Abstract Background: Acute liver failure (ALF) represents a critical medical condition marked by the abrupt onset of hepatocyte damage, commonly induced by etiological factors such as hepatic ischemia/reperfusion injury (HIRI) and drug-induced hepatotoxicity. Across various types of liver injury, oxidative stress, heightened inflammatory responses, and dysregulated hepatic retinol metabolism are pivotal contributors, particularly in the context of excessive reactive oxygen species (ROS). Methods: C-dots were combined with Cu[5.4]O USNPs to synthesize a cost-effective nanozyme, Cu[5.4]O@CNDs, which mimics the activity of cascade enzymes. The in vitro evaluation demonstrated the ROS scavenging and anti-inflammatory capacity of Cu[5.4]O@CNDs. The therapeutic potential of Cu[5.4]O@CNDs was evaluated in vivo using mouse models of hepatic ischemia/reperfusion injury and LPS/D-GalN induced hepatitis, with transcriptome analysis conducted to clarify the mechanism underlying hepatoprotection. Results: The Cu[5.4]O@CNDs demonstrated superoxide dismutase (SOD) and catalase (CAT) enzyme activities, as well as hydroxyl radical (·OH) scavenging capabilities, effectively mitigating ROS in vitro. Furthermore, the Cu[5.4]O@CNDs exhibited remarkable targeting efficacy towards inflammation cells induced by H[2]O[2] and hepatic tissues in murine models of hepatitis, alongside exhibiting favorable biocompatibility in both in vitro and in vivo settings. Moreover, it has been demonstrated that Cu[5.4]O@CNDs effectively scavenged ROS, thereby enhancing cell survival in vitro. Additionally, Cu[5.4]O@CNDs exhibited significant therapeutic efficacy in mice models of HIRI and lipopolysaccharide-induced acute lung injury (LPS-ALI). This efficacy was achieved through the modulation of the ROS response and hepatic inflammatory network, as well as the amelioration of disruptions in hepatic retinol metabolism. Conclusions: In summary, this study demonstrates that Cu[5.4]O@CNDs exhibit significant potential for the treatment of various acute liver injury conditions, suggesting their promise as an intervention strategy for clinical application. Keywords: C-dots nanozymes, Cu[5.4]O nanoparticles, Reactive oxygen species, Hepatic ischemia-reperfusion injury, Acute liver injury Introduction Acute liver failure (ALF) is a clinical syndrome characterized by extensive hepatocellular necrosis, hepatic hypoplasia, and multiorgan dysfunction [45]^1^, [46]^2. Clinical symptoms of ALF usually include hepatic dysfunction, abnormalities in liver biochemical indices, and coagulation dysfunction [47]^3. Despite the relatively low morbidity rate, up to half of all cases may present with multiorgan failure and death, with a mortality rate of up to 30%. Consequently, there is an urgent requirement to devise effective therapeutic strategies addressing the causes of acute liver failure [48]^4^, [49]^5. The etiology of ALF includes hepatic ischemia-reperfusion injury (HIRI), viral infections, autoimmune hepatitis, and various other triggers of acute liver injury (ALI) [50]^6. Several recent studies suggested that high levels of oxidative stress and inflammatory responses in vivo played a vital role in all types of liver injury, with reactive oxygen species (ROS) being critical [51]^7^, [52]^8. Hepatocytes are rich in mitochondria. Disruption of the mitochondrial electron transport chain by hepatotoxic compounds, their active metabolites, and factors like hypoxia and reoxidation leads to excessive ROS production [53]^9. Due to its powerful oxidizing properties, ROS are important inflammatory mediators and cause cellular damage at high concentrations. The main reactive oxygen species (ROS) are superoxide anion radical (O[2]^•-), hydroxyl radical (·OH), and hydrogen peroxide (H[2]O[2]) [54]^10. In addition, inflammation is triggered by hepatic injury, followed by the derivation of ROS mainly from activated inflammatory cells and hepatic sinusoidal endothelial cells (LSECs), which further dysregulate hepatic redox homeostasis [55]^10^, [56]^11, creating a vicious cycle of continuous stimulation. The liver plays a crucial role in metabolism and detoxification. Numerous enzymes in the liver have overlapping substrate specificities and are typically classified as phase I (oxidative) or phase II (conjugative) drug-metabolizing enzymes. The cytochrome P450 (CYP) superfamily accounts for nearly 90% of phase I metabolic processes [57]^12^, [58]^13. In addition, hepatic stellate cells (HSCs) store 50%-95% of human vitamin A [59]^14, which consists of a series of retinol compounds, including retinol, retinoic acid, and retinaldehyde [60]^15. CYP450 enzymes play an essential role in retinol metabolism, mainly involving the CYP1, CYP2C, CYP3A, and CYP26 families [61]^16. The current study suggests that regulation of retinol metabolic homeostasis is a defining feature of hepatic stellate cells (HSCs) in both healthy and injured livers [62]^14. Targeting excess ROS to alleviate oxidative stress, regulate inflammation, inhibit persistent stimuli, and stabilize retinol metabolism could effectively treat acute liver injury. A robust intracellular antioxidant enzyme system, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), is recognized for its ability to scavenge ROS [63]^11^, [64]^17. However, this system is insufficient to address liver injury effectively [65]^18^, [66]^19. Antioxidant nanozymes, for example, noble metal (Au, Pt, Pd, etc.) nanoparticles, metal oxide (CeO[2], Mn[3]O[4], Co[3]O[4], etc.) [67]^20^-[68]^24, synthesized from nanomaterials, are promising therapeutic agents due to their multifunctionality, including diverse antioxidant enzyme activities, catalytic activity modulation, and liver-targeted delivery through structural, compositional, and size adjustments. Our research indicates that carbon dots (C-dots) nanozymes possess SOD-like activity, effectively scavenging free radicals and offering therapeutic advantages for hepatic ischemia/reperfusion (I/R) injury [69]^25. However, C-dots, which have only one type of enzymatic activity and generate toxic H[2]O[2] after scavenging O[2]^•-, still have not completely solved the problem of reactive oxygen overload. It has been shown that the coupling of noble metal nanoparticles with carbon materials allows hybridized nanozymes to achieve higher catalytic activity due to charge transfer at the metal-carbon interface and facilitated adsorption on the carbon substrate [70]^26. For example, our previous study reported that Pt@CNDs nanocomposites have higher SOD and CAT catalytic activities than individual CNDs and PtNPs [71]^27. Cu is an essential trace element that plays an important role in many enzymes such as tyrosinase and Cu - Zn SOD. Therefore, Cu-based nanomaterials are often developed as nanozymes with the ability to scavenge reactive oxygen species. For example, Cu NPs have excellent catalytic activity and can scavenge H[2]O[2] and O[2]^•-, but cannot eliminate ·OH simultaneously [72]^28. Cu[2]O NPs have good catalytic activity to inactivate H[2]O[2] or ·OH, thus partially mimicking peroxidase [73]^29. Thus, Cu[5.4]O USNPs are designed to achieve both broader-spectrum enzyme catalytic performance and antioxidant activity by combining Cu[2]O and Cu nanocrystals [74]^30. It was shown that the synergistic catalytic activity of Cu^0 and Cu^1+ arises from matching the rates of activation of the reactants, i.e., the rate of activation of H[2] by Cu^0 and the rate of activation of carbonyl-containing reactants by Cu^1+ [75]^31. Very interestingly, in our previous studies, we found that the hydroxyl group on the surface of CNDs binds to O[2]^•-, which can serve as a catalytic binding site, and the carbonyl group serves as the center of its catalytic activity, i.e., the SOD-like enzyme activity of CNDs is dependent on the coupling of the carbonyl group on its surface with the π- system. Therefore, combining these two nanozymes to synthesize hybrid nanozymes can have the advantages of both high catalytic activity and SOD-CAT cascade nanozymes, and the raw materials are more economical and stable. This study synthesized Cu[5.4]O@CNDs with cascade mimetic enzyme activity, which protected the liver from ROS-induced stress and inflammation and improved retinol metabolism disorders, resulting in effective therapeutic outcomes (Scheme [76]1). It demonstrated that Cu[5.4]O@CNDs could enhance the cellular status and survival of THLE-2 and Raw264.7 cells under oxidative stress and inflammatory conditions in vitro. In models of HIRI and LPS-induced ALI, the administration of Cu[5.4]O@CNDs effectively scavenged ROS, reduced the expression of inflammatory cytokines, and exhibited significant therapeutic effects. Transcriptome sequencing revealed that Cu[5.4]O@CNDs provide hepatoprotection by modulating the ROS response and hepatic inflammatory network, disrupting the hepatic retinol metabolism pathway, and inhibiting apoptosis. The study highlights Cu[5.4]O@CNDs as a promising intervention for acute liver injury, supporting its potential for clinical application. Scheme 1. [77]Scheme 1 [78]Open in a new tab Schematic representation of Cu[5.4]O@CNDs for the treatment of HIRI and LPS-ALI diseases. (A) Synthesis route of Cu[5.4]O@CNDs with multiple enzymatic activities. (B) Cu[5.4]O@CNDs were injected into mice via the tail vein to scavenge excess ROS as free radical scavengers and to treat various acute liver injury diseases through anti-inflammatory, antioxidant, and regulation of retinol metabolism. Materials and methods Synthesis of Cu[5.4]O@CNDs The C-dots were synthesized following the previously reported methodology [79]^25. Initially, 0.5 g of activated carbon was introduced into a boiling 50 mL mixed acid solution (:=1:1) and maintained for 1.5 h. After cooling to room temperature, the C-dots samples were neutralized using a NaHCO[3] solution. The neutralized solution was filtered using a 0.22 μm aqueous membrane and dialyzed 4-5 times daily for a week. The dialyzed C-dots solution was further filtered through a 0.22 μm aqueous membrane to remove any remaining insoluble materials. The filtrate was ultrafiltered with a 100 kDa molecular retention ultrafiltration tube. After separation, C-dots were concentrated and lyophilized for utilization in the subsequent experiments. The Cu[5.4]O USNPs were synthesized according to the methodology reported previously. CuCl[2] powders (10 mM) were dissolved in 50 mL of deionized water and stirred magnetically in an oil bath at 80 °C for 10 min. The CuCl[2] solution was incrementally combined with a 50 mL aqueous solution of L-ascorbic acid (100 mM). After that, a NaOH solution (1 M) was used to bring the solution's pH down to 8.0-9.0. The mixture was continuously stirred for 12 h at 80 °C. Centrifugation (6,500 × g, 15 min) was used to remove the bigger aggregates following the reaction. The supernatant was dialyzed against water using a 10,000 Da molecular weight cutoff for 2 days to eliminate small molecules [80]^30^, [81]^32. According to the previous report, the synthesis pathway of Cu[5.4]O@CNDs has some modifications. Initially, 15 mM CuCl[2] powder was dissolved in a 50 mL C-dots aqueous solution (3 mg/mL) and stirred magnetically at 80 °C for 10 min in an oil bath. Subsequently, a 300 mM L-AA aqueous solution (50 mL) was gradually added to the resulting CuCl[2] solution. Afterward, the solution's pH was then brought down to 7.0-8.0 with a 1 M NaOH solution. For 10 h, the mixture was continuously stirred and maintained at 80 °C. Post-reaction, larger aggregates were eliminated via centrifugation at 6,577 × g for 15 min. After dialyzing against water for 2 days (Mw cutoff: 3,500 Da), the supernatant was lyophilized in preparation for further experiments. Meanwhile, CNDs-A, CNDs-B, Re-CNDs-A, CNDs-Cy5.5 were used to replace CNDs to obtain Cu[5.4]O@CNDs-A, Cu[5.4]O@CNDs-B, Cu[5.4]O@Re-CNDs-A or Cu[5.4]O@CNDs-Cy5.5. The SOD-like activity of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs To evaluate the SOD-like activity, a commercial SOD assay kit was used, in which the reaction substrate WST reacted with O[2]^•- to generate a water-soluble formazan that exhibited a distinctive absorption peak at 450 nm. When the disproportionation of O[2]^•- was impeded, indicating the presence of SOD-like activity in the test sample, the enzymatic activity of SOD was determined through colorimetric analysis of the WST product. The O[2]^•- scavenging activity of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs The scavenging activity of O[2]^•- was assessed using NBT reduction, a method that produces a blue-colored formazan in the presence of O[2]^•-. When C-dots with SOD-like activity are present, they competitively scavenge O[2]^•-, resulting in a lighter or absent blue color. Samples with six concentrations (0-200 μg/mL) were combined with NBT (0.05 mM), L-met (13 mM), and riboflavin (20 μM) in a PBS buffer (pH 7.4, 25 mM) for investigation. The mixture was then irradiated by LED for 5 min. The scavenging activity of O[2]^•- was assessed using ESR assay. Hypoxanthine/xanthine oxidase (HYP/XOD) produces O[2]^•- by mixing DMPO, DTPA, HYP, XOD, and Cu[5.4]O@CNDs in PBS (pH = 7.3), and the ESR spectra of BMPO/⋅OOH were recorded after 2 min. The CAT-like activity of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs O[2] production from H[2]O[2] scavenging by different materials was determined using dissolved oxygen. A reaction system with a final volume of 15 mL of ultrapure water containing 60 μL of 30% H[2]O[2] and various concentrations of different materials was then kinetically measured using a dissolved oxygen meter (JPSJ-605F, Ray Magnet) for 15 min. The H[2]O[2] scavenging activity of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs The Hydrogen Peroxide Detection Kit was used to evaluate the H[2]O[2] scavenging capacity of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs. An absorbance peak at 405 nm is observed when ammonium molybdate reacts with H[2]O[2]. Samples were incubated at 37 °C for 2 h with different concentrations of 2 mM H[2]O[2], following the manufacturer's instructions. The scavenging activity of H[2]O[2] was assessed using ESR assay. Different 100 µg/mL materials catalyzed the degradation of H[2]O[2] to generate O[2]. The ESR spin probe CTPO was employed to capture the O[2], and the ESR spectra of CTPO exhibited a proton hyperfine structure in a control nitrogen-saturated solution. Increasing O[2] concentration elevated the collision frequency between oxygen and nitrogen-oxygen radicals, leading to a broadened ESR triplet state spectrum and reduced resolution of the proton hyperfine structure. The ⋅OH scavenging activity of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs The ⋅OH scavenging activity was evaluated via the TMB assay, utilizing solutions of 10 µM FeCl[2], 50 µM H[2]O[2], and 300 µM TMB, all prepared with ultrapure water. The three solutions were then mixed homogeneously. The mixtures were prepared by thoroughly combining with sample solutions ranging from 0 to 200 µg/mL and allowed to react for 30 min at room temperature, followed by absorbance measurement at 645 nm. The scavenging activity of ⋅OH was assessed using an ESR assay. The Fenton reaction produced ⋅OH by combining 100 mM DMPO, 1.0 mM FeSO[4], 1.0 mM H[2]O[2], and 100 µg/mL Cu[5.4]O@CNDs in ddH[2]O. ABTS^•+ radical scavenging activity of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs Following instructions, the ABTS^•+ radical scavenging capacity of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs was assessed using the T-AOC analysis kit (S0119, Beyotime). The ABTS^•+ and oxidant solution were combined in a 1:1 volume ratio to create a fresh working fluid, which was then stored at ambient temperature and shielded from light for 12-16 h before utilization. The working mix was subsequently diluted according to the kit instructions, and the samples were analyzed at various concentrations of different materials (0-200 μg/mL) as specified. Cell culture THLE-2 cells were maintained at 37 °C in a humidified incubator with 5% CO[2] using a cell-specific medium. Raw 264.7 cells were maintained in DMEM (Gibco) with 10% fetal bovine serum and antibiotics at 37°C in a 5% CO[2] humidified incubator. Cell targeting Preparation of Cy5.5-loaded Cu[5.4]O@CNDs for cellular uptake studies. THLE-2 and Raw 264.7 cells were each incubated for 24 h before being cocultured with Cu[5.4]O@CNDs for an additional 4 h. Subsequently, the cells were fixed, stained with DAPI, and washed multiple times. The cells were then imaged with a fluorescence microscope. In vitro oxidative stress and inflammation model To examine the capacity of Cu[5.4]O@CNDs to scavenge ROS in cells, firstly, the cells underwent 12 h of nutrient deprivation. Then, an oxidative stress model was established in vitro. After a 24 h incubation using the specified cell culture method, cells were treated with Cu[5.4]O@CNDs at concentrations ranging from 0 to 80 µg/mL for 8-12 h. They were then exposed to either 1 mM H[2]O[2] or 1 µg/mL LPS and incubated at 37 °C for an additional 1 or 8 h. In vitro ROS scavenging using Cu[5.4]O@CNDs nanozymes After the treatment, the cells were gently rinsed with a serum-free medium. Cells were incubated in darkness at 37 °C for 30 min in a serum-free medium containing 10 μM of 2,7-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE). Following incubation, cells underwent three washes to eliminate surplus DCFH-DA and DHE probes. Then, the cells were imaged using fluorescence microscopy, and flow cytometry analysis was performed to quantify intracellular ROS levels. Furthermore, the treated cells were stained with 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) for 30 min. Fluorescence microscopy was employed to assess cell protection by Cu[5.4]O@CNDs. In vitro anti-inflammatory using Cu[5.4]O@CNDs nanozymes After the incubation of Raw 264.7, the cells were separately incubated with Cu[5.4]O@CNDs for 8 h, followed by overnight incubation with 1 μg/mL lipopolysaccharide (LPS). Cells were stained with CD 80 marker cells followed by flow cytometry to assess the extent of macrophage polarization. THLE-2 and Raw 264.7 cells were incubated in a 6-well plate and stained with JC-1 for 30 min. Following incubation, the cells underwent three washes to remove any residual JC-1 probe. Fluorescence microscopy was employed to assess cell protection by Cu[5.4]O@CNDs. THLE-2 and Raw 264.7 cells were incubated Cu[5.4]O@CNDs for 8 h, followed by an overnight incubation with 1 μg/mL LPS. Cells were collected, RNA was extracted, and mRNA expression levels of inflammatory factors IL-1β, IL-6, IL-12, and TNF-α were measured using an rt-QPCR kit. The primer sequences of related genes are shown in [82]Table S1. In vivo biodistribution Initially, Cy5.5-containing nanosystems were prepared at a dose of 0.5 mg/kg. Subsequently, BALB/C mice were randomly divided into two groups: healthy mice and those with hepatitis. Each group was randomized into two groups: the Cy5.5-labeled group, and the Cu[5.4]O@CNDs group. The in vivo distribution of nanomaterials was assessed using a near-infrared small animal imager within 0-4 h following tail vein injection. At the endpoint, mice were euthanized, and their heart, liver, spleen, lung, and kidney tissues were collected for ex vivo fluorescence imaging. Biocompatibility of C-dots nanozymes Cell viability was evaluated via the MTT assay after co-incubation with Cu[5.4]O@CNDs nanozymes at concentrations ranging from 0 to 100 μg/mL for 12 and 24 h, following the specified cell culture protocol. The in vivo biocompatibility of Cu[5.4]O@CNDs was assessed by intravenously administering 0.5 mg/kg of Cu[5.4]O@CNDs to BALB/c mice. Control mice were injected with PBS. Controls were mice injected with PBS. Blood samples were collected for complete blood cell analysis and serum biochemical tests after 1 day and 7 consecutive days of injection. The mice were euthanized, and their major organs were collected for histological examination using H&E staining. HIRI model in mice Male BALB/c mice aged 6-8 weeks were selected [83]^25. Anesthesia was administered using a small animal anesthesia machine. A midline abdominal incision was made to expose the liver, and noninvasive vascular clips were used to occlude the left branch of the hepatic artery, the left hepatic duct, and the portal vein. The left and middle lobes of the liver appeared white, indicating 70% partial ischemia. After 1 h, the clamps were removed, and the liver tissue became red and moist, this confirms that circulation has been restored. The abdominal incision was then sutured. Mice were euthanized 24 h after blood flow was restored. In vivo therapeutic effect of Cu[5.4]O@CNDs on HIRI BALB/c mice with an established HIRI model were randomly assigned to six groups (n = 5): Sham-operated (control), HIRI, and Cu[5.4]O@CND therapeutic doses of 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, and 2.5 mg/kg. Mice weight changes in each group were monitored 24 h post-treatment. 24 h after injection, mice were euthanized, and blood samples were collected to quantify the levels of albumin transaminase (AST) and alanine transaminase (ALT). Liver tissues were collected and homogenized to determine the transcription levels of pro-inflammatory factors. The primer sequences of related genes are shown in [84]Table S1. At the same time, a portion of the liver tissue was paraffin-embedded for H&E and TUNEL staining. The other portion of liver tissue was freeze-embedded and cryosectioned. The frozen liver sections were stained with DAPI and DCFH/DHE. Fluorescence microscopy is used to qualitatively evaluate ROS levels in liver tissue sections. LPS-ALI model in mice Male BALB/c mice, aged 6-8 weeks, were selected and underwent a 15 h period of food and water deprivation. They were injected intraperitoneally with 30 μg/mL LPS, 200 mg/kg D-gal, and euthanized after 12 h. In vivo therapeutic effect of Cu[5.4]O@CNDs on LPS-ALI BALB/c mice with an established LPS-ALI model were randomly assigned to three groups (n = 5): a PBS control group, an LPS-ALI group, and a treatment group receiving 0.5 mg/kg of Cu[5.4]O@CNDs. Mice were executed after 12 h, serum was taken, liver tissue was removed, and the same validation experiments as in the HIRI model were performed. The primer sequences of related genes are shown in [85]Table S1. Transcriptome analysis HIRI and LPS-ALI model mice were divided into three groups: Con, modeling group, and treatment group, respectively. Liver tissues were collected from mice post-experiment and kept on ice. RNA was isolated from liver tissue, and its purity and concentration were assessed using a NanoDrop 2000 spectrophotometer. Sequencing libraries were constructed using high-quality RNA samples meeting the criteria: OD[260/280] = 1.8-2.2, RIN ≥ 6.5, 28S: 18S ≥ 1.0, > 2 μg. The library underwent bipartite sequencing using the Illumina NovaseqTM 6000 (LC Bio-Technology CO., Ltd., Hangzhou, China), following standard procedures, with a sequencing mode of PE150. Western blotting Liver tissues were lysed using RIPA buffer (WB3100, NCM Biotech, China) supplemented with a protease inhibitor cocktail (P001, NCM Biotech, China). Protein concentrations were determined using a BCA protein assay kit (WB6501, NCM Biotech, China). Equal amounts of protein were then separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% milk in TBST for 1 h, followed by incubation with primary antibodies overnight at 4 °C. After 1 h of incubation with secondary antibodies, the membranes were visualized using an enhanced chemiluminescence system, and the images were quantified using ImageJ software. Details of the antibodies and their dilutions are provided in [86]Table S2. Statistical analysis The GraphPad Prism 9 software was used to analyze data. A two-tailed Student's t-test was employed for comparing two groups, while one-way ANOVA was utilized for comparisons involving multiple groups. Statistical significance was assessed with thresholds: ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Results and Discussion Transcriptomic sequencing of HIRI and LPS-ALI to analyze the potential pathogenesis The RNA sequencing of acute liver injury disease by the GEO database was analyzed to find an effective treatment for acute liver injury disease. Sample correlation analysis between patients and healthy controls was performed, and the results of PCA analysis suggested that the screened [87]GSE112713 database and [88]GSE38941 database had good intragroup clustering, and the gene expression differences between the healthy control group and the disease group were significant enough to allow for subsequent analysis (Figure [89]1A). GSEA analysis of the two databases separately showed that the ROS metabolic process [90]^8^, [91]^20, inflammatory response pathway [92]^7^, [93]^33, and retinol metabolism [94]^12 were significantly abnormal in patients with HIRI and ALI, which might be an effective therapeutic target (Figure [95]1B). Meanwhile, to verify the consistency of the pathogenesis of acute liver injury at the mice level with that in humans, mice models of HIRI and LPS-ALI were established, and the H&E results suggested that apparent inflammatory cell infiltration and tissue necrosis could be observed (Figure [96]1C). Tissue RNA sequencing revealed statistically significant gene expression differences between control and modeling groups in HIRI and LPS-ALI models, confirming successful disease model establishment for further analysis (Figure [97]1D). Then, based on the sequencing results, GO analysis was based on the differential genes of the two disease models (HIRI and LPS-ALI), and the top 15 metabolic processes were listed. The results suggested that both disease processes were highly correlated with inflammatory responses, ROS metabolic processes, oxidative stress, and retinol metabolism pathways (Figure [98]1E). To further clarify the differential gene expression profiles of our pathways of interest, a heatmap analysis of the differential genes related to the ROS metabolic process, inflammatory response pathway, and retinol metabolic pathway was performed. Significant differences in gene expression between the disease models (HIRI and LPS-ALI) and control mice are evident within the same metabolic pathway (Figure [99]1F-H). The above results demonstrated that ROS metabolic processes, inflammatory response pathways, and retinol metabolism were highly relevant in the pathogenesis of two acute liver injury diseases, HIRI and LPS-ALI. Figure 1. [100]Figure 1 [101]Open in a new tab Transcriptomic sequencing of HIRI and LPS-ALI to analyze potential pathogenesis. (A) PCA analysis was done using the [102]GSE112713 database and the [103]GSE38941 database. (B) GSEA analysis of ROS metabolic process, inflammatory response pathway, and retinol metabolism was done using the [104]GSE112713 database and [105]GSE38941 database. (C) H&E staining of Con, HIRI, and LPS-ALI. (D) Volcano plots showed the identified upregulated and downregulated genes by Cu[5.4]O@CNDs. (E) GO (Biological Process) analysis. The 15 most significantly enriched pathways are shown. (F) Differential gene heat maps associated with Response to ROS (fold change ≥ 2 and P < 0.01). (H) Differential gene heat maps associated with Retinol metabolic process (fold change ≥ 2 and P < 0.01). (G) Differential gene heat maps associated with Inflammatory response (fold change ≥ 2 and P < 0.01). Synthesis and characterization of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs Prior research has demonstrated that C-dots nanozymes serve as free radical scavengers and significantly ameliorate HIRI injury by influencing hepatic inflammation and retinol metabolism pathways [106]^25. Cu[5.4]O USNPs, known for their enzyme mimicry and broad-spectrum ROS scavenging capabilities, have been shown to aid in treating various ROS-related diseases, including acute kidney and liver injuries, by regulating ROS-related genes and modulating multiple classical inflammatory pathways [107]^30. Based on this, a cascade mimetic enzyme Cu[5.4]O@CNDs was designed by combining the two to synthesize green and economical nanozymes. Cu[5.4]O@CNDs, C-dots, and Cu[5.4]O USNPs were synthesized using a simple, efficient one-pot method (Figure [108]2A). The optimal synthesis conditions were determined by adjusting the Cu^2+ to L-ascorbic acid (AA) ratio, C-dots input concentration, pH, and final Cu^2+ concentration to evaluate their impact on the catalytic activity of the resulting materials [109]^30^, [110]^34. The catalysis activity of the synthesized Cu[5.4]O USNPs was almost the same at Cu^2+ to AA feed ratios of 1:5, 1:10, 1:40, and 1:60. The optimal feeding ratio was 1:20, leading to a Cu^2+ to AA molar ratio of 1:20, with a Cu^2+ concentration of 15 mM, C-dots at 3 mg/mL, and a pH range of 7.0-8.0 ([111]Figure S1). Figure 2. [112]Figure 2 [113]Open in a new tab Synthesis and characterization of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (A) Synthesis path diagram of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. The figure was created partially with BioRender.com. (B-D) TEM and HR-TEM images of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs. (E) Zeta potential of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (F) XRD patterns of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (G) FTIR spectra of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (H) XPS patterns of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (I-J) C 1s split-peak fitting profiles of C-dots and Cu[5.4]O@CNDs. (K-L) Cu 2p split-peak fitting profiles of Cu[5.4]O USNPs and Cu[5.4]O@CNDs. Transmission electron microscopy (TEM) analysis revealed that C-dots were homogeneously and monodisperse distributed, with a mean particle size of 2.39 ± 0.50 nm, as depicted in the statistical graph of the particle size distribution. The lattice of 0.21 nm corresponds to the (100) face of graphite [114]^27 (Figure [115]2B and [116]Figure S2A). TEM analysis of Cu[5.4]O USNPs revealed uniformly spherical nanoparticles with an average size of 2.06 ± 0.50 nm, as depicted in the particle size distribution graph. The lattice of 0.24 nm corresponds to the (111) face of Cu[2]O, and the lattice of 0.19 nm corresponds to the (111) face of Cu(0) [117]^35^, [118]^36 (Figure [119]2C and [120]Figure S2B). Cu[5.4]O USNPs and C-dots were synthesized into the polymer, with TEM analysis indicating uniformly spherical nanoparticles and an average particle size of 5.87 ± 0.94 nm. The lattice of 0.21 nm in Cu[5.4]O@CNDs could be ascribed to the (100) facet of graphite observed in C-dots, while the lattice of 0.24 nm could be ascribed to the (111) facet of Cu[2]O observed in Cu[5.4]O USNPs, which can indicate that Cu[5.4]O@CNDs was successfully synthesized by C-dots and Cu[5.4]O USNPs (Figure [121]2D and [122]Figure S2C). The zeta potential of C-dots was -51.7 ± 0.9 mV, and it increased to -60.47 ± 0.67 mV upon the addition of Cu[5.4]O USNP[S], indicating the successful integration of C-dots with Cu[5.4]O USNPS (Figure [123]2E). X-ray powder diffractograms (XRD) results of C-dots, Cu[5.4]O USNP[S], and Cu[5.4]O@CNDs showed that the C-dots had prominent diffraction peaks at 2θ at 25-30° and 42°, and the Cu[5.4]O USNP[S] had obvious 2θ at 30°, 42° and 50° diffraction peaks at 2θ at 25-30°, 42°, 50°. All appeared relative to C-dots and Cu[5.4]O USNP[S], indicating the successful synthetization of Cu[5.4]O@CNDs (Figure [124]2F). The FI-IR results showed that the characteristic peaks at 3300-3500 cm^-1 were attributed to the O-H stretching vibration, while the absorption peaks in the range of 2870-2980 cm^-1 were attributed to the aliphatic hydrocarbon C-H stretching vibration. The characteristic peaks at 1650-1750 cm^-1 and 1350-1450 cm^-1 for the C-dots and Cu[5.4]O@CNDs are attributed to the stretching vibrations of C=O and C-O, respectively. The absorption peaks at 530-622 cm^-1, 774-1075 cm^-1, and 1219 cm^-1 for Cu[5.4]O USNP[S] and Cu[5.4]O@CNDs are characteristic peaks of Cu[2]O. Therefore, the results of FI-IR experiments suggested that Cu[5.4]O@CNDs possessed the characteristic bands of both C-dots and Cu[5.4]O USNPs, which proved the successful synthesis of Cu[5.4]O@CNDs (Figure [125]2G). X-ray photoelectron spectroscopy (XPS) maps are commonly employed to examine the elemental bonding in composites. The total XPS spectra showed the elemental peaks of Na 1s, O 1s, and C 1s in C-dots, and Cu 2p and Cu LM peaks in Cu[5.4]O USNPs, while the elemental peaks of Na 1s, Cu 2p, Cu LM, O 1s, and C 1s were detected in Cu[5.4]O@CNDs, which proved the successful integration of C-dots and Cu[5.4]O USNPS (Figure [126]2H). C 1s fractional peak fitting maps, including graphitic carbon at 284.8 eV, alcoholic carbon at 286.6 eV, carbonyl carbon at 288.1 eV, and carboxy carbon at 288.9 eV, were detected in C-dots and Cu[5.4]O@CNDs, which showed that the characteristic peaks of C-dots appeared in Cu[5.4]O@CNDs (Figure [127]2I-J). The Cu 2p split-peak fitting profiles of Cu[5.4]O USNPS and Cu[5.4]O@CNDs confirmed the presence of Cu^0 and Cu^1+ in Cu[5.4]O@CNDs (Figure [128]2K-L). The above results suggested that the Cu[5.4]O@CNDs were successfully synthesized and retained the respective characteristic structures and characterization of C-dots and Cu[5.4]O USNP[S]. Enzymatic characterization of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs O[2]^•- is a well-known and common type ROS, and the level of SOD-like enzyme activity of the material can be assessed by testing its scavenging rate (Figure [129]3A). Firstly, the NBT reduction method can indirectly detect the SOD-like enzyme activity of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs. In this system, O[2]^•- can make the NBT coloration produce blue formazen. Suppose the material has SOD activity that can remove O[2]^•-. In that case, the coloration of NBT will be weaker and, at the same time, indirectly indicate that there is SOD-like enzyme activity. Under a series of concentration gradients, Cu[5.4]O@CNDs clearly showed superior O[2]^•- scavenging efficiency than Cu[5.4]O USNP[S], which was basically in line with C-dots (Figure [130]3B). The SOD-like enzyme activities of C-dots, Cu[5.4]O USNP[S], and Cu[5.4]O@CNDs were measured using a SOD assay kit. The study found that C-dots exhibited SOD-like enzyme activity of 9609 U/mg, whereas Cu[5.4]O USNPs demonstrated an activity of 3063 U/mg. The SOD-like enzyme activity of Cu[5.4]O@CNDs was 11310 U/mg, superior to that of the other two materials described (Figure [131]3C). In addition, the scavenging ability of Cu[5.4]O@CNDs for O[2]^•- was assessed by ESR, which detected the changes in the intensity of free radical signals. It showed that no ESR signal was present in DMPO alone. In contrast, a distinct signal peak appeared when both DMPO and O[2]^•- were present, indicating that DMPO successfully captured O[2]^•- and exhibited vigorous signal intensity. The addition of 100 μg/mL of C-dots, Cu[5.4]O USNP[S], and Cu[5.4]O@CNDs significantly weakened the signal peak intensity, suggesting that all three materials can remove O[2]^•-. Among them, the signal peaks were weakened to a greater extent after the addition of C-dots and Cu[5.4]O@CNDs, which proved that Cu[5.4]O@CNDs could effectively scavenge O[2]^•- and possess a good antioxidant capacity (Figure [132]3D). Figure 3. [133]Figure 3 [134]Open in a new tab Enzymatic characterization of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (A) Schematic diagram of O[2]^•- scavenging by SOD-like enzymes. (B) The O[2]^•- the scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (C) The WST-1 kit evaluated the SOD-like activity of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs. (D) ESR assay for O[2]^•- scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (E) The schematic diagram for the scavenging of H[2]O[2] by CAT-like enzymes. (F) Ultraviolet absorption test H[2]O[2] scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (G) The dissolved oxygen level of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs reacted with H[2]O[2] detected by an oxygen sensor. (H) ESR assay for H[2]O[2] scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (I) The schematic diagram for the scavenging of ⋅OH and ABTS^•+ radicals. (J) The ⋅OH scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (K) ESR assay for ⋅OH scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. (L) The ABTS^•+ radical scavenging capability of C-dots, Cu[5.4]O USNPs, Cu[5.4]O@CNDs. Data represent means ± s.d. from three independent replicates. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. H[2]O[2], as the product of disproportionation catalyzed by SOD, is also a kind of toxic ROS, and to achieve the purpose of cascade scavenging of ROS, H[2]O[2] needs to be decomposed into non-toxic oxygen and water, which can be catalyzed by catalase (Figure [135]3E). Firstly, in different concentrations of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs in solutions of H[2]O[2] (10 mM), C-dots had no H[2]O[2] scavenging ability, Cu[5.4]O USNPs and Cu[5.4]O@CNDs possessed concentration-dependent decreases in absorption values at 240 nm, and Cu[5.4]O@CNDs had a higher H[2]O[2] scavenging rate than Cu[5.4]O USNPS (Figure [136]3F). Then, the CAT activities of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs were measured by the dissolved oxygen concentration detected by a dissolved oxygen meter. The results showed that in the C-dots group, almost no O[2] was produced, and the Cu[5.4]O@CNDs group produced twice as much O[2] as the Cu[5.4]O USNPs group, which indicated that C-dots had no H[2]O[2] scavenging ability. The rate of H[2]O[2] decomposition by Cu[5.4]O@CNDs could reach about two times that of Cu[5.4]O USNPs. The oxygen production capacity of Cu[5.4]O USNPs and Cu[5.4]O@CNDs increased in a dose-dependent manner (Figure [137]3G). In addition, the generation of O[2] catalyzing the degradation of H[2]O[2] was tested using ESR test materials. The ESR spin probe CTPO was employed to capture oxygen, revealing a proton hyperfine structure in a nitrogen-saturated control solution. As oxygen concentration increased, the collision frequency between oxygen and nitrogen-oxygen radicals rose, leading to a broadening of the ESR triplet state spectrum and a reduction in the resolution of the proton hyperfine structure. The results showed that the fine peak of CTPO did not change after the addition of C-dots, which verified that C-dots do not have H[2]O[2] scavenging ability and only have SOD enzyme activity. When Cu[5.4]O USNPs and Cu[5.4]O@CNDs were added, the CTPO fine peak signals appeared attenuated. Among them, the addition of Cu[5.4]O@CNDs showed a large decrease in the CTPO fine peak signal, which proved that Cu[5.4]O@CNDs had a significantly better H[2]O[2] scavenging ability than the other two materials (Figure [138]3H). ·OH is another important ROS with oxidizing solid properties, so ·OH scavenging can also effectively protect cells from oxidative damage (Figure [139]3I). The TMB method was employed to indirectly evaluate the hydroxyl radical scavenging effect using the Fenton reaction (Fe^2+/H[2]O[2]) to produce hydroxyl radicals, which catalyzed TMB to form soluble blue products. The addition of C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs altered the color, and measurements were taken using an enzyme marker to calculate the scavenging rate, achieving over 80% clearance at 25 μg/mL. Cu[5.4]O@CNDs significantly outperformed the hydroxyl radical scavenging ability of the other two materials mentioned above (Figure [140]3J). Meanwhile, the ·OH scavenging ability was detected with the help of ESR to detect the intensity of free radical signaling. The system utilized the Fenton reaction to generate ·OH radicals, which were subsequently captured by DMPO to form DMPO/·OH spin adducts. When Fenton's reagent was mixed with DMPO, a tetra-linear eigen peak with apparent signal intensity appeared, which proved that a large amount of ·OH was generated in this mixture system. In contrast, the intensity of the tetra-linear eigen peak was reduced when C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs were added. The intensity of the tetra-linear eigen peak of Cu[5.4]O@CNDs was almost reduced. The four-linear state characteristic peaks of Cu[5.4]O@CNDs nearly disappeared, indicating that Cu[5.4]O@CNDs can effectively scavenge ·OH (Figure [141]3K). The above experiments describe the practical scavenging ability of Cu[5.4]O@CNDs in the presence of one free radical alone. The ABTS^•+ method was employed using a total antioxidant capacity kit to assess the radical scavenging ability of Cu[5.4]O@CNDs (Figure [142]3I). They were diluting the master batch according to the kit's instructions and testing the samples at different concentrations according to the requirements. The results showed that C-dots, Cu[5.4]O USNPs, and Cu[5.4]O@CNDs had good total antioxidant levels, the antioxidant capacity was concentration-dependent, and the total antioxidant capacity of Cu[5.4]O@CNDs was significantly better than the others; more than 80% clearance can be achieved at 25 μg/mL (Figure [143]3L). The above results exhibited that Cu[5.4]O@CNDs not only possessed excellent SOD and CAT activities but also were able to scavenge hydroxyl radicals, as well as possessed high antioxidant capacity, which indicated that Cu[5.4]O@CNDs were expected to treat the ROS-related injuries at cellular and animal levels. The mechanism of synergy effect between Cu[5.4]O USNPs and CNDs To investigate why Cu[5.4]O@CNDs have better SOD and CAT-like enzyme activities than C-dots and Cu[5.4]O USNPs, we synthesized Cu[5.4]O@CNDs by replacing CNDs with passivating the hydroxyl, carboxyl, and carbonyl groups on the surface of CNDs, then we investigated the synergistic mechanism between Cu[5.4]O USNPs and CNDs and the catalytic mechanism of their activity. First, to investigate the contribution of hydroxyl groups on the surface of CNDs to the formation of Cu[5.4]O@CNDs, we selectively deactivated the carboxyl and hydroxyl groups using 1,3 -propanesulfonic acid ketone (PS). PS reacted with the carboxyl and hydroxyl groups on the surface of the CNDs to form an ester and an ether, respectively, as demonstrated in our previous work. Esters can be hydrolyzed under alkaline conditions, whereas ethers cannot. Hydrolysis of CNDs in 0.5 M sodium hydroxide solution (reaction with PS) yielded hydroxyl-only passivated CNDs- A ([144]Figure S3A). To investigate the contribution of carboxyl groups on the surface of CNDs to the formation of Cu[5.4]O@CNDs, the carboxyl groups on the surface of CNDs were first converted to amides, and the amino groups were exposed on the surface of CNDs. Then, the aminated CNDs were reacted with PS, in which the amino and hydroxyl groups of the resulting CNDs (CNDs-B) were passivated ([145]Figure S3A). To investigate the contribution of carbonyl groups on the surface of CNDs to the formation of Cu[5.4]O@CNDs, sodium borohydride (NaBH[4]) was used to reduce the carbonyl groups to form hydroxyl groups on the surface of CNDs. The obtained Re-CNDs were reacted with PS followed by a hydrolysis process in 0.5 M sodium hydroxide solution. As a result, when the carbonyl group of CNDs was converted to hydroxyl groups, the hydroxyl group on the surface of Re-CNDs was converted to ether again, which led to the passivation of the carbonyl groups [146]^25^, [147]^27. Next, Cu[5.4]O@CNDs were synthesized by replacing CNDs in synthesis with optimal doses of CNDs-A, CNDs-B, and Re-CNDs-A. The lyophilized Cu[5.4]O@CNDs-A and Cu[5.4]O@Re-CNDs-A were similar in character and were a dense black powder, in contrast to Cu[5.4]O@CNDs-B, which was sparse in texture and showed a cotton-like appearance ([148]Figure S3B). Finally, we tested the radical scavenging catalytic activities of the synthesized Cu[5.4]O@CNDs-A, Cu[5.4]O@CNDs-B, and Cu[5.4]O@Re-CNDs-A, respectively, by the WST-1 kit. The results showed that the SOD enzyme activity of Cu[5.4]O@CNDs-A synthesized from CNDs-A with passivated hydroxyl groups was 1673 U/mg ([149]Figure S3C), The SOD enzyme activity of Cu[5.4]O@CNDs-B synthesized from CNDs-B with passivated hydroxyl and carboxyl groups was 2914 U/mg ([150]Figure S3D), The SOD enzyme activity of Cu[5.4]O@Re-CNDs-A synthesized from Re-CNDs-A after passivating the carbonyl group was 1635 U/mg ([151]Figure S3E). It was evidenced that passivation of the hydroxyl group alone makes the synthesis of Cu[5.4]O@CNDs blocked, and that the hydroxyl group has a positive contribution to the synthesis of Cu[5.4]O@CNDs; The enzyme activity of Cu[5.4]O@CNDs increased when the carboxyl group was simultaneously substituted, with partial recovery, suggesting that the presence of the carboxyl group forms a negative contribution to the synthesis of Cu[5.4]O@CNDs; The enzyme activity of Cu[5.4]O@CNDs decreased after passivation of the carbonyl group, suggesting a positive contribution of the carbonyl group to the formation of Cu[5.4]O@CNDs. These results indicate that carbonyl and hydroxyl groups promote Cu[5.4]O@CNDs, while the opposite is true for carboxyl groups. Therefore, we hypothesized that the hydroxyl and carboxyl groups on the surface of CNDs could form covalent coordination and ionic coordination bonds with Cu^2+, which contributed to Cu^2+ complexation on the surface of CNDs. When L-AA was added, the Cu^2+ located on the surface of CNDs underwent a reduction reaction, and the Cu[5.4]O USNPs aggregated by Cu^0 and Cu^1+ were promoted to grow on the surface of CNDs by the adjustment of pH. We also believe that the SOD-like activity of CNDs is dependent on the carbonyl group coupled to the π-system and that electron transfer between the Cu[5.4]O USNPs and the carbonyl group of CNDs may occur during catalytic superoxide anion disproportionation. It was shown that the synergistic catalytic activity of Cu^0 and Cu^1+ arises from matching the rates of activation of the reactants, i.e., the rate of activation of H[2] by Cu^0 and the rate of activation of carbonyl-containing reactants by Cu^1+. Where the carbonyl group is adsorbed at the center of Cu^1+, the -CH[2]OH group attached to C=O will have strong electrostatic interactions with the oxygen vacancy center, causing the intermediate to form a downward adsorption conformation, elongating the C=O bond length and lowering the subsequent hydrogenation energy barrier [152]^31. In summary, Cu[5.4]O USNPs grow on the surface of CNDs through the combination of hydroxyl and carbonyl groups to generate Cu[5.4]O@CNDs. Cu[5.4]O USNPs make up for the deficiency of single enzyme activity of C-dots, which makes Cu[5.4]O@CNDs possess cascade nano-enzymatic activity. Moreover, compared with Cu[5.4]O USNPs and C-dots alone, the SOD-like enzyme activities of CNDs were greatly enhanced due to the synergistic activation of carbonyl-containing reactants by Cu^0 and Cu^1+, which lowered the hydrogenation barrier of the carbonyl group of the catalytic activity centers on the surface of CNDs. In addition, the binding of Cu^0-Cu^1+ to the carbonyl group increases its electron-hole separation, which makes the Cu[2]O coating on Cu NPs much more stable, which also contributes to the enhancement of ROS scavenging ability of Cu[5.4]O@CNDs [153]^37. Cellular uptake and organ-targeted distribution capacity of Cu[5.4]O@CNDs To assess the cellular uptake of Cu[5.4]O@CNDs under increased ROS conditions, THLE-2 and Raw264.7 cells were treated with 100 μM H[2]O[2]. The results indicated a time-dependent uptake of the drug, reaching a maximum after 4 h of incubation (Figure [154]4A-B). Figure 4. [155]Figure 4 [156]Open in a new tab Cellular uptake and biodistribution of Cu[5.4]O@CNDs. (A) Fluorescence images of THLE-2 cells incubated with Cu[5.4]O@CNDs for 4 h. (B) Fluorescence images of Raw264.7 cells incubated with Cu[5.4]O@CNDs for 4 h. (C) Fluorescence imaging of healthy mice and organs at specified time points after intravenous injection of Cu[5.4]O@CNDs. (D) Fluorescence imaging of hepatitis mice and organs at specified time points after intravenous injection of Cu[5.4]O@CNDs. (E-F) Distribution of Cu[5.4]O@CNDs in major organs at different time points. (G) Difference of liver fluorescence level between healthy mice and hepatitis mice after intravenous injection of Cu[5.4]O@CNDs. (H) Average cellular fluorescence signals of blood at different time points after intravenous injection of Cu[5.4]O@CNDs. Data represent means ± s.d. from three independent replicates. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. The biodistribution and targeting capabilities of Cu[5.4]O@CNDs in healthy and damaged livers were further investigated in vivo. Cu[5.4]O@CNDs (0.5 mg/kg) were intravenously injected into both healthy and hepatitis mice, and their accumulation was monitored using fluorescence imaging at 0 to 4 h. A significant fluorescence signal was detected in vivo 5 min after injection and gradually diminished over the next 4 h. This indicated that Cu[5.4]O@CNDs circulated within the mice and were progressively eliminated from the body (Figure [157]4C-D). Additionally, isolated organs were collected from the euthanized mice for further examination. Fluorescence imaging of isolated organs showed a rapid increase in Cu[5.4]O@CNDs accumulation in the liver 30 min post-injection, followed by a decline (Figure [158]4C). In hepatitis model mice, Cu[5.4]O@CNDs rapidly accumulated in the liver 20 min after injection, followed by a decline (Figure [159]4D), which demonstrated that Cu[5.4]O@CNDs could be uptaken by the damaged liver tissues more rapidly. Quantitative analysis of fluorescence data corroborated earlier findings, indicating that Cu[5.4]O@CNDs accumulation in the liver peaked 20 to 30 min post-injection. This suggests that administering Cu[5.4]O@CNDs prior to hepatic ischemia-reperfusion injury (HIRI) modeling offers an effective targeting strategy and a swift therapeutic window (Figure [160]4E-F). Additionally, ex vivo fluorescence quantification of liver tissues showed that the fluorescence intensity of Cu[5.4]O@CNDs in damaged liver tissues was significantly higher than that in healthy mice, and the fluorescence intensity of the hepatitis model mice cleared faster than that of healthy mice (Figure [161]4G). The above results demonstrated that Cu[5.4]O@CNDs contributes to its uptake and accumulation by the liver due to its properties of small size and negative charge. Importantly, owing to the proliferation of liver tissues and endothelial dysfunction of hepatic sinusoids in acute hepatitis, the uptake of Cu[5.4]O@CNDs by the liver in hepatitis was further promoted, which facilitated its entry into hepatocytes through hepatic sinusoids to play a role. Meanwhile, the increased GSH content in acute hepatitis liver tissues promoted GSH-mediated biotransformation, which led to an enhanced efficiency of Cu[5.4]O@CNDs clearance by the acute hepatitis liver (Figure [162]4G). Finally, the pharmacokinetic study revealed that the blood fluorescence intensity gradually decreased following injection, with a half-life (t[1/2]) of 119.6 min, which also indicated that Cu[5.4]O@CNDs were readily excreted from the body within 24 h (Figure [163]4H). In conclusion, Cu[5.4]O@CNDs exhibited excellent targeting and rapid accumulation in the liver. Anti-oxidative stress properties of Cu[5.4]O@CNDs in vitro Hepatic ischemia-reperfusion injury and acute liver injury not only lead to oxidative stress in hepatocytes but also can activate macrophages that have long been present in liver tissue [164]^38. Hydrogen peroxide was utilized as an exogenous stimulus to quickly induce large amounts of ROS in THLE-2 and Raw264.7 cells (Figure [165]5A). First, Cu[5.4]O@CNDs were evaluated for their protective capacity against oxidative stress cells. The JC-1 staining reagent accumulates in mitochondria based on membrane potential, displaying green fluorescence as monomers in early apoptotic cells and shifting to red fluorescence as J-aggregates in healthy cells [166]^39. A decreased red/green fluorescence intensity ratio indicates early apoptosis. JC-1 staining indicated red fluorescence in both the blank THLE-2 and Raw264.7 cell groups. However, upon H[2]O[2]-induced oxidative stress, a significant increase in green fluorescence was observed, confirming ROS generation, mitochondrial membrane damage, and early apoptosis in the modeled cells (Figure [167]5B). In the group pre-incubated with Cu[5.4]O@CNDs (0 ~ 80 μg/mL), intracellular green fluorescence signals significantly decreased as the concentration of Cu[5.4]O@CNDs increased (Figure [168]5B). Quantitative fluorescence staining results confirmed that Cu[5.4]O@CNDs protect cells and mitigate oxidative stress-induced damage (Figure [169]5C -D). Following H[2]O[2] exposure, apoptotic cells constituted approximately 68.73 ± 4.45% in THLE-2 cells, whereas pretreatment with 80 μg/mL Cu[5.4]O@CNDs reduced this percentage to 0.58 ± 0.24% (Figure [170]5C). The apoptotic cells accounted for about 91.4 ± 4.26% after H[2]O[2] exposure in Raw.264.7 cells and 14.90 ± 7.30% in the 80 μg/mL Cu[5.4]O@CNDs pretreatment group (Figure [171]5D). Figure 5. [172]Figure 5 [173]Open in a new tab Anti-oxidative stress properties of Cu[5.4]O@CNDs in vitro. (A) Schematic representation of Cu[5.4]O@CNDs treatment of Oxidative stress cells. The figure was created partially with BioRender.com. (B) Representative JC-1 staining of THLE-2 and Raw264.7 cells under different treatment conditions. (C) Quantitative analysis of JC-1 fluorescent staining of THLE-2 cells. (D) Quantitative analysis of JC-1 fluorescent staining of Raw264.7 cells. (E) The quantitative flow cytometry results show cell apoptosis and necrosis distribution of THLE-2 cells under different treatment conditions. (F) The quantitative flow cytometry results show cell apoptosis and necrosis distribution of Raw264.7 cells under different treatment conditions. (G) Representative DHE staining of THLE-2 and Raw264.7 cells under different treatment conditions. (H) Quantitative analysis of DHE fluorescent staining of THLE-2 cells. (I) Quantitative analysis of DHE fluorescent staining of Raw264.7 cells. (J) Representative DCFH-DA staining of THLE-2 and Raw264.7 cells under different treatment conditions. (K) Quantitative analysis of DCFH-DA fluorescent staining of THLE-2 cells. (L) Quantitative analysis of DCFH-DA fluorescent staining of Raw264.7 cells. (M) The results by flow cytometry to ROS levels of THLE-2 cells under the indicated treatment conditions. (N) The results by flow cytometry to ROS levels of Raw264.7 cells under the indicated treatment conditions. (O) Quantitative analysis of flow cytometry to ROS levels of THLE-2 cells under the indicated treatment conditions. (P) Quantitative analysis of flow cytometry to ROS levels of Raw264.7 cells under the indicated treatment conditions. Data represent means ± s.d. from three independent replicates. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. Subsequently, apoptosis experiments were performed to determine the preventive effect of Cu[5.4]O@CNDs on apoptosis. Similar to the results of JC-1, the live cells accounted for about 62.67 ± 1.52% after H[2]O[2]exposure in THLE-2 cells, while the percentage of apoptotic cells increased to 89.30 ± 1.11% in the 80 μg/mL Cu[5.4]O@CNDs pretreatment group (Figure [174]5E). The live cells accounted for about 35.00 ± 2.39% after H[2]O[2] exposure in Raw.264.7 cells, and 79.07 ± 0.35% in the 80 μg/mL Cu[5.4]O@CNDs pretreatment group (Figure [175]5F). Dihydroethidium (DHE), a red fluorescent probe specific for O[2]^•-, was employed to evaluate the O[2]^•- scavenging capability of Cu[5.4]O@CNDs [176]^40. DHE staining revealed no red fluorescence in the control group for both THLE-2 and Raw264.7 cells. In contrast, the H[2]O[2]-treated group showed prominent red fluorescence, which was notably diminished in the Cu[5.4]O@CNDs group in a concentration-dependent manner (Figure [177]5G). Fluorescence staining analysis revealed that H[2]O[2] exposure increased DHE production by 8.61 ± 0.20-fold in THLE-2 cells and 194.23 ± 27.88-fold in Raw264.7 cells compared to the control group. Following treatment with Cu[5.4]O@CNDs at 80 μg/mL, DHE levels in THLE-2 and Raw264.7 cells were reduced to 1.10 ± 0.08 and 2.23 ± 0.93 times the control levels, respectively (Figure [178]5H-I). Intracellular ROS levels were assessed using the oxidation-sensitive fluorescent dye 2′,7′-dichlorofluorescein (DCFH-DA). DCFH-DA is a non-fluorescent compound that permeates cell membranes and hydrolyzes into DCFH. Intracellular ROS oxidizes non-fluorescent DCFH to generate fluorescent DCF. Thus, the fluorescence of DCF intensity reflects the amount of intracellular ROS [179]^41. The DCFH-DA staining showed that the blank cell group exhibited little green fluorescence. In contrast, a very bright green fluorescence appeared in the hydrogen peroxide-induced group, indicating that a large amount of ROS was successfully induced in the cells (Figure [180]5J). Pre-incubation with Cu[5.4]O@CNDs (0-80 μg/mL) for 6 h significantly decreased intracellular green fluorescence intensity in a concentration-dependent manner, suggesting a substantial reduction in intracellular ROS content (Figure [181]5J). Fluorescence staining analysis revealed that H[2]O[2] exposure increased ROS production by 421.68 ± 38.19 times in THLE-2 cells and 19.41 ± 0.32 times in Raw264.7 cells compared to controls. Treatment with 80 μg/mL Cu[5.4]O@CNDs reduced ROS levels to 3.83 ± 0.88 times in THLE-2 cells and 1.78 ± 1.28 times in Raw264.7 cells relative to controls (Figure [182]5K-L). Flow cytometry analysis indicated a concentration-dependent reduction in intracellular ROS levels in cells treated with varying concentrations of Cu[5.4]O@CNDs (0-80 μg/mL) compared to H[2]O[2]-stimulated cells (Figure [183]5M-N). Quantitative statistics of the flow cytometry results suggested that 39.63 ± 2.14% of the THLE-2 cells induced by H[2]O[2] cells generated ROS, and Raw264.7 cells induced 70.53 ± 1.00% (Figure [184]5O-P). Following treatment with 80 μg/mL Cu[5.4]O@CNDs, ROS ratios were reduced to 13.67 ± 1.06% in THLE-2 cells and 14.07 ± 2.33% in Raw264.7 cells. It also indicated that Cu[5.4]O@CNDs had a significant concentration-dependent ROS scavenging effect (Figure [185]5O-P). Anti-inflammatory properties of Cu[5.4]O@CNDs in vitro Inflammatory responses often occur during oxidative stress, and lipopolysaccharide (LPS) activates macrophages through cell signaling systems, inducing a variety of cytokines and inflammatory mediators, which leads to an inflammatory state in cells [186]^42 (Figure [187]6A). The protective effect of Cu[5.4]O@CNDs on inflammatory cells was assessed using JC-1 in both THLE-2 and Raw264.7 cell lines. In the control group, cells exhibited red fluorescence. However, upon LPS-induced inflammation, significant green fluorescence was observed, indicating mitochondrial membrane damage and early apoptosis in the modeled cells (Figure [188]6B). After pre-incubation with Cu[5.4]O@CNDs, the intensity of green fluorescence was reduced, proving the improvement of cell status (Figure [189]6B). Quantitative analysis revealed that in THLE-2 cells, the apoptotic cell ratio was 79.83 ± 2.56% in the LPS group, which significantly decreased to 1.98 ± 1.76% with 80 μg/mL Cu[5.4]O@CNDs pretreatment (Figure [190]6C). The apoptotic cells ratio was 66.77 ± 5.57% after LPS exposure in Raw.264.7 cells, and 8.93 ± 5.49% in the 80 μg/mL Cu[5.4]O@CNDs pretreatment group (Figure [191]6D). Figure 6. [192]Figure 6 [193]Open in a new tab Anti-inflammatory properties of Cu[5.4]O@CNDs in vitro. (A) Schematic representation of Cu[5.4]O@CNDs treatment of LPS-induced inflammatory cells. The figure was created partially with BioRender.com. (B) Representative JC-1 staining of THLE-2 & Raw264.7 cells under different treatment conditions. (C) Quantitative analysis of JC-1 fluorescent staining of THLE-2 cells. (D) Quantitative analysis of JC-1 fluorescent staining of Raw264.7 cells. (E) The results by flow cytometry to CD80 levels of Raw264.7 cells under different treatment conditions. (F) Quantitative analysis of flow cytometry to CD80 levels of Raw264.7 cells. (G) Relative expression of mRNAs for inflammatory cytokines of IL-1β, IL-6, IL-12, and TNF-α. Data represent means ± s.d. from three independent replicates. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. Macrophage activation and polarization into various subpopulations are influenced by distinct microenvironments, with LPS induction typically transforming macrophages into M1-type, which enhances inflammation [194]^43. The impact of Cu[5.4]O@CNDs on macrophage polarization was confirmed by examining cytomorphological changes in M1 macrophages, indicated by the overexpression of surface CD80. Flow cytometry analysis indicated that Cu[5.4]O@CNDs suppressed the conversion of pro-inflammatory M1 macrophages, leading to decreased expression of inflammatory mediators and a reduced cellular inflammatory response (Figure [195]6E). Quantitative statistics showed that LPS cells induced 57.80 ± 0.70% conversion of Raw264.7 cells into M1 macrophages (Figure [196]6F). Real-time quantitative polymerase chain reaction (qPCR) was employed to measure the relative transcription levels of TNFα, IL-6, IL-12, and IL-1β. The study demonstrated a significant increase in cellular inflammatory factors following LPS induction, which was notably reduced after pre-incubation with Cu[5.4]O@CNDs (Figure [197]6G). Biocompatibility evaluation of Cu[5.4]O@CNDs Since biocompatibility is important for the potential clinical applications of nanomaterials, the toxicity of Cu[5.4]O@CNDs was evaluated in vitro. Cell viability in THLE-2 cells exposed to various concentrations of Cu[5.4]O@CNDs for 24 h was assessed using the MTT assay, revealing nearly 100% viability even at 100 μg/mL (Figure [198]7A). When the incubation time reached 48 h, the cell viability was still higher than 80%, which proved the good safety of Cu[5.4]@CNDs in vitro (Figure [199]7B). Next, the hemolysis assay was used to detect the biocompatibility of Cu[5.4]O@CNDs, and the results showed that upon the concentration was 160 μg/mL, the hemolysis rate was still below 5%, which suggested that the nanomaterials didn't cause hemolysis of blood cells and were safe for intravenous injection (Figure [200]7C). Figure 7. [201]Figure 7 [202]Open in a new tab Biocompatibility of Cu[5.4]O@CNDs. (A) THLE-2 cell viability after incubation for 24 h with Cu[5.4]O@CNDs. (B) THLE-2 cell viability after incubation for 48 h with Cu[5.4]O@CNDs. (C) The ratio of hemolysis in the subgroups. (D) Schematic diagram of biocompatibility experiment. The figure was created partially with BioRender.com. (E) Weight variation of normal mice at 7 days after treatment with Cu[5.4]O@CNDs. (F) Evaluation of in vivo toxicity of Cu[5.4]O@CNDs to major organs (heart, liver, spleen, lung, and spleen) at 1 day and 7 days after intravenous administration. (G) Serum levels of liver function indicators: alanine transaminase (ALT) and aspartate transaminase (AST). Serum levels of kidney function indicators: blood urea nitrogen (UREA) and creatinine (CRE). (H) Blood parameters in normal mice (control group) and mice intravenously injected with Cu[5.4]O@CNDs. Data represent means ± s.d. from three independent replicates. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. In addition, 0.5 mg/kg was used as the concentration for all in vivo biocompatibility evaluation experiments. The in vivo biocompatibility of Cu[5.4]O@CNDs was assessed in healthy mice by examining blood chemistry and major organ histopathology (Figure [203]7D). Following a 7-day regimen of Cu[5.4]O@CNDs injections, samples from major organs (heart, liver, spleen, lungs, and kidneys), serum, and plasma were collected on days 1 and 7, with body weight monitored throughout the study. The body weight record results exhibited that Cu[5.4]O@CNDs did not affect the status and body weight of the mice (Figure [204]7E). H&E staining results showed no obvious signs of damage in the tissues, suggesting that Cu[5.4]O@CNDs had good biocompatibility (Figure [205]7F). Serum biochemical analysis showed that liver function indicators (AST and ALT) and kidney function indicators (UREA and CRE) in the Cu[5.4]O@CNDs injected group were similar to the control group, indicating liver and kidney biocompatibility (Figure [206]7G). Whole blood cell analysis in mice revealed no statistically significant differences from the control group (Figure [207]7H). The studies uniformly demonstrated that synthesized Cu[5.4]O@CNDs exhibited minimal in vivo toxicity both in the short and long term. In vivo therapeutic efficacy of Cu[5.4]O@CNDs on HIRI mice Based on the anti-oxidative stress and anti-inflammatory effects of Cu[5.4]O@CNDs in vitro, the protective effects of Cu[5.4]O@CNDs in the mouse model of HIRI were further investigated. According to the results analyzed above and the current consensus, excessive oxidative stress and inflammation contribute to the pathology of HIRI [208]^34. Firstly, the HIRI mice model was established (Figure [209]8A). Then, the optimal treatment dose of Cu[5.4]O@CNDs was determined. Liver tissues were photographed, showing that some tissue damage surfaces were visible, and the liver status of the 0.5 mg/kg pretreatment group was better than that of the disease modeling group (Figure [210]8B). Meanwhile, the H&E staining images of mouse liver tissues showed that the HIRI modeling group's cells appeared to punctate hepatocyte necrosis, some intracapillary cholestasis, and inflammatory cell infiltration (Figure [211]8C). Hepatic histopathological manifestations were significantly improved in the pre-injected Cu[5.4]O@CNDs group, and minimal areas of hepatocyte necrosis and cytolysis were observed in the HIRI mouse model treated with 0.5 mg/kg of Cu[5.4]O@CNDs (Figure [212]8C). Liver function tests in mice revealed elevated alanine transaminase (ALT) and aspartate aminotransferase (AST) levels in the HIRI model group compared to controls. However, pre-injection with Cu[5.4]O@CNDs significantly reduced ALT and AST levels, indicating a protective effect on liver function (Figure [213]8D). At a concentration of 0.5 mg/kg, Cu[5.4]O@CNDs resulted in liver function indices in mice that closely matched those of the control group, suggesting this as the optimal therapeutic dose (Figure [214]8D). In summary, the Cu[5.4]O@CNDs demonstrated a protective effect on hepatic function in the HIRI mice model, with 0.5 mg/kg identified as the optimal therapeutic concentration for further studies. Figure 8. [215]Figure 8 [216]Open in a new tab In vivo therapeutic efficacy of Cu[5.4]O@CNDs on HIRI & LPS-ALI mice. (A) Schematic illustration of the establishment and treatment schedule of HIRI mice. The figure was created partially with BioRender.com. (B) Optical image of liver tissue. (C) H&E staining (Scale bar: 100 μm & 50 μm) of liver tissues. (D) Serum levels of ALT and AST in HIRI mice at 24 h after different treatments. (E) TUNEL assay, DCFH-DA, and DHE staining (Scale bar: 25 μm) of liver tissues. (F) Relative expression of mRNAs for cytokines of IL-1β, IL-6, IL-12, TNF-α. (G) Schematic illustration of the establishment and treatment schedule of LPS-ALI mice. The figure was created partially with BioRender.com. (H) H&E staining (Scale bar: 200 μm & 100 μm) of liver tissues. (I) Serum levels of ALT and AST in LPS-ALI mice at 12 h after different treatments. (J) TUNEL assay, DCFH-DA, and DHE staining (Scale bar: 25 μm) of liver tissues. (K) Relative expression of mRNAs for cytokines of IL-1β, IL-6, IL-12, TNF-α. Data represent means ± s.d. from three independent replicates. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. TUNEL staining of frozen tissue sections was used to evaluate hepatocyte necrosis and apoptosis. The results showed a significant increase in green fluorescence in the HIRI group, while no notable difference was observed between the pretreated and control groups, suggesting that Cu[5.4]O@CNDs effectively protect hepatocytes and hepatic function (Figure [217]8E). The in vivo ROS removal capability of Cu[5.4]O@CNDs was assessed using the ROS-specific fluorescent dyes DCFH-DA and DHE for imaging. The green fluorescence of DCFH-DA was significantly increased in liver tissues after hepatic ischemia-reperfusion, and the ROS level was reduced in the tissues stained by Cu[5.4]O@CNDs pre-injection (Figure [218]8E). Meanwhile, DHE was also seen to increase the intensity of red fluorescence in the modeling group and recover the intensity of staining fluorescence in the pretreatment group (Figure [219]8E). The results indicated that Cu[5.4]O@CNDs could alleviate liver function injury and hepatocyte apoptosis by removing ROS in liver ischemia-reperfusion injury disease. In addition, mouse liver tissues were processed to extract the relevant RNA to verify the changes in the transcription levels of common pro-inflammatory cytokines. After successful modeling, levels of the inflammatory factors IL-1β, IL-6, IL-12, and TNF-α significantly increased. However, in the group pre-injected with Cu[5.4]O@CNDs, inflammation levels remained comparable to those in healthy control mice (Figure [220]8F). The findings demonstrated that Cu[5.4]O@CNDs effectively mitigated the inflammatory response by scavenging ROS, thereby preventing oxidative stress and inflammation in hepatic ischemia-reperfusion injury. In vivo therapeutic efficacy of Cu[5.4]O@CNDs on LPS-ALI mice To verify that Cu[5.4]O@CNDs can be applied to other acute liver injury diseases, an LPS-induced acute liver injury model (LPS-ALI) was established. Cu[5.4]O@CNDs (0.5 mg/kg) was administered 6 h post LPS + D-gal induction, followed by euthanasia 12 h later to collect liver tissues and serum for liver function analysis (Figure [221]8G). Similar to the HIRI model, the H&E staining images of mouse liver tissues showed that in the LPS-induced hepatitis model, the hepatocytes showed apparent balloon-like lesions accompanied by a large number of inflammatory cell infiltrations. The hepatic histopathological manifestations of the Cu[5.4]O@CNDs-treated group were markedly improved, and the inflammatory symptoms were reduced (Figure [222]8H). Biochemical tests on mouse serum revealed elevated liver function indexes in LPS-induced hepatitis mice, indicating severe liver injury. In contrast, the Cu[5.4]O@CNDs treatment group showed reduced liver function indexes, approaching those of the control group, demonstrating a significant therapeutic effect (Figure [223]8I). TUNEL staining of frozen tissue sections was conducted to evaluate hepatocyte necrosis and apoptosis. The modeling group exhibited significantly increased TUNEL green fluorescence, while no notable difference was observed between the pretreatment and control groups, indicating that Cu[5.4]O@CNDs possess a strong anti-inflammatory effect (Figure [224]8J). To evaluate the ROS scavenging ability in the ALI-affected liver of Cu[5.4]O@CNDs, frozen sections were stained with DCFH-DA and DHE. LPS-induced hepatitis showed a significant increase in DCFH-DA green fluorescence, while Cu[5.4]O@CNDs treatment reduced ROS levels (Figure [225]8J). Meanwhile, high intensity of red fluorescence in hepatitis tissues was seen by DHE, and the fluorescence intensity of the tissues decreased after treatment. The results suggested that Cu[5.4]O@CNDs can achieve relief of inflammation by scavenging ROS in LPS-induced acute hepatitis disease (Figure [226]8J). Mouse liver tissues were processed to extract RNA for verifying changes in common pro-inflammatory cytokine expression levels. The study found that four common cytokines were significantly elevated following successful modeling, while inflammation levels in the Cu[5.4]O@CNDs-injected group were comparable to those in healthy control mice (Figure [227]8K). The study indicates that Cu[5.4]O@CNDs can mitigate oxidative stress and inflammation in acute hepatitis by eliminating ROS in vivo. Therapeutic mechanisms of Cu[5.4]O@CNDs on HIRI & LPS-ALI To further elucidate the potential therapeutic mechanism, after the mice were executed and the frozen liver tissues were obtained, RNA extraction and purification, RNA fragmentation and reverse transcription, construction of cDNA libraries, high-throughput sequencing and other experimental procedures were carried out, and the results were subjected to gene and protein differential expression analyses, which were able to reveal the comprehensive gene expression information of the liver tissues of all the groups in the Cu[5.4]O@CNDs intervention model, and analyze the changes of gene expression under different conditions. It can reveal the comprehensive gene expression information of each group in the Cu[5.4]O@CNDs intervention model, analyze the expression changes of differentially expressed genes under different conditions, and screen the differentially expressed genes and corresponding signaling pathways in the HIRI group and the Cu[5.4]O@CNDs prevention group, and the LPS-ALI group and the Cu[5.4]O@CNDs treatment group, respectively. Firstly, the graphs were made by transcriptomics PCA analysis which showed that there was a high reproducibility between samples within the experimental groups, the healthy mice in the control group had a significant difference and low correlation with the mice in the modeling group, and the mice in the treatment or prevention group had a high correlation with the healthy group (Figure [228]9A-B). It proved that HIRI and LPS-ALI modeling was successful. Differential gene expression (DGE) analysis remains an essential application of RNA-seq as a traditional research tool. The Wayne plots suggested that Cu[5.4]O@CNDs were associated with a total of 166 genes related to the treatment of HIRI disease ([229]Figure S4A), and Cu[5.4]O@CNDs were associated with a total of 1978 genes related to the treatment of LPS-ALI disease ([230]Figure S5A). The volcano map analysis revealed that in the Cu[5.4]O@CNDs and HIRI group, there were 900 up-regulated and 450 down-regulated genes, while in the Cu[5.4]O@CNDs and LPS-ALI group, there were 1,421 up-regulated and 2,719 down-regulated genes ([231]Figures S4B and S5B). Gene clustering and analysis revealed differential expression levels between the two disease models, as well as variations in the expression of the same genes across control, model, and treatment groups ([232]Figures S4C and S5C). GO and KEGG analyses were conducted following the sequencing results. First, according to GO analysis, significant differences in metabolic processes related to redox, immune system, inflammatory response, and defense against viruses were found between the HIRI disease and treatment models ([233]Figure S4D-E), and significant differences in metabolic processes related to redox, immune system, and cholesterol were found between the LPS-ALI disease and treatment models ([234]Figure S5D-E). The findings indicate that Cu[5.4]O@CNDs could be involved in modulating hepatic oxidative stress and inflammatory pathways, potentially aiding in the prevention and treatment of liver diseases. KEGG analysis identified enriched pathways among differential genes in the control-disease-treatment groups, including PI3K-Akt, MAPK, Rap1 signaling, and retinol metabolism pathways, which are classical inflammatory and apoptotic pathways ([235]Figures S4F and S5F). And Bubble plots suggested that treating acute liver injury disease by Cu[5.4]O@CNDs may be associated with multiple inflammatory signaling pathways, and retinol metabolism pathways ([236]Figure S4G and S5G). The above evidence and results demonstrated that Cu[5.4]O@CNDs could regulate hepatitis inflammation and oxidative stress processes in the prevention and treatment of liver diseases. Figure 9. [237]Figure 9 [238]Open in a new tab Therapeutic mechanisms of Cu[5.4]O@CNDs on HIRI & LPS-ALI. (A) PCoA of the DEGs between the Sham group, HIRI group, and Cu[5.4]O@CNDs group (n = 4), each point represented one mouse. (B) PCoA of the DEGs between the Sham group, LPS-ALI group, and Cu[5.4]O@CNDs group (n = 4), each point represented one mouse. (C) Differential gene heat maps associated with Response to ROS (fold change≥2 and P< 0.01). (D) Differential gene heat maps related to inflammatory response (fold change≥2 and P< 0.01). (E) Differential genes with the same trend in HIRI and LPS-ALI. (F) Differential gene heat maps with the same trend in HIRI and LPS-ALI. (G) KEGG pathway enrichment analysis. The 20 most significantly enriched pathways were shown. (H) Differential gene heat maps associated with Retinol metabolic process (fold change≥2 and P< 0.01). (I) Expression difference of CYP4a14 and RDH11 genes in HIRI and LPS-ALI diseases, respectively. (J) Liver protein expression of CYP4a14 and RDH11 (n = 3). The above RNA sequencing analysis of the modeling group and healthy mice demonstrated that ROS metabolic processes, inflammatory response pathways, and retinol metabolism are highly correlated in the pathogenesis of two acute liver injury diseases, HIRI and LPS-ALI. Accordingly, Cu[5.4]O@CNDs were designed to target their pathogenesis. To assess the therapeutic efficacy, differential gene heat map clustering analysis was conducted on ROS and inflammation response-related genes. The analysis revealed distinct gene expression patterns among the control, model, and treatment groups, demonstrating that Cu[5.4]O@CNDs effectively modulate genes influencing our target genes (Figure [239]9C-D). To further analyze the common therapeutic mechanism of Cu[5.4]O@CNDs in HIRI and LPS-ALI, the two RNA sequencing models were screened in the order of control-model-treatment to find out the differential genes that "increase-decrease" or "decrease-increase". The results indicated that both HIRI and LPS-ALI models exhibited four differential genes with an 'ascending-descending' pattern in the control-model-treatment group sequence. In the HIRI and LPS-ALI models, 18 genes showed a "decrease-increase" in the control-model-treatment group (Figure [240]9E). Therefore, the total number of differential genes with the same trend was 22. At the same time, these 22 genes were clustered and analyzed, which can visualize the expression level and enrichment degree of different genes among different groups (Figure [241]9F). Subsequently, based on the screening results, KEGG analysis of the target genes was also performed, and the enrichment results showed that the lipid metabolism-related pathways such as "cholesterol metabolism, retinol metabolism", the classical inflammatory response pathways such as "MAPK signaling pathway", drug metabolism pathway, etc. were highly enriched, which proved that the expression level of different genes in different groups was highly enriched. The results showed that lipid metabolism-related pathways such as "cholesterol metabolism, retinol metabolism", classical inflammatory response pathways such as "MAPK signaling pathway", and drug metabolism pathways were highly enriched, which demonstrated that the therapeutic mechanism of Cu[5.4]O@CNDs was common to HIRI and LPS-ALI. Interestingly, the pathogenesis common to both acute liver injury diseases, HIRI and LPS-ALI, i.e., is highly associated with disorders of ROS metabolic processes, inflammatory response pathways, and retinol metabolism. The pathogenesis corresponds to the therapeutic mechanism, demonstrating that the stabilization of the retinol metabolic pathway may be a therapeutic pathway specific for Cu[5.4]O@CNDs, which showed that there is a difference in the expression of the same genes in the control-model-treatment group for the retinol metabolic pathway (Figure [242]9G). Notably, there were 3 genes associated with the retinol metabolic pathway among the 22 differential genes, and the gene CYP4a14 was screened based on enrichment, differential level, and gene function (Figure [243]9H). CYP4a14 is a retinoic acid degradation-associated gene [244]^44. Up-regulation of CYP4a14 expression and down-regulation of rate-limiting enzymes of retinol metabolism, such as RDH11, and retinol dehydrogenase, resulted in retinol metabolism disorders characterized by tissue retinol overload and reduction of all-trans-retinol [245]^45. This promoted metabolic disorders in liver tissues and tissue toxicity (Figure [246]9I). Based on relative mRNA expression, we further explored the protein level of CYP4a14 and RDH11. The results demonstrated that both HIRI and LPS-ALI diseases were associated with up-regulation of the retinol metabolism gene CYP4a14 and its protein expression, accompanied by down-regulation of RDH11. The intervention of Cu[5.4]O@CNDs reversed retinol metabolism disorders, resulting in the restoration of the CYP4a14 and RDH11 genes and their protein expression (Figure [247]9J and [248]Figure S6). The results of the above RNA sequencing analyses and immunoblotting results demonstrated that Cu[5.4]O@CNDs could not only remove excessive ROS through its cascade nano-enzymatic activity but also prevent and alleviate ROS infiltration by regulating ROS-responsive pathway-related genes; at the same time, Cu[5.4]O@CNDs could ameliorate the metabolic disorders of retinol metabolism and tissue retinol overload through the down-regulation of CYP4a14; furthermore, Cu[5.4]O@CNDs could improve the metabolic disorders of retinol metabolism by down-regulation of CYP4a14; and Cu[5.4]O@CNDs simultaneously modulated multiple classical inflammatory pathways and attenuated the inflammation level in liver tissues and body forces. The above evidence and results demonstrate that Cu[5.4]O@CNDs can regulate hepatic retinol metabolism, hepatitis inflammation, and oxidative stress processes in the prevention and treatment of liver diseases. Conclusion In summary, the synthesized Cu[5.4]O@CNDs nanozyme particles exhibit cascade-amplified enzyme activity, are cost-effective and straightforward to produce, and can treat various acute liver injury diseases. And it showed good biocompatibility. Cu[5.4]O@CNDs can be enriched in the mitochondrial membrane of damaged liver-targeted cells by scavenging oxidative stress products. Transcriptome sequencing was employed to explore the specific mechanisms of Cu[5.4]O@CNDs in treating acute liver injury diseases. This treatment involves down-regulating CYP4a14, correcting retinol metabolism disorders and overload, regulating ROS-responsive pathway genes, mitigating ROS infiltration, and modulating inflammatory responses. In conclusion, multiple acute liver injury disease models were established to explore specific regulatory pathways, provide new therapeutic targets for oxidative stress-related diseases, and construct a new type of nano-enzymatic material, providing a promising biomedical therapeutics strategy. Supplementary Material Supplementary materials and methods, figures and tables. [249]thnov15p5592s1.pdf^ (2.2MB, pdf) Acknowledgments