distributed in living organisms and possesses inherent biocompatibility [35–37]. Owing to its abundant catechol and amine groups, melanin exhibits excellent antioxidant activity and strong metal ion chelation capacity [38–40]. Recent studies have shown that melanin can effectively mitigate oxidative stress-related diseases such as acute kidney injury [41]. Compared with naturally extracted melanin, artificially synthesized melanin-like materials, such as polydopamine, not only retain these bioactivities but also offer advantages including controllable synthesis and lower cost, making them widely applicable in areas such as antioxidation, cancer therapy, radioprotection, and anti-infection [42–45]. Based on these properties, in recent years, researchers have integrated SeNPs with multifunctional melanin-like materials into a single nanoplatform for the treatment of various disease models. For example, Park et al. used polydopamine-coated SeNPs to exert therapeutic effects in a rat model of osteoarthritis through antioxidant, anti-inflammatory, and cartilage-repair capabilities [46]. Fang et al. developed an oral delivery system comprising methacrylated hyaluronic acid hydrogel microspheres encapsulating polydopamine-coated SeNPs (CS-Se@PDA) for antioxidant and immunomodulatory therapy of ulcerative colitis [47]. However, there have been few reports on strategies that integrate the redox homeostasis regulation of Se with the iron-chelating and reactive oxygen species (ROS)-scavenging capabilities of melanin-like materials for mitigating oxidative stress and ferroptosis during LIRI.

In this study, we developed a biocompatible and multi-bioactive nanoplatform Se@MelP to alleviate oxidative stress and ferroptosis in IR-induced liver injury (Fig. 1). Se@MelP comprised a selenium nanoparticle (SeNP) core encapsulated within a melanin-like shell, exhibiting broad-spectrum ROS scavenging activity and iron chelating capability. In vitro experiments demonstrated that Se@MelP effectively protected AML-12 cells from oxidative stress and ferroptosis damage by reducing ROS levels, suppressing iron overload, and preserving mitochondrial integrity. Moreover, the assessment of histopathological changes combined with the measurement of serum and tissue homogenate indicators confirmed that Se@MelP markedly attenuated liver injury, alleviated oxidative stress and inflammation, and inhibited ferroptosis in the LIRI model. Further mechanistic studies revealed that the protective effect of Se@MelP against LIRI was primarily associated with the amelioration of oxidative stress, the regulation of iron dyshomeostasis, and the activation of the AMPK/Nrf2 pathway to enhance the endogenous antioxidant defenses and anti-ferroptotic capacity. In summary, Se@MelP serves as an effective and safe nano‑platform against LIRI, offering a promising strategy for clinical translation.

Fig. 1.

Schematic illustration of the preparation and protective effect of Se@MelP against LIRI. Specifically, Se was incorporated into selenoproteins to regulate redox reactions, while the melanin-like shell simultaneously scavenged ROS and chelated Fe²⁺, thereby synergistically alleviating liver injury. Mechanistically, the protective effect of Se@MelP against LIRI was primarily associated with the amelioration of oxidative stress, the regulation of iron dyshomeostasis, and the activation of the AMPK/Nrf2 pathway

Results and discussion

Characterization of Se@Mel and Se@MelP

The synthesis method of Se@MelP is shown in Fig. 2A. Briefly, SeNPs were first synthesized as the core via a literature method [48]. Then, under alkaline conditions, the surface of SeNPs was co-assembled with dopamine and bovine serum albumin (BSA) to form a melanin-like shell and subsequently functionalized with polyethylene glycol (PEG) to enhance physiological stability. In Fig. S1, the synthesized SeNPs displayed a spherical structure and had an average diameter of about 60.3 nm. Upon the self-assembly of dopamine and BSA, a uniform polymer shell was observed on the surface of SeNPs, confirming successful preparation of Se@Mel (Fig. 2B). Furthermore, it was observed that Se@Mel had an average size of about 90.6 nm and remained well dispersed (Fig. 2B, C), facilitating hepatic accumulation post-intravenous injection. The element mapping analysis showed that the elements C, N, O, S, and Se were distributed in Se@Mel. As expected, the element Se was mainly distributed in the core of Se@Mel (Fig. 2D). In addition, the presence of a Se 3d peak in Se@Mel, as confirmed by X-ray photoelectron spectroscopy (XPS) survey scan, further confirming the successful encapsulation of SeNPs (Fig. 2E). Then, high-resolution XPS analysis of the Se 3d region was conducted. The spectrum of Se@Mel exhibited two prominent peaks at 55.3 eV and 56.2 eV, corresponding to Se 3d_5/2 and Se 3d_3/2, respectively (Fig. 2F). These binding energies are consistent with those of elemental selenium (Se⁰), indicating that the Se in Se@Mel exists in the zero-valent state [49]. The UV–vis absorption spectra further confirmed the successful preparation of Se@Mel, as the spectrum of Se@Mel showed a combination of the characteristic ~ 263 nm peak of SeNPs and the broad absorption background of MelNPs (Fig. 2G). After PEG modification, the hydrodynamic size and zeta potential of Se@MelP were then characterized. As shown in Fig. 2H, the SeNPs had a hydrodynamic diameter of 105.9 ± 1.5 nm. A gradual size increase was observed upon the successive formation of melanin-like shell and subsequent PEG modification, resulting in a final diameter of 132.0 ± 0.8 nm for Se@MelP. Successful melanin-like coating was further demonstrated by a zeta potential shift from − 13.8 ± 0.6 mV to -17.6 ± 0.7 mV, and the zeta potential of the final product Se@MelP was − 19.0 ± 0.3 mV (Fig. 2I). Finally, we assessed the physiological stability of Se@MelP. The Se@MelP maintained a consistent particle size without significant precipitation over 7 days, providing a prerequisite for effective in vivo delivery and function (Fig. 2J, K).

Fig. 2.

Characterization of Se@Mel and Se@MelP. (A) Schematic illustration of the synthesis of Se@MelP. (B) TEM image of Se@Mel. Scale bar: 200 nm. The inset is an enlarged TEM image. Scale bar: 50 nm. (C) SEM image of Se@Mel. Scale bar: 200 nm. (D) Element mapping of Se@Mel. Scale bar: 50 nm. (E) XPS spectrum of Se@Mel. (F) High-resolution XPS spectrum of Se 3d in Se@Mel. (G) The UV-vis absorption spectra of SeNPs, MelNPs, and Se@Mel. (H) Hydrodynamic diameter of SeNPs, Se@Mel, and Se@MelP. (I) Zeta potential of SeNPs, MelNPs, Se@Mel, PEG, and Se@MelP. (J) The hydrodynamic diameter of Se@MelP was monitored in different media over 7 days. (K) Stability of Se@MelP at 0 and 7 days in various media. Data are mean ± SD (n = 3)

ROS and iron overload play pivotal roles in the occurrence and development of oxidative stress and ferroptosis during LIRI, making their suppression a key therapeutic strategy to alleviate damage (Fig. 3A). After verifying the successful synthesis, the antioxidant efficacy and Fe²⁺ chelation capacity of Se@MelP were further investigated. To evaluate the ROS scavenging ability of Se@MelP, hydroxyl radical (·OH) and superoxide anion (O₂^⁻) were selected. As displayed in Fig. 3B and C, Se@MelP exhibited concentration-dependent free radical scavenging capacity. At a concentration of 100 µg/mL, the scavenging rates of Se@MelP against ·OH and O₂^⁻ reached 82.8 ± 1.3% and 70.0 ± 1.1%, respectively. To further assess the antioxidant capacity of Se@MelP, classical DPPH and ABTS radical scavenging assays were conducted. The DPPH assay detects antioxidant activity by monitoring the color change from purple to yellow as DPPH radicals are scavenged (Fig. 3D) [50]. Figure 3E and F showed a concentration-dependent change, with the solution color fading and its characteristic absorbance peak diminishing. At 100 µg/mL, Se@MelP achieved a high scavenging rate of 92.5 ± 1.9% for DPPH radical (Fig. 3G). A similar trend was observed for the ABTS radical scavenging activity of Se@MelP, which increased with concentration and reached a scavenging rate of 94.1 ± 2.1% at 100 µg/mL, demonstrating its effectiveness as a free radical scavenger (Fig. 3H). Subsequently, the chelating ability of Se@MelP for Fe²⁺ was tested. Under acidic conditions, Fe²⁺ chelates with tripyridyltriazine to form a blue-colored tripyridyltriazine-Fe²⁺ compound [42]. Increasing the concentration of Se@MelP led to a gradual fading in the blue color and a reduction in characteristic absorbance of tripyridyltriazine-Fe²⁺, confirming the Fe²⁺ chelating capability of Se@MelP (Fig. 3I-K). Collectively, these results demonstrated that Se@MelP possessed broad-spectrum ROS scavenging activity and Fe²⁺ chelating capacity, which were beneficial for alleviating oxidative stress and ferroptosis during LIRI.

Fig. 3.

In vitro antioxidant and Fe²⁺ chelation capacity of Se@MelP. (A) Illustration of the ROS elimination and Fe²⁺ chelation capacity of Se@MelP. (B) ·OH scavenging ability of Se@MelP. (C) O₂^⁻ scavenging ability of Se@MelP. (D) Schematic diagram of the DPPH radical scavenging assay. (E) Photographs showing the DPPH solution after incubation with varying concentrations of Se@MelP. (F) UV–vis absorption spectra from DPPH radical scavenging assay. (G) DPPH radical scavenging ability of Se@MelP. (H) ABTS radical scavenging ability of Se@MelP. (I) Photographs of the tripyridyltriazine-Fe²⁺ compound solution after incubation with Se@MelP at various concentrations. (J) Absorbance spectra of tripyridyltriazine-Fe²⁺ compound after the addition of Se@MelP. (K) Fe²⁺ chelation ability of Se@MelP. Data are mean ± SD (n = 3)

Cytotoxicity and cellular uptake of Se@MelP

To ensure the validity and safety of subsequent in vitro cellular experiments, it is essential to evaluate the biosafety of Se@MelP. The cytotoxicity of Se@MelP in AML-12 cells was assessed with the CCK-8 assay. As shown in Fig. 4A and Fig. S2, no significant cytotoxicity was observed for SeNPs, MelNPs, or Se@MelP across a range of concentrations, demonstrating their excellent biosafety and paving the way for their use in subsequent experiments. Subsequently, AML-12 cells were incubated with FITC-labeled Se@MelP and observed at different time points to monitor the nanoparticle internalization. After 2 h co-incubation, weak green fluorescence was detected in the cytoplasm. Subsequently, the intracellular fluorescence signal became strong after 4 h co-incubation, indicating time-dependent and efficient cellular uptake of Se@MelP (Fig. 4B).

Fig. 4.

Cytotoxicity, cellular uptake, and protective effect of Se@MelP. (A) Viability of AML-12 cells treated with Se@MelP for 24 h. (B) Cellular uptake of FITC-labeled Se@MelP in AML-12 cells at 0, 2, and 4 h. Scale bar: 50 μm. (C) Illustration of anti-apoptotic effect of Se@MelP. (D) Viability of AML-12 cells exposed to 400 µM TBHP and co-treated with SeNPs, MelNPs, or Se@MelP for 12 h. (E) Representative Calcein-AM staining images of live AML-12 cells under different treatment conditions. Scale bar: 200 μm. (F) Flow cytometry analysis of apoptotic cell distribution in AML-12 cells. (G) Representative fluorescence images showing intracellular ROS levels in AML-12 cells. Scale bar: 100 μm. (H) Fluorescence images of AML-12 cells stained with JC-1, indicative of MMP. Scale bar: 50 μm. Data are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Cytoprotection of Se@MelP

The pathogenesis of LIRI is characterized by a burst of ROS, which triggers severe oxidative stress and initiates a cascade of cellular damage [51]. Encouraged by the excellent physicochemical properties and biosafety of Se@MelP, we further assessed its protective effects against oxidative stress-induced cell injury. Here, tert-butyl hydroperoxide (TBHP) was employed to establish an in vitro oxidative stress model (Fig. 4C). TBHP stimulation significantly reduced the viability of AML-12 cells to 68.6 ± 4.2%. Cells co-incubated with TBHP and SeNPs, MelNPs, or Se@MelP exhibited a marked increase in viability, with the greatest protection observed in the Se@MelP group (93.8 ± 1.6%) (Fig. 4D). The Calcein-AM staining provided further evidence of the protective effect of Se@MelP in TBHP-stimulated AML-12 cells. Upon entering live cells, the non-fluorescent probe Calcein-AM is enzymatically converted into green-fluorescent Calcein, enabling the specific staining of live cells [52]. A significant increase in live cell numbers was observed in the Se@MelP group, nearly comparable to the control group (Fig. 4E). Finally, to quantify apoptosis, AML-12 cells were analyzed by flow cytometry after the indicated treatments. Following TBHP stimulation, early-apoptotic cells accounted for 11.2% and late-apoptotic cells for 32.6%. All treatment groups showed a marked improvement in cell survival. Notably, the Se@MelP group exhibited the lowest level of apoptosis, with early-apoptotic cells at 5.58% and late-apoptotic cells at 11.2% (Fig. 4F). These results suggested that Se@MelP provided a potent protective effect against oxidative stress-induced cell injury.

Then, intracellular ROS levels in different groups were detected and analyzed using the fluorescent probe DCFH-DA. As shown in Fig. 4G, TBHP stimulation significantly elevated intracellular ROS levels, whereas treatment with SeNPs, MelNPs, or Se@MelP effectively reduced them. Notably, Se@MelP administration showed the most significant reduction in ROS levels. Quantitative analysis of the fluorescence signals further indicated that Se@MelP could effectively scavenge ROS generated by TBHP stimulation (Fig. S3). Next, we measured TBHP-induced mitochondrial membrane potential (MMP) damage using the JC-1 fluorescent probe as an indicator. JC-1 forms red-fluorescent J-aggregates in normal mitochondria, while shifting to green monomers upon depolarization, serving as a sensitive indicator of MMP. Intense green fluorescence in TBHP-stimulated cells confirmed significant mitochondrial depolarization. This damage was effectively mitigated by Se@MelP treatment, as evidenced by markedly reduced green fluorescence and quantitative analysis of JC-1 fluorescence ratio, underscoring its potent mitochondria-protective capacity (Fig. 4H and Fig. S4). Bio-TEM imaging revealed that TBHP triggered mitochondrial damage in AML-12 cells, including mitochondria shrinkage, membrane thickening, and a reduction in cristae. Notably, these ultrastructural changes were effectively mitigated by Se@MelP treatment (Fig. S5). Taken together, these findings demonstrated that Se@MelP effectively reduced apoptosis, eliminated intracellular ROS, and preserved mitochondrial function, thereby attenuating TBHP-induced oxidative stress damage in cells.

Anti-ferroptotic effect of Se@MelP

Anti-ferroptotic activity of Se@MelP was explored using an RSL3-induced injury model in AML-12 cells. RSL3, a classical ferroptosis inducer, triggers ferroptosis by binding to and inhibiting the key antioxidant enzyme GPX4 [53]. As shown in Fig. 5A and B, RSL3 stimulation induced intense green fluorescence in AML-12 cells, indicative of oxidative stress. The Se@MelP treatment inhibited the increase in fluorescence intensity caused by RSL3, indicating that Se@MelP effectively reduced RSL3-induced ROS production. Then, to evaluate lipid peroxidation levels, we performed fluorescence imaging using BODIPY 581/591 C11 probe. As shown in Fig. S6, the RSL3 group exhibited significantly increased oxidized lipids (green fluorescence), which was restored to near-normal levels by Se@MelP treatment. Fe²⁺ accumulation serves as a pivotal trigger for ferroptosis by driving the Fenton reaction, which generates harmful lipid peroxides [41]. Here, the Fe²⁺-selective fluorescent probe FerroOrange was employed to monitor intracellular labile iron levels. RSL3 stimulation increased intracellular Fe²⁺ levels, as indicated by intense red fluorescence. As expected, Se@MelP treatment markedly attenuated this fluorescence signal, demonstrating its ability to reduce labile iron levels in AML-12 cells (Fig. 5C). We next evaluated the protective effects of Se@MelP against mitochondrial damage. Figure 5D and E showed that RSL3 stimulation resulted in intense green fluorescence, indicating MMP depolarization. As anticipated, Se@MelP treatment effectively preserved the MMP. Similarly, MitoSOX staining showed that RSL3 stimulation caused excessive mitochondrial ROS production, whereas Se@MelP treatment significantly reduced its generation (Fig. 5F and Fig. S7). Collectively, the results demonstrated that Se@MelP protected AML-12 cells against RSL3-induced ferroptosis by mitigating ROS accumulation and lipid peroxidation, reducing labile Fe²⁺, and maintaining mitochondrial integrity.

Fig. 5.

The anti-ferroptotic effect of Se@MelP against RSL3-induced injury in AML-12 cells. DCFH-DA staining (A) and quantification (B) of AML-12 cells treated with 1 µM RSL3 and SeNPs, MelNPs, or Se@MelP for 12 h. Scale bar: 100 μm. (C) Labile iron detection in AML-12 cells using FerroOrange staining and quantitative analysis. Scale bar: 50 μm. Fluorescent images (D) and quantification (E) of MMP in AML-12 cells. Scale bar: 50 μm. (F) Fluorescent images of MitoSOX-stained in AML-12 cells. Scale bar: 50 μm. Data are mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

To further evaluate the protective effect of Se@MelP, a hypoxia/reoxygenation (H/R) model was established in AML-12 cells to mimic an in vitro LIRI model. Following H/R induction, AML-12 cells exhibited increased ROS levels, Fe²⁺ accumulation, and MMP depolarization. Notably, Se@MelP treatment effectively suppressed these alterations (Fig. S8). These results indicated that Se@MelP exerted a potent protective effect against oxidative stress, iron metabolism dysregulation, and mitochondrial dysfunction in AML-12 cells after H/R stimulation.

Distribution study and biosafety of the Se@MelP

To visualize in vivo distribution, ICG-labeled Se@MelP was intravenously administered to normal mice and monitored in real-time. As shown in Fig. 6A and B, Se@MelP exhibited a pronounced liver enrichment trend as early as 2 h post-injection and reached peak fluorescence intensity at 4 h, indicating its rapid and significant accumulation in the liver. Ex vivo fluorescence imaging of major organs harvested at 24 h further confirmed the passive liver targeting capability of Se@MelP, with the liver exhibiting a significantly higher fluorescence signal compared to other organs (Fig. 6C). These results demonstrated that Se@MelP could effectively accumulate in the liver, providing a possibility for its subsequent therapeutic intervention in LIRI.

Fig. 6.

In vivo distribution and biosafety of the Se@MelP. In vivo fluorescence images (A) and quantification (B) of ICG-labeled Se@MelP biodistribution following intravenous administration. (C) Ex vivo fluorescent images and quantification of major organs. (D) Hemolysis analysis of Se@MelP. (E) The blood routine analysis of mice after intravenous injection of saline and Se@MelP. (F) H&E staining images of the major organs harvested from mice after indicated treatment. Scale bar: 200 μm. Data are mean ± SD (n = 3)

Next, the biosafety of Se@MelP was comprehensively evaluated. As shown in Fig. 6D, the hemolysis rate remained below 5% even at a concentration of 400 µg/mL, indicating the excellent blood compatibility of Se@MelP. To evaluate in vivo safety, mice were administered Se@MelP and subsequently euthanized for the collection of blood samples and major organs. Blood routine analysis showed that all parameters of the whole blood cells remained within the normal physiological range, with no detectable abnormalities (Fig. 6E). Meanwhile, histological examination of the major organs by H&E staining showed that all tissues appeared normal, with no obvious pathological changes (Fig. 6F). In summary, Se@MelP exhibited excellent biosafety and biocompatibility, supporting its potential for subsequent in vivo therapeutic applications.

In vivo protective effect of Se@MelP in LIRI model

To further evaluate the hepatoprotective effect of Se@MelP against LIRI, a mouse model was successfully established by occluding blood flow to the left and middle liver lobes for 0.5 h. The clinically used hepatoprotective drug Silybin, known for its antioxidant and anti-inflammatory properties, was used as a positive control, as recent studies have also demonstrated its ability to inhibit ferroptosis-related injury [54, 55]. Following injection with saline, SeNPs, MelNPs, Se@MelP, or Silybin, serum and liver tissue samples were collected from LIRI mice for subsequent analysis (Fig. 7A). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), critical clinical biomarkers, served as the primary indicators for assessing the extent of liver injury. The IR group exhibited a significant elevation in serum ALT and AST levels, reaching 10,957 U/L and 19,901 U/L, respectively, indicating severe liver injury (Fig. 7B and C). While SeNPs, MelNPs, and Silybin treatments resulted in a modest reduction, Se@MelP treatment significantly attenuated the serum ALT and AST levels caused by IR. Subsequently, H&E staining of liver tissues was performed to visually assess the liver injury induced by IR and the therapeutic effect of each treatment. As shown in Fig. 7D, severe structural damage and extensive necrotic areas were observed in the IR group. Treatment with SeNPs, MelNPs, or Silybin afforded partial mitigation of liver injury and reduced the necrotic areas. Notably, the Se@MelP group exhibited the smallest area of liver damage, confirming its superior hepatoprotective efficacy. Similarly, TUNEL staining revealed significant hepatocellular damage in the liver of the IR group. Se@MelP treatment most significantly reduced the number of TUNEL-positive cells, indicating hepatocyte apoptosis was markedly inhibited (Fig. 7E). These results collectively provided evidence that Se@MelP effectively protected against hepatocyte injury during LIRI.

Fig. 7.

In vivo liver protective effect of Se@MelP. (A) Schematic of the therapeutic protocol in LIRI mice. LIRI mice were injected with saline, SeNPs, MelNPs, Se@MelP, or Silybin, after which serum and liver samples were collected for further analysis. Assessment of serum levels of ALT (B) and AST (C) (n = 6). (D) Representative H&E staining images of liver tissues from each group. The injured regions are outlined by the dashed line. Scale bar: 200 μm. (E) TUNEL staining (green) indicates apoptotic cells in liver tissues. Scale bar: 200 μm. (F-H) Levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in liver tissue (n = 6). Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Given that inflammatory infiltration is a pathological hallmark of tissue damage in LIRI [2], the expression of pro-inflammatory cytokines in liver tissues was assessed. As can be seen from Fig. 7F-H, the levels of IL-1β, IL-6, and TNF-α were markedly elevated following IR but significantly reduced upon Se@MelP treatment, closely approaching control levels. This reduction confirmed the effective attenuation of liver inflammation by Se@MelP.

Then, analysis of antioxidant enzyme activity and DHE staining in liver tissue further revealed the critical antioxidative role of Se@MelP in the LIRI model. GPX and superoxide dismutase (SOD), key antioxidant enzymes in vivo, play an essential role in protecting against oxidative damage. As shown in Fig. 8A and B, the levels of GPX and SOD were significantly decreased in the IR group, indicating a severe oxidative stress state. SeNPs, MelNPs, or Silybin treatments increased the levels of GPX and SOD in the IR injured liver tissue, whereas Se@MelP treatment most effectively restored their levels. Moreover, DHE staining provided direct visualization of oxidative stress. As expected, the fluorescence intensity was markedly suppressed by Se@MelP treatment compared to the IR group (Fig. 8C and D), indicating effective alleviation of IR-induced oxidative stress.

Fig. 8.

Antioxidant and anti-ferroptotic effects of Se@MelP in LIRI mice. Assessment of levels of GPX (A) and SOD (B) in liver tissues (n = 6). (C-D) Representative DHE staining images and quantification (n = 3). Scale bar: 100 μm. (E) ACSL4 staining in liver sections. Scale bar: 100 μm. (F) 4-HNE staining in liver sections, indicating lipid peroxidation. Scale bar: 50 μm. Assessment of levels of MDA (G) and GSH (H) in liver tissues (n = 6). (I) Immunofluorescence images of GPX4 in liver tissues from each group. Scale bar: 50 μm. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Ferroptosis triggers extensive lipid peroxidation of cell membranes, characterized by Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) upregulation and the generation of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) as byproducts [56]. ACSL4 plays a critical role in the biosynthesis of polyunsaturated fatty acid-containing phospholipids and serves as a key positive regulator of ferroptosis. In the IR group, the ACSL4 level was markedly higher than that in the control group. After different treatments, ACSL4 levels were reduced to varying degrees in all groups, with the most pronounced reduction observed in the Se@MelP group (Fig. 8E). Subsequently, we investigated the expression levels of 4-HNE and MDA. As shown in Fig. 8F and G, the IR group exhibited significant accumulation of 4-HNE and MDA, indicating the activation of ferroptosis. Se@MelP treatment effectively reduced the accumulation of these lipid peroxides. Another hallmark of ferroptosis is the depletion of glutathione (GSH) and the inactivation of GPX4. In Fig. 8H-I and Fig. S9, GSH levels and GPX4 expression were significantly decreased in the IR group. After Se@MelP treatment, both GSH levels and GPX4 expression were restored most prominently. Notably, Se@MelP was superior to the positive control drug Silybin in inhibiting ACSL4 levels and lipid peroxides, as well as in increasing GSH levels and GPX4 expression. Collectively, these results suggested that Se@MelP effectively inhibited ferroptosis during LIRI.

Therapeutic mechanisms of Se@MelP in LIRI model

To investigate the underlying therapeutic mechanism of Se@MelP, RNA-seq was performed on liver tissues of mice in the IR group and IR + Se@MelP group. Clear separation between the two groups was observed by principal component analysis (PCA), indicating significant transcriptional differences (Fig. 9A). A total of 2584 differentially expressed genes (DEGs) were identified between IR and IR + Se@MelP mice via volcano plot analysis, with 1393 upregulated and 1191 downregulated (Fig. 9B). Furthermore, the gene expression pattern of liver tissues from the IR + Se@MelP group was distinctly different from that of the IR group in the hierarchical clustering analysis (Fig. 9C). Gene Ontology (GO) analysis showed that the DEGs were enriched in biological processes related to lipid metabolism and oxidative stress, such as unsaturated fatty acid metabolic process, lipid oxidation, oxidoreductase activity (Fig. 9D). Notably, iron ion binding was also among the enriched pathways. These findings suggested that the protective effect of Se@MelP against IR injury may be mediated through the coordinated modulation of cellular redox balance and iron homeostasis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further revealed significant enrichment in the ferroptosis and glutathione metabolism pathways (Fig. 9E). Interestingly, the AMPK signaling pathway was also found to be enriched. Recent studies have shown that AMPK can phosphorylate and further activate Nrf2, a master regulator of the antioxidant response, thereby modulating downstream proteins to enhance endogenous antioxidant defenses and anti-ferroptotic capacity [57, 58]. Here, western blot analysis was employed to investigate whether the AMPK/Nrf2 pathway mediated the therapeutic effect of Se@MelP against LIRI. As shown in Fig. 9F and G, the IR group exhibited a marked reduction in the p-AMPK/AMPK ratio and Nrf2 level, accompanied by increased expression of Keap1, which acts as a negative regulator by promoting Nrf2 degradation [59]. These changes collectively indicated suppression of the AMPK/Nrf2 pathway during LIRI progression, which was effectively reversed by Se@MelP. It is worth noting that, compared with SeNPs, MelNPs, and Silybin, Se@MelP more effectively upregulated p-AMPK and Nrf2 while downregulating Keap1, indicating its enhanced ability to modulate the AMPK/Nrf2 pathway. Furthermore, Se@MelP most significantly upregulated the expression of ferroptosis regulator GPX4 and the antioxidant enzyme heme oxygenase‑1 (HO‑1) via Nrf2 activation. To further elucidate the molecular mechanism of Se@MelP in regulating the AMPK/Nrf2 pathway, the AMPK inhibitor Dorsomorphin (Dor) and the Nrf2 inhibitor ML385 were used at the cellular level. TBHP treatment decreased AMPK phosphorylation levels and Nrf2 expression, and increased Keap1 expression. However, Se@MelP reversed these changes and increased the levels of GPX4 and HO-1 (Fig. S10). Notably, Dorsomorphin treatment abolished the increases in p-AMPK and Nrf2 levels induced by Se@MelP, and increased Keap1 expression. Furthermore, ML385 did not affect AMPK phosphorylation levels but blocked Se@MelP-induced upregulation of GPX4 and HO-1. Collectively, the results suggested that Se@MelP regulated the AMPK/Nrf2 pathway to enhance antioxidant defenses and anti‑ferroptotic capacity against IR‑induced liver damage (Fig. 9H).

Fig. 9.

Therapeutic mechanisms of Se@MelP on LIRI. (A) PCA of liver tissues between the IR and IR + Se@MelP groups. (B) Volcano plot of DEGs. (C) Hierarchical clustering analysis of gene expression patterns. (D) GO enrichment analysis of DEGs. (E) KEGG pathway enrichment analysis of DEGs. Western blot analysis (F) and quantification (G) showing liver levels of p-AMPK/AMPK, Nrf2, Keap1, GPX4, and HO-1 (n = 3). (H) Regulatory pathway activation by Se@MelP to enhance endogenous antioxidant defenses and anti-ferroptotic capacity. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Limitations

We acknowledge that this study lacked further investigation into the in vivo degradation behavior of Se@MelP (e.g., degradation rate, degradation pathway). Given that our work focused on validating the feasibility of Se@MelP for LIRI and elucidating its protective mechanism, investigation of this degradation behavior was not performed here and will be the focus of our subsequent translational research.

Conclusions

In summary, a multi-bioactive nanoplatform Se@MelP was developed to alleviate oxidative stress and iron dyshomeostasis during LIRI. Se@MelP exhibited excellent biosafety and broad-spectrum ROS scavenging capacity, including ·OH, O₂⁻, DPPH·, and ABTS·⁺. Moreover, melanin-like shell endowed Se@MelP with effective iron chelating capability. Then in vitro experiments showed that Se@MelP treatment protected cells from oxidative stress and ferroptosis damage by eliminating intracellular ROS, reducing Fe²⁺ overload, and preserving mitochondrial integrity. Upon intravenous injection, Se@MelP was passively enriched in the liver, and in the LIRI model, it markedly improved liver function, alleviated oxidative stress and inflammatory infiltration, and inhibited ferroptosis. Further mechanistic investigation revealed that Se@MelP exerted its protective effects by activating the AMPK/Nrf2 pathway to upregulate the expression of HO‑1 and GPX4, thereby enhancing antioxidant defenses and anti‑ferroptotic capacity. This study demonstrates that Se@MelP represents a promising therapeutic strategy against LIRI, highlighting its potential for further development in clinical settings.

Experimental section

Detailed materials and experimental protocols are available in the Supporting Information.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

J.D., J.H.S. and R.P.Z. designed and supervised the study. J.D., P.Q.Z., C.F.G., Q.W., J.Z., Y.H.H., and R.F. performed all experiments and conducted data analysis and interpretation. J.D., P.Q.Z., C.F.G.,and J.H.S. participated in data analysis and result interpretation. J.D. drafted the manuscript. T.F.C., J.H.S. and R.P.Z. provided critical feedback. All authors reviewed and approved the final version of the manuscript.

Funding

Funding sources are inserted in acknowledgment part.

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

The eight-week-old male ICR mice were purchased from the Experimental Animal Center of Shanxi Medical University. All animal experiments were approved by the Institutional Animal Experiment Committee of Shanxi Medical University (Approval No. 2022-025, Taiyuan, China).

Consent for publication

All authors consent for publication.

Competing interests

The authors declare no competing interests.