
==== Front
J Adv Res
J Adv Res
Journal of Advanced Research
2090-1232
2090-1224
Elsevier

S2090-1232(23)00315-6
10.1016/j.jare.2023.10.011
Medicine
Ceria nanozyme coordination with curcumin for treatment of sepsis-induced cardiac injury by inhibiting ferroptosis and inflammation
Jiang Chenxiao a1
Shi Qianzhi b1
Yang Jing c1
Ren Hao c
Zhang Lu b
Chen Shan d
Si Jiayi b
Liu Yihai e
Sha Dujuan tbwen0912@126.com
bd⁎
Xu Biao xubiao62@njmu.edu.cn
e⁎
Ni Jie nijie@njglyy.com
bf⁎
a Department of Pharmacy, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu 210008, China
b Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210008, China
c School of Pharmaceutical Science, Nanjing Tech University, Nanjing, Jiangsu 211816, China
d Department of General Medicine, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu 210008, China
e Department of Cardiology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu 210008, China
f Department of Emergency Medicine, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu 210008, China
⁎ Corresponding authors at: Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210008, China (J. Ni and D. Sha); Department of Cardiology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, Jiangsu 210008, China (B. Xu). tbwen0912@126.comxubiao62@njmu.edu.cnnijie@njglyy.com
1 These authors contributed equally to this work.

21 10 2023
9 2024
21 10 2023
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2 8 2023
12 10 2023
20 10 2023
© 2024 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

Schematic illustration of CeCH for sepsis-induced myocardial injury. (A) Preparation of CeCH with self-assembled by HSA. (B) The mechanism of CeCH to show protective effect for sepsis-induced cardiac injury by inhibiting ferroptosis and inflammation.

Highlights

• CeCH was successfully developed by a green self-assembled method with human serum albumin to increase the water solubility and poor bioavailability of curcumin.

• CeCH performed SOD-like and CAT-like activities to eliminate ROS generation and inhibit ferroptosis in H9C2 cells.

• CeCH reduced the secretion of inflammatory factors by M1 macrophages to suppress the inflammation.

• CeCH protected the heart against sepsis-induced cardiac injury and reversed cardiac dysfunction in vivo.

• A promising strategy with Cur and nanozyme for septic cardiomyopathy by inhibiting ferroptosis and inflammation in clinical application.

Introduction

Sepsis-induced cardiac injury is the leading cause of death in patients. Recent studies have reported that reactive oxygen species (ROS)-mediated ferroptosis and macrophage-induced inflammation are the two main key roles in the process of cardiac injury. The combination of ferroptosis and inflammation inhibition is a feasible strategy in the treatment of sepsis-induced cardiac injury.

Objectives

In the present study, ceria nanozyme coordination with curcumin (CeCH) was designed by a self-assembled method with human serum albumin (HSA) to inhibit ferroptosis and inflammation of sepsis-induced cardiac injury.

Methods and results

The formed CeCH obtained the superoxide dismutase (SOD)-like and catalase (CAT)-like activities from ceria nanozyme to scavenge ROS, which showed a protective effect on cardiomyocytes in vitro. Furthermore, it also showed ferroptosis inhibition to reverse cell death from RSL3-induced cardiomyocytes, denoted from curcumin. Due to the combination therapy of ceria nanozyme and curcumin, the formed CeCH NPs could also promote M2 macrophage polarization to reduce inflammation in vitro. In the lipopolysaccharide (LPS)-induced sepsis model, the CeCH NPs could effectively inhibit ferroptosis, reverse inflammation, and reduce the release of pro-inflammatory factors, which markedly alleviated the myocardial injury and recover the cardiac function.

Conclusion

Overall, the simple self-assembled strategy with ceria nanozyme and curcumin showed a promising clinical application for sepsis-induced cardiac injury by inhibiting ferroptosis and inflammation.

Keywords

Sepsis-induced cardiac injury
Ferroptosis
Inflammation
Ceria nanozyme
Curcumin
==== Body
pmcIntroduction

Sepsis is caused by a dysregulated response to an infection, which results in organ dysfunction, particularly cardiac injury. Clinical studies have proved that the increased mortality rate of sepsis-induced cardiac injury is approximately 80 %, posing a serious threat to human health worldwide [1], [2]. There is substantial evidence that ferroptosis is closely associated with sepsis and plays a crucial role in sepsis-induced cardiac injury [3], [4], [5]. As known to us, ferroptosis is an iron-dependent form of regulated cell death characterized by increased lipid reactive oxygen species (ROS), which causes lipid peroxidation and cell membrane damage [6]. Ferroptosis has been linked to myocardial injury involving increased levels of free iron, which are associated with ferroptosis. Previous studies have confirmed that polydopamine or melanin nanoparticles (NPs) could not only scavenge ROS but also inhibit intracellular iron accumulation, resulting in ferroptosis suppression and improved cardiac function in cardiac injury [7], [8], [9]. Consequently, inhibition of ferroptosis in cardiomyocytes is a promising strategy to enhance the therapeutic efficacy in sepsis-induced cardiac injury.

Apart from ferroptosis-induced ROS, the overproduction of ROS such as hydrogen peroxide (H2O2), hydroxyl radicals, and nitrogen species have also been considered as a major mechanism leading to the development of cardiac dysfunction in sepsis [10]. The increased ROS released from the endothelium promotes endothelial deterioration and increases vascular permeability, thereby accelerating the procession of septic heart failure. Furthermore, the reduced availability of antioxidant enzymes disrupts the balance between ROS production and the antioxidant capacity of cardiac cells, resulting in oxidative damage [11]. In addition, sepsis is regarded as a systemic inflammatory response syndrome for infection, which activates the innate immune system to produce excessive inflammation. It attracted M1 macrophages and other leukocytes to produce pro-inflammatory cytokines [12]. The M1 macrophages could produce nitric oxide to induce myocardial depression [13]. Additionally, after myocardial injury, the released cell fragments could also activate resident cardiac immune cells and promote the inflammatory response [14]. Previous studies have also confirmed that excessive ROS generation plays a crucial role in amplifying and sustaining inflammation, particularly in influencing macrophage differentiation to the M1 phenotype in sepsis-induced cardiac injury [15]. Therefore, scavenging ROS is an effective treatment to improve myocardial function and reduce inflammation to enhance the therapeutic efficacy of sepsis-induced cardiac injury.

Recently, with the development of nanotechnology, nanozymes with natural enzyme-like properties have been widely applied to scavenge ROS as an antioxidant compared to substitute traditional enzymes due to their low cost, high stability, and facile preparation [16]. Among various nanozymes, nano-ceria (CeO2) mimics the actions of superoxide dismutase (SOD) and catalase as an excellent ROS scavenger for various inflammatory diseases [17], [18]. The catalytic property of nano-ceria is attributed to the presence of two reversible oxidation states (Ce3+ or Ce4+) on the surface [19]. Extensive studies have demonstrated that nano-ceria showed protective effects against cardiac injury by reducing oxidative stress and cardiac inflammation [20], [21], [22]. It can also attenuate sepsis-induced organ damage and death by inhibiting the release of pro-inflammatory cytokines and macrophage differentiation [23], [24]. However, the insufficient efficiency of nano-ceria and unsatisfactory capacity to scavenge ROS especially ferroptosis limited its successful application in sepsis-induced cardiac injury. Therefore, it is urgent to develop a combination strategy that enhances the ability of ROS scavenging and inhibits ferroptosis for sepsis treatment. This may require the modification of nano-ceria to improve its catalytic efficiency, exploring potential synergistic effects when combined with other nanozymes or antioxidants, or the integration of additional therapeutic agents aimed at inhibiting ferroptosis pathways. Through the amalgamation of nanotechnology and a deeper comprehension of the pathogenesis of sepsis-induced cardiac injury, researchers have the opportunity to pioneer innovative approaches for augmenting the therapeutic efficacy of nano-ceria. This approach holds the potential to overcome the challenges associated with scavenging ROS and suppressing ferroptosis inhibition, thereby advancing the treatment of sepsis.

Curcumin (Cur) is a natural polyphenol extracted from the rhizome of turmeric, which plays a key role in resisting oxidative stress and inhibiting inflammation for the treatment of sepsis [25]. This effect is underpinned by its robust suppression of pro-inflammatory factors, including interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) [26], [27]. Previous studies have shown that curcumin ameliorates cardiac fibrosis, reduces cardiac cell apoptosis and myocardial infarct size, and decreases inflammation to improve cardiac function [28], [29]. In addition, it acts as a superb H-atom donor to activate and enhance the enzyme activity of nanoceria [30], [31]. More interestingly, due to its polyphenolic structure, curcumin has the property of an iron chelator to inhibit ferroptosis by strikingly modulating proteins of iron metabolism [32]. Previous studies have further confirmed that it could increase the expression of GPX4 and HO-1 to attenuate ferroptosis-induced myocardial injury by activating the Nrf2/ARE pathway [33]. Therefore, the combination of curcumin and nanoceria is suitable to enhance the ROS scavenging ability of nanoceria and inhibit ferroptosis [34]. However, the unsatisfactory water solubility and poor bioavailability of Cur limited the practical applications [35]. In this study, human serum albumin was applied as a carrier to co-deliver curcumin and nanoceria, termed CeCH. The CeCH nanozyme performed both superoxide dismutase (SOD)-like and catalase (CAT)-like activities to exhibit ROS scavenging capabilities, which is better than nanoceria alone. In vitro and in vivo experiments have demonstrated that our formed CeCH NPs can reduce oxidative damage and inhibit ferroptosis of cardiac organs. We also found that the formed CeCH NPs can enhance the ability of antioxidants to reduce the secretion of inflammatory factors by M1 macrophages, resulting in the suppression of inflammation (Fig. 1). Taken together, our findings provide a novel and feasible strategy for sepsis-induced cardiac injury by amplifying the antioxidant capacity of nanozymes with curcumin.Fig. 1 Schematic illustration of CeCH for sepsis-induced myocardial injury. (A) Preparation of CeCH with self-assembled by HSA. (B) The mechanism of CeCH to show a protective effect for sepsis-induced cardiac injury by inhibiting ferroptosis and inflammation.

Materials and methods

Materials

Curcumin was provided by Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Cerium nitrate hexahydrate and salicylic acid（SA）were obtained from Shanghai Aladdin Bio-chem Technology Co., Ltd (Shanghai, China). HSA solution was purchased from Octapharma. The fluorometric hydrogen peroxide assay kit and lipopolysaccharide (LPS) were provided by Sigma-Aldrich. (St. Louis, MO, USA). The cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Griess assay, dihydroethidium (DHE), Mito-Tracker Red CMXRos, MDA, GPX4, IL-1β, IL-10, and TNF-α ELISA kits were provided by Beyotime Institute of Biotechnology (Nanjing, China). Anti-mouse-CD206-APC was bought from BioLegend (San Diego, CA, USA). All chemicals used in this study were of analytical grade and without further purification.

Synthesis and characteristics of CeCH NPs

The Cur encapsulated in HSA nanoparticles (CH) was obtained by self-assembled methods. The Cur dissolved in ethanol (3 mg/ml) was added to HSA (20 mg/ml) by adjusting the pH to 9 with 0.1 M NaOH under stirring conditions for 1 h. After that, 0.44 mL of 0.1 M aqueous solution of Ce(NO2)3-6H2O was added to the obtained CH NPs and stirred for another 15 min. The reaction was carried out in a water bath heated at 37 °C with a speed of 500 rpm to obtain CeCH NPs.

The particle size, size distribution, and zeta potential of the obtained CeCH NPs were determined by Zetasizer Nano ZS-90 (Malvern Instruments, Malvern, UK). After lyophilization, the chemical structure of CeCH was characterized by FT-IR spectroscopy. The morphology and structure were evaluated by transmission electron microscopy (TEM) images. A portion was taken for characterization by X-ray diffraction analysis (XRD) and X-ray photoelectron spectroscopy (XPS), and for determination of the concentration of cerium dioxide with inductively coupled plasma emission spectrometry. To determine the concentration of Cur, the obtained nanoparticles containing CeCH and CH were dissolved in dimethylsulfoxide (DMSO) and then centrifuged at 3500 rpm for 10 min. The content of Cur in the samples was then determined using a UV spectrophotometer and calculated based on the standard curve.

Assay of superoxide dismutase activity

SOD-like activity was measured by photochemical reduction of nitroblue tetrazolium chloride (NBT) inhibition. 400 µL EDTA (0.1 mmol/L), 150 µL nitrotetrazolium blue (2 mmol/L), and100 µL riboflavin (1.2 mmol/L) were dissolved in 5.8 mL of deionized water solution to form the assay solution. The assay solution was then mixed with CeH NPs (HSA nanoparticles delivering nanoceria) and CeCH NPs containing 25 µg/mL of CeO2. The enzymatic reaction was initiated by irradiating the reaction mixture with a 27 W lamp for 2 min at room temperature, and the SOD-like activity of the samples was determined based on the peak at 560 nm.

Assay of peroxidase activity

As H2O2 is another important product of ROS that can damage cells, the scavenging capacity of H2O2 was also evaluated by the addition of formed CeCH. The scavenging capacity of H2O2 was determined using a fluorescent hydrogen peroxide assay kit (Sigma, MAK165). The principle is that the peroxidase substrate reacts with hydrogen peroxide to produce a red fluorescent product (λex/λem = 540/590 nm), and the peroxidase activity can be measured by the intensity of the red fluorescence. Briefly, CeH and CeCH with 25 µg/mL of CeO2 were added to the reaction buffer containing hydrogen peroxide (4.0 μM) and incubated for 5 min. After that, Amplex Red Reagent/HRP working solution (50 μL) was added and incubated for 30 min in the dark. Finally, the fluorescence intensity of the red fluorescence was measured by luminescence digestion.

Assay of hydroxyl radical scavenging activity

Hydroxyl radical (•OH) is another important ROS, and its scavenging will help to protect cells or living organisms more effectively from damage caused by ROS. To investigate whether CeCH NPs can scavenge •OH, UV–Vis and electron spin resonance (ESR) were used. Spectroscopy was used to detect •OH levels in the presence of CeCH NPs. The Fenton reaction of the Fe2+ /H2O2 system was used to produce •OH. The treatments of CeH and CeCH NPs containing 50 µg/mL of CeO2 were added to mix the Fenton reaction system, respectively. After 10 min of reaction, salicylic acid (1.8 mM) was added for color development, and a clear absorption peak was observed at 510 nm. 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin trap to form DMPO/•OH spin adducts and determined by ESR. The DMPO (0.2 mg/ml) was added to the above reaction system and ESR analysis was performed 30 min later.

Assay of DPPH scavenging activity

Free radical scavenging activity was also determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging photometric assay. Different treatments of CeH and CeCH with 12.5 µg/mL of CeO2 were added to the DPPH solution (2 mL, 50 µM) and mixed for 90 min. The absorbance of DPPH radical at 517 nm was determined using a UV–Vis spectrophotometer.

Cytotoxicity assay of CeCH in H9C2 cardiomyocytes

The cytotoxicity of CeCH was evaluated in H9C2 cells. The cells were inoculated in 96-well plates at a density of 5x103 cells/well for 48 h. Then, different concentrations of CH, CeH, and CeCH (Cur, 2.5 μM, 10 μM, and 15 Μm) were incubated with H9C2 for 24 h respectively. After that, H9C2 cells were washed twice with PBS and incubated with CCK-8 reagent for 2 h. The OD values of each well were measured on an ELISA at 450 nm. The relative cell activity was calculated.

Anti-oxidant of CeCH in H9C2 cardiomyocytes

Cardiomyocytes were treated with serum-free Dulbecco's Modified Eagle Medium (DMEM) diluted with different concentrations of H2O2 (50, 100, 200, 400, 800, 1000, 1200, and 1400 μM) for 12 h. According to the cell viability, the concentration of H2O2 (1200 μM) was selected for subsequent experiments. The different concentrations of CH, CeH, and CeCH with the same concentrations of curcumin (10 μM, 20 μM, 30 μM) were incubated with H9C2 cells for 1 h. Then, H2O2 (1200 μM) was added and incubated with H9C2 cells for 12 h. Finally, the cells were washed twice with PBS, and CCK-8 was added and incubated at 37 °C for a further 4 h. The absorbance of each group of cells at 450 nm was measured using an enzyme marker.

ROS was assessed using a fluorescent probe (DHE), which can be dehydrogenated by intracellular superoxide anions after uptake by living cells to produce ethidium, which can bind to RNA or DNA to produce red fluorescence. After co-incubation of 400 μM H2O2 with 60 μM sample for 1 h, the cells were washed twice with phosphate-buffered saline (PBS). Then, 10 μM DHE was incubated with cells in different treatments for 30 min. Finally, after being washed twice, fluorescence imaging was performed using fluorescence microscopy. The level of malondialdehyde (MDA), as a lipid peroxidation biomarker, was determined according to the standard protocols [36].

Ferroptosis inhibition of CeCH in H9C2 cardiomyocytes

The cardiomyocytes were treated for 12 h with serum-free DMEM medium diluted with RSL3 and prepared in a gradient of 2, 4, 6, 8, 12, 16, 18, and 20 μM concentrations. The 20 μM RSL3 was selected for subsequent experiments to induce the ferroptosis in H9C2 cells. The treatments of CH, CeH and CeCH with the same concentration of curcumin (20 μM and 40 μM) and RSL3 were incubated together with H9C2 cells for 12 h. After washed twice with PBS, CCK-8 was added and the incubation continued for 4 h. The absorbance of each group of cells at 450 nm was measured by an enzyme marker. Glutathione peroxidase (GPX) activity was measured using the Cellular Glutathione Peroxidase Assay Kit with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Beyotime, China). All the steps were performed according to the manufacturer's instructions.

Mito-Tracker Red CMXRos is a fluorescent probe that can cross the cell membrane by passive transport to specifically label biologically active mitochondria in cells. After co-incubation of RSL3 (5 μM) and treatments (curcumin, 40 μM) for 6 h, the cells were washed twice with PBS and then incubated with 50 nM Mito-Tracker Red CMXRos for 30 min. Fluorescence imaging was then captured by fluorescence microscopy.

Cytotoxicity assay of CeCH in macrophages

RAW264.7 cells were inoculated in 96-well plates at a density of 1x104 cells/well for 24 h. After incubation with CH, CeH and CeCH containing curcumin at concentrations of 0.25 μM, 0.5 μM and 1 μM, respectively, for 24 h, the cells were washed twice with PBS and incubated with CCK-8 reagent for 4 h. The results were measured at 450 nm using an enzyme marker.

Anti-inflammatory activity of CeCH in macrophages

LPS was applied to polarize macrophages to release inflammatory factors. Briefly, the RAW264.7 cells were incubated overnight in 96-well plates at a density of 1x104 cells per well. The treatments of CH, CeH and CeCH with the same concentrations of Cur (0.25 μM, 0.5 μM and 1 μM) were added and incubated for 1 h. After that, LPS (5 μg/mL) was added to incubate for another 24 h. Then, the cell supernatants were centrifuged to evaluate the levels of ·NO and H2O2 using a nitric oxide assay kit (Beyotime, China) and a fluorescent hydrogen peroxide assay kit (Sigma, MAK165), respectively. In addition, the gene expression factors relevant to M1 (IL-1β) and M2 phenotype (IL-10) were determined by Q-PCR (Agilent Tech.). The primer sequences used for qPCR were obtained from GenScript. All experiments were repeated six times.

M2 macrophage polarization of CeCH

The RAW264.7 cells were inoculated in 6-well plates at a density of 2x105 cells per well and incubated for 12 h. After incubation with CeCH (Cur, 1 μM) for 1 h, LPS (5 μg/mL) was added to co-incubated for 24 h. Then, the cells were washed twice with PBS and repeatedly blown to make a cell suspension. At last, the cells were stained with CD206 antibody (BioLegend, San Diego, CA, USA) to label M2 cells and analyzed using a flow cytometry.

Animals treatments

C57BL/6 mice (male, 8 weeks) were purchased from the Model Animal Research Center of Nanjing University. The animals were maintained on standard laboratory chow with free access to food and water and housed in a temperature (22 ± 1 ℃) and humidity (65–70 %)-controlled room with a 12-h light–dark cycle. C57BL/6 mice were subjected to cecal ligation and perforation.

The animals were randomly divided into five groups: the sham group, LPS induced model group, LPS with CH group, LPS with CeH group and LPS with CeCH group. LPS (10 mg/kg) was intraperitoneally injected to establish septic cardiomyopathy models. After 2 h, the different formulations of CH, CeH and CeCH were intravenously injected with the same concentration of Cur and Ce (2 mg/kg and 0.6 mg/kg) to septic mice. Then, echocardiography was performed on mice under anesthesia to evaluate the cardiac function (heart rate, LV ejection fraction and LV fractional shortening) after 24 h of treatment. Finally, the heart was collected and performed the H&E staining and immunohistochemical staining for 4HNE, GPX4, IL-1β, IL-10, and TNF-α to evaluate the therapeutic efficacy. The staining was performed according to standard protocols [37], [38]. In addition, the secretion of MDA, GPX4, IL-1β, IL-10, and TNF-α in homogenates of heart tissue were determined by ELISA kits (Beyotime, China). The specific steps were performed according to the manufacturer’s instructions.

Ethics statement

All experiments involving animals were approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital (Approval no. 201921A011) and were performed in accordance with the Care and Use of Laboratory Animals published by the National Institutes of Health (Eighth Edition).

Statistical analysis

All the data were presented as mean ± standard deviation (SD) or mean ± standard error (SE) of at least three samples. For multiple comparisons, one-way analysis of variance (ANOVA) and Tukey’s post-doc tests were applied. All data were analyzed using GraphPad PRISM 5 software. Unpaired Student’s t-test (two-tailed) was performed for the comparison of two groups. Statistical significance was defined as *p < 0.05, **p < 0.01.

Results and discussion

Synthesis and characterization of CeCH NPs

CeCH NPs were synthesized according to our previous methods [39]. The CH NPs were self-assembled by simply mixing Cur with HSA. Then, the Ce3+ was absorbed to CH and formed CeCH under an alkaline environment. The surface morphology was characterized by TEM, which showed good monodisperses (Fig. 2A). Also, CeCH clearly showed the formation of lattice stripes of the nanoparticles with 0.31 Å, consistent with the reported CeO2 nanostructure. The average size of CeCH NPs was 51.2 ± 4.8 nm, which was similar as CeH NPs (48.3 ± 6.4 nm). They were both larger than that of Cur NPs (2.7 ± 0.2 nm), attributing to the loading of cerium dioxide (Fig. 2B). The size distribution of CeCH NPs lied in the range of 30 nm to 200 nm which was determined by dynamic light scattering (Fig. 2C). The zeta potential of CeCH NPs was about −17 mV, which was lower than that of CH NPs and CeH NPs, indicating that CeCH NPs were more stable than them (Fig. 2D). As shown in Fig. 2E, the CeCH showed an absorption at 420 nm, which is similar of CH. These indicated that the successful encapsulation of Ce and Cur in the prepared nanoparticles. In addition, the loading capacity of Cur was calculated to be 12.4 % by a calibration curve of the absorbance (A) at 428 nm versus the concentration (C) of Cur, which can be expressed as A = 0.05176 × C −0.0011.Fig. 2 Characterization of CeCH. (A) TEM images, scale bars, 200 nm, (B) Particle size, (C) Size distribution, and (D) Zeta potential of CeCH. (E) UV spectra of different formulations. (F) EDS spectrum of CeCH. (G) Full XPS analysis, (H) XPS spectra and (I) XRD of CeCH and CeH.

The energy dispersive spectra (EDS) of Cur-Ce NPs indicated the presence of O, S, and Ce elements (Fig. 2F). The sulfur (S 2p) signal was detected by X-ray photoelectron spectroscopy (XPS) analysis, indicating the successful binding of CH NPs with CeO2 (Fig. 2G). In addition, the corresponding binding energy peaks of Ce3+ (880.20, 885.00, 899.50, and 903.50 ev) and Ce4+ (882.10, 888.10, 898.00, 900.90, 906.40 and 916.35 ev) were present in both of CeCH and CeH NPs (Fig. 2H). The structural characteristic peaks of cerium dioxide crystals 111, 220, 311, 331 were evaluated by XRD (Fig. 2I). All these characterization results confirmed that the formed CeCH NPs successfully encapsulated CeO2.

In vitro free radical scavenging capacity of CeCH NPs

Since oxygen radicals (•O2–) are the primary ROS that initiate cascade reactions to further generate ROS, scavenging for •O2– is the initial step in the anti-ROS cascade reactions [40], [41]. Previous study has reported that the coexistence of Ce3+ and Ce4+ oxidation states on the surface of CeO2 NPs contributes to their antioxidant activity [4]. In this study, we systematically evaluated the SOD-like activities of CeH NPs and CeCH NPs using NBT as an •O2– sensitive indicator, to monitor their capability to eliminate •O2–. As shown in Fig. 3A, both CeH NPs and CeCH NPs effectively suppressed the generation of •O2– induced by riboflavin (RF), demonstrating excellent SOD-like activity, and CeCH NPs exhibited even higher SOD-like activity than CeH NPs.Fig. 3 ROS elimination of CeCH. (A) SOD-like activity of CeCH to eliminate O2–. (B) CAT-like activity to scavenge H2O2 by fluorescence intensity. (C) DPPH elimination of CeCH. (D) UV spectra and (E) ESR of •OH scavenging activity by CeCH.

Hydrogen peroxide (H2O2) is another common oxidant and a downstream product of •O2– catabolism. Catalyzing H2O2 into H2O and O2 by CAT is the second key step in the ROS scavenging cascade system [42]. To further investigate the CAT-like activity of CeCH NPs, we evaluated the levels of H2O2 after adding CeCH NPs into a H2O2 (4 µM) solution using a fluorometric hydrogen peroxide assay. As shown in Fig. 3B, the strong fluorescence peak at 590 nm indicated the presence of H2O2, and was significantly attenuated after the addition of CeH NPs or CeCH NPs. These results indicated that both CeH NPs and CeCH NPs possessed excellent CAT-like activities for scavenging H2O2.

The free radical scavenging activity was measured by the DPPH scavenging assay. The maximum absorbance at 517 nm declined after the addition of CeH NPs or CeCH NPs. The absorbance of the CeCH NPs group declined more significantly than that of the CeH NPs group. These results indicated that CeCH NPs exhibited higher DPPH radical scavenging activity than CeH NPs (Fig. 3C), which could be attributed to the action of curcumin on DPPH radicals [3].

Among all types of ROS, hydroxyl radical (•OH) is considered to be one of the most reactive substances, capable of attacking biomolecules and causing irreversible oxidative damage [43], [44]. To investigate the elimination of •OH, levels of •OH were detected using its specific probe, salicylic acid (SA), through absorption spectroscopy and EPR spectroscopy. The generated •OH exhibited a clear absorption peak at 510 nm. The absorbance of the CeCH NPs group decreased more significantly than that of the CeH NPs group, indicating that CeCH NPs possessed higher •OH scavenging activity than CeH NPs (Fig. 3D). Additionally, the •OH scavenging activity of CeCH NPs was further confirmed by EPR, as the signal of DMPO/•OH was reduced in the presence of CeCH NPs, consistent with the results obtained using the SA method (Fig. 3E). The above results clearly demonstrated that CeCH NPs exhibited excellent ROS scavenging activity, which will be further tested in the subsequent cell experiments.

ROS inhibition of CeCH in H9C2 cells

The cytotoxicity of CH, CeH, and CeCH NPs at different concentrations (2.5 μM, 10 μM, 15 μM of Cur), was first evaluated in H9C2 cardiomyocytes by CCK-8 assay. No obvious cytotoxicity was observed (Fig. 4A), demonstrating the safeness of the NPs. We then moved to investigate the ability of CeCH NPs to protect cardiomyocytes from ROS damage. An H2O2 oxidative stress model was established by incubating H9C2 cardiomyocytes with 1200 μM of H2O2 for 12 h, which led to 40 % cell viability. The addition of CeCH NPs (30 μM) significantly decreased ROS-induced cell death and improved the survival rate by up to 80 %, while CeH NPs showed a lower protective effect as evidenced by a survival rate of ∼60 % (Fig. 4B). Moreover, CH NPs barely showed a protective effect in preventing ROS-induced cell death, suggesting the anti-oxidant activity of CeO2 nanozymes mainly contributed to the protective effect, which might be promoted by the combination of CeO2 and Cur in CeCH. However, reducing the concentration of the NPs would make CeCH comparably ineffective as CeCH in protecting H2O2-caused cell death.Fig. 4 ROS inhibition of CeCH in H2O2-treated H9C2 cells. (A) Cell viability of different formulations and concentrations in H9C2 cells. (B) Protective effect of CeCH with different concentrations in H2O2-treated H9C2 cells. (C) Representative fluorescence images and (D) mean fluorescence intensity of ROS after various treatments in H2O2-treated H9C2 cells. Scale bar = 200 μm. (E) MDA levels in the supernatant of H2O2-treated H9C2 cells after various treatments. All data were presented as mean ± SD (n = 3, **p＜0.01 CeCH vs. other treatments).

To confirm whether the protective effect of CeCH NPs is a consequence of ROS scavenging, the DHE probe, an indicator of oxidative stress, was then used in cardiomyocytes to monitor the cellular oxidative stress. As shown in Fig. 4C, the increased ROS release triggered by H2O2 was effectively inhibited upon the addition of NPs as evidenced by the attenuated red fluorescence. Among them, the red fluorescence was almost quenched in CeCH NPs, indicating that CeCH NPs had a good inhibitory effect on ROS generation in the H2O2 oxidative stress model. The quantitative analysis of the DHE fluorescence intensity indicated the best performance of CeCH NPs by scavenging almost 70 % of the H2O2-induced ROS generation (Fig. 4D). Moreover, the level of MDA could also be effectively suppressed by CeCH NPs, suggesting the potential to suppress ferroptosis (Fig. 4E). Taken together, the above results confirmed the ability of CeCH to protect H9C2 cardiomyocytes from ROS damage by scavenging ROS generation.

In vitro inflammation inhibition of CeCH

Cardiomyocytes' inflammatory response is also one of the characteristic pathological signs in septic cardiomyopathy, which is unfavorable as the inflammation would promote mitochondrial ROS generation and ferroptosis [45]. Macrophages play a key role in inducing local inflammation, especially M1 macrophages [46]. After proving the low cytotoxicity of the NPs towards RAW264.7 macrophages (Fig. 5A), we established an in vitro inflammation model by adding LPS (5 μg/mL) into RAW264.7 cells to stimulate the transformation of M1 macrophage and the production of ROS and reactive nitrogen species (NOS). As shown in Fig. 5B and C, all of the CH NPs, CeH NPs, and CeCH NPs exhibited the ability to significantly reduce NO and H2O2 levels in RAW264.7 macrophages. Meanwhile, high concentration (1 μM) of CeCH NPs showed the best performance in the inhibition of LPS leading to ROS/RNS production than the other treatments. This is consistent with the ROS-eliminating results discussed above, which could be due to the synergistic effect between Cur and CeO2 in scavenging extracellular free radicals and inhibiting the excessive production of intracellular oxidants [44].Fig. 5 Inflammation reverse of CeCH in LPS-treated macrophages. (A) Cell viability after different treatments in RAW 264.7 macrophages. (B) NO level and (C) H2O2 level in the supernatant of LPS-treated macrophages incubated with different formulations. (D) In vitro mRNA expression of IL-1β and (E) IL-10 with the treatment of LPS and formulations in the RAW 264.7 macrophages. (F) Quantification of CD206-positive cells (M2) with different treatments by flow cytometry analysis. All data were presented as mean ± SD (n = 3, **p＜0.01 CeCH vs. other treatments).

The generation of M1 and M2-related inflammatory cytokines (IL-1β and IL-10) was determined by incubating CeH and CeCH in LPS-induced RAW264.7 cells. As shown in Fig. 5D, CeCH NPs were found to significantly alleviate the inflammation response by eliminating the expression of inflammatory cytokine IL-1β stimulated by LPS. We further found that CeCH NPs could markedly elevate the expression of IL-10 in LPS-treated RAW264.7 macrophages by approximately 4-fold than the LPS-treatment group (Fig. 5E), indicating the reprogramming of M1 macrophages to M2 macrophages, which led to reduced secretion of pro-inflammatory cytokines. Furthermore, the flow cytometry results confirmed the high proportion of M2 phenotype macrophages in the CeCH NPs treated cells (Fig. 5F). In comparison, CeH NPs exhibited much lower capability in decreasing the LPS-induced inflammation responses. All these results indicated that CeCH NPs can effectively induce the polarization of macrophages toward M2 phenotype cells, thus reducing inflammation.

Protective effect of CeCH in RSL3-induced ferroptosis of H9C2 cells

As one of the major cell death pathways in sepsis-induced cardiac injury, ferroptosis is also initiated by the accumulation of ROS [3], [47], [48]. After proving the ROS scavenging ability of CeCH, the inhibition of ferroptosis by CeCH was further investigated. The ferroptosis in H9C2 cells was first induced by RSL3 [49]. As shown in Fig. 6A, different concentrations of RSL3 were added to H9C2 cells. The cell survival rate reached about 45 % at the concentration of RSL3 (20 μmol/L) for 12 h, which was suitable for establishing the RSL3 ferroptosis model and for subsequent studies. Similar to the results in the ROS inhibition section, CeCH exhibited better performance in preventing cell death caused by RSL3-induced ferroptosis than CeH (Fig. 6B). Especially, at the concentration of 40 μM of Cur, CeCH almost entirely inhibited cell ferroptosis. We also found that the CeH group showed no significant difference from the RSL3 group, which indicated that CeO2 showed no effect in the ferroptosis of H9C2 cells. CH showed comparable activity in inhibiting ferroptosis as CeCH, suggesting Cur could be the main contributor in inhibiting cell ferroptosis. The protective effect of CeCH against ferroptosis was further verified by staining H9C2 cells with Mito-Tracker Red CMXRos. Compared with the control group, the RSL3 group exhibited disordered mitochondrial arrangement, indicating that RSL3 could induce a ferroptosis model in H9C2 cells. Conversely, the CeCH group exhibited reduced myocardial mitochondrial damage and maintained membrane integrity (Fig. 6D), attenuating mitochondrial damage resulting from ferroptosis.Fig. 6 Ferroptosis inhibition of CeCH in RSL3-induced H9C2 cells. (A) Cell viability treated with different concentrations of RSL3 to form ferroptosis model in H9C2 cells. (B) Protective effect of CeCH with different concentrations in 20 μM RSL3-induced ferroptosis model in vitro. (C) GPX4 activity in the supernatant of RSL3-induced H9C2 cells after different treatments. (D) Representative fluorescence images of mitochondrial membrane potential (red fluorescence) with different treatments. Scale bar = 200 μm. All data were presented as mean ± SD (n = 3, **p＜0.01 CeCH vs. other treatments).

The down-regulation of key enzyme GPX4 which regulates the ratio of the reduced and oxidized forms of glutathione is recognized as the classic pathway of ferroptosis [50], [51], [52]. As shown in Fig. 6C, the RSL3 reduced the expression of GPX significantly to induce ferroptosis in H9C2 cells; meanwhile, the addition of the three NPs recovered the activity of GPX4. Still, CeCH-treated H9C2 cells exhibited the highest expression of GPX, which was close to that of the control group (Fig. 6C), demonstrating the advantage of the CeO2/Cur combination in facilitating the GPX4 expression. CH-treated H9C2 cells showed higher expression of GPX-4 than that of CeH-treated ones, which was consistent with the cell viability results and confirmed the main role of Cur in inhibiting ferroptosis. These results revealed that CeCH NPs had an excellent protective effect against ferroptosis of cardiomyocytes.

CeCH protected the hearts against LPS-induced acute myocardial injury and cardiac dysfunction

Holding excellent anti-ferroptosis and anti-inflammation activities, we are encouraged to investigate the therapeutic potential of CeCH NPs on sepsis-induced myocardial injury. Male C57BL/6J mice were exposed to LPS (10 mg/kg, i.p.) to establish the septic cardiomyopathy animal model. After 24 h-post injection of LPS, echocardiography results revealed that the LPS-treated mice demonstrated a sharp decrease in Left Ventricular Ejection Fraction (LVEF) and Fraction Shortening (FS) by ∼50 %, confirming the acute septic cardiomyopathy (Fig. 7A). Compared with the LPS-treated group, all treatment groups appeared to increase LVEF and FS (Fig. 7B and C). Notably, CeCH treatment markedly enhanced LVEF and FS, which even reached the normal level when compared to the control group. These results implied that CeCH NPs mitigated heart injury and preserved heart contractile function.Fig. 7 Reverse of cardiac dysfunction by CeCH in LPS-induced septic cardiomyopathy. (A) Representative M−mode echocardiographs of septic mice exposed to different treatments were collected at 24 h after LPS injection. (B) Quantitative analysis of the ejection fraction and (C) fractional shortening after treatments. All data were presented as mean ± SD (n = 6, **p＜0.01, *p＜0.05, CeCH vs. other treatments).

Further biological/biochemical analysis was performed to clarify the protective effect of CeCH in vivo. H&E staining showed the myocardial cells were sparse and disordered, with interstitial edema and inflammatory cell infiltration in the LPS-treated group; in contrast, treatment with CeCH NPs ameliorated the structural abnormalities in the heart of LPS-treated mice and reduced inflammatory cell infiltration, demonstrating effective protection (Fig. 8A). In addition, to confirm that the in vivo protective effect also relies on the reduction of oxidative stress, the expressions of antioxidant enzymes, 4-HNE, and GPX4, were analyzed. As shown in Fig. 8B and G, the expression of 4-HNE was significantly increased by LPS treatment and decreased after the administration of CeCH. Moreover, the immunohistochemical staining of GPX4 showed that CeCH could significantly elevate its expression, facilitating the inhibition of ferroptosis (Fig. 8C, D and E). The GPX activity was also determined, which was consistent with the expression results (Fig. 8F). In addition, suppressed MDA level was detected in the CeCH group, further confirming the mechanism of reducing oxidative stress to inhibit ferroptosis (Fig. 8H). Collectively, the in vivo evidence demonstrated the potential of CeCH NPs in protecting hearts against LPS-induced acute myocardial injury and improving cardiac function via scavenging ROS/RNS and inhibiting ferroptosis.Fig. 8 Therapeutic effects of CeCH in LPS-induced septic cardiomyopathy by inhibiting ROS-related ferroptosis. (A) H&E staining images of heart tissue after various treatments. (B) Immunohistochemical staining images of 4HNE and (C) GPX4 after various treatments. (D) Western blotting analysis of GPX4 levels. GAPDH used as the control. (E) Relative analysis of GPX4 and GAPDH levels after various treatments. (F) GPX4 activity in the heart after various treatments. (G) Mean intensity of 4HNE with various treatments. (H) MDA level after various treatments. All data were presented as mean ± SD (n = 6, **p＜0.01 CeCH vs. other treatments). Scale bar = 200 μm.

Inflammation and M2 polarization of CeCH in septic cardiomyopathy

Local inflammation in the heart exerts a detrimental influence on septic cardiomyopathy. Macrophages, particularly M1 macrophages, are pivotal contributors to the imitation of local inflammation [46]. To evaluate the anti-inflammation performance of CeCH in septic cardiomyopathy, we monitored the macrophage polarization by immunofluorescence staining CD86 + macrophages (M1, red fluorescence) and CD206 + macrophages (M2, green fluorescence). As shown in Fig. 9A and B, the population of M1 macrophages increased significantly under the stimulation of LPS as evidenced by the bright red fluorescence; whereas, the red fluorescence disappeared along with the enhancement of green fluorescence after the administration of CeCH, indicating the macrophage M2 polarization in the septic myocardium. Especially, the M2 macrophages were at the same level as the control group, which was much higher than that of the CH group or CeH group, demonstrating the great ability of CeCH to regulate macrophage polarization.Fig. 9 Therapeutic effects of CeCH in LPS-induced septic cardiomyopathy by inhibiting inflammation in vivo. (A) Immunofluorescence staining images of M1 and M2 macrophages after treatments. (B) Quantitative analysis of CD206-positive cells (M2) in heart tissues after treatments. (C-E) Immunohistochemical staining images and determination of IL-1β and TNF-α after various treatments. (F-G) The determination of IL-10 in heart tissues after various treatments. All data were presented as mean ± SD (n = 6, **p＜0.01 CeCH vs. other treatments). Scale bars = 200 μm.

In order to confirm the results, the expression of proinflammatory factors (IL-1β and TNF-α) and anti-inflammatory factors (IL-10) were also evaluated by immunohistochemical staining and determination [53]. As shown in Fig. 9C-F, consistent with the in vitro evaluation, we also found that CeCH effectively alleviated the expression of IL-1β and TNF-α promoted by LPS (Fig. 9C-E), while greatly raising the level of IL-10 (Fig. 9G and F), exhibiting superior anti-inflammation potential than other NPs. Together, all these data confirmed the anti-inflammatory effect of CeCH NPs in septic cardiomyopathy, contributing to heart protection against LPS-induced acute myocardial injury.

Conclusion

In summary, a self-assembled CeO2 nanozyme coordination with Cur based on an HSA template (CeCH NPs) was developed to enhance the therapeutic efficacy of septic cardiomyopathy. In vitro studies showed that CeCH NPs possessed SOD-like and CAT-like activities as they scavenged the ROS generation in H9C2 cells, and hence, protected H9C2 cells by inhibiting RSL3-induced ferroptosis. Anti-inflammation experiments showed that CeCH NPs could alleviate inflammation response by inhibiting the production of inflammatory cytokines and promoting M2 macrophage polarization. More importantly, we further showed that CeCH could effectively protect the hearts against acute myocardial injury and reverse cardiac dysfunction using the LPS-induced septic cardiomyopathy mice model. Inhibition of ferroptosis and anti-inflammation were found to be the major mechanisms of the protective effect, which is in line with in vitro characterizations. In addition, CeCH NPs exhibited superior performance in ferroptosis inhibition and anti-inflammation when compared to CH or CeH NPs, suggesting the synergistic effect between Cur and CeO2. Taken together, this study provided a novel strategy of combining CeO2 with Cur for septic cardiomyopathy via inhibiting ferroptosis and inflammation, which showed promise in clinical applications.

CRediT authorship contribution statement

Chenxiao Jiang: Data curation, Writing – original draft, Writing – review & editing, Funding acquisition. Qianzhi Shi: Data curation, Writing – original draft, Writing – review & editing. Jing Yang: Visualization, Investigation. Hao Ren: Visualization, Investigation. Lu Zhang: Writing – review & editing. Shan Chen: Supervision. Jiayi Si: Writing – review & editing. Yihai Liu: Data curation, Writing – original draft, Writing – review & editing. Dujuan Sha: Conceptualization, Methodology, Software. Biao Xu: Conceptualization, Methodology, Software. Jie Ni: Conceptualization, Methodology, Software, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by grants of the National Natural Science Foundation of China (82100398); the Nanjing Medical Science and Technique Development Foundation (YKK21068); Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2023-LCYJ-PY-24); the Jiangsu Research Hospital Association for Precision Medication (JY202120); the Jiangsu Pharmaceutical Association for Jinpeiying Project (J2021001); China Postdoctoral Science Foundation (2022M721576).
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