Mackinawite nanozymes as reactive oxygen species scavengers for acute kidney injury alleviation

Background Iron sulfide nanomaterials have been successfully employed as therapeutic agents for bacterial infection therapy and catalytic-ferroptosis synergistic tumor therapy due to their unique structures, physiochemical properties, and biocompatibility. However, biomedical research and understanding of the biological functions of iron sulfides are insufficient, and how iron sulfide nanomaterials affect reactive oxygen species (ROS) in diseases remains unknown. Acute kidney injury (AKI) is associated with high levels of ROS, and therefore nanomedicine-mediated antioxidant therapy has emerged as a novel strategy for its alleviation. Results Here, mackinawite nanozymes were synthesized from glutathione (GSH) and iron ions (Fe3+) (denoted as GFeSNs) using a hydrothermal method, and then evaluated as ROS scavengers for ROS-related AKI treatment. GFeSNs showed broad-spectrum ROS scavenging ability through synergistic interactions of multiple enzymes-like and hydrogen polysulfide-releasing properties. Furthermore, both in vitro and in vivo experiments demonstrated that GFeSNs exhibited outstanding cytoprotective effects against ROS-induced damage at extremely low doses and significantly improved treatment outcomes in AKI. Conclusions Given the synergetic antioxidant properties and high biocompatibility, GFeSNs exhibit great potential for the treatment of AKI and other ROS-associated diseases. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-023-02034-7.


Background
Acute kidney injury (AKI) is a prominent clinical disease characterized by a rapid decrease in renal excretory function accompanied by decreased urine output and increased nitrogen metabolism accumulation [1,2].AKI can be caused by a variety of injuries, including ischemia-reperfusion, sepsis [3,4], hypotension [5,6], and overuse of antibiotics and chemotherapeutics [7], and is a growing healthcare concern worldwide [8].AKI is one of the most common clinical complications, occurring in nearly 46% of hospitalized patients with COVID-19 [9,10], a higher rate than in non-COVID-19 patients.AKI patients with COVID-19 also face a reduced likelihood of renal function recovery and increased risk of death [2,11,12].However, apart from hemodialysis and kidney transplantation, there is currently a lack of effective therapies to prevent the development of AKI or promote its reversal [13,14].
Pathological analysis of AKI suggests that reactive oxygen species (ROS) can trigger oxidative stress and inflammation, causing damage to lipids, nucleic acids, and proteins, which are closely related to the occurrence of AKI [15,16].While low levels of ROS generated during renal metabolism in healthy kidneys are beneficial for physiological redox signaling to promote cell survival [17,18], proliferation, and growth, excessive ROS can cause oxidative stress damage in cells [19], further stimulating the activation of interstitial fibroblasts, leading to renal fibrosis, chronic kidney disease [20], and eventually irreversible and permanent damage to renal function [21].Therefore, effective ROS clearance may be the key to AKI prevention and treatment [22,23].
Iron-based nanomaterials have been widely employed in biomedicine due to their biocompatibility and biological function [38][39][40].For instance, iron oxide nanoparticles (ferumoxytol) have been approved by the Food and Drug Administration (FDA) as iron supplement for treating iron deficiency, and as contrast agents for magnetic resonance imaging [41][42][43].Furthermore, iron oxide and iron sulfide nanoparticles have been widely used in antibacterial and antitumor therapy via intrinsic enzyme-like activity, accompanied by ferroptosis-like properties [44,45].We recently found that iron sulfide nanomaterials (FeSNs) can continuously and steadily release hydrogen polysulfides, with strong anti-inflammatory and antioxidant properties [46].Polysulfides rapidly scavenge the biological oxidant hydrogen peroxide (H 2 O 2 ) and inhibit mitochondrial ROS production by modulating mitochondrial redox signaling [47].The hydrogen polysulfide donor Na 2 S 4 is also a promising therapeutic agent for the treatment of cisplatin-induced nephrotoxicity and as an adjuvant to cisplatin for cancer therapy [48].
Here, we screened several active sulfides, including L-cysteine, diallyl trisulfide (DATS), glutathione (GSH), and sodium thiosulfate, to hydrothermally synthesize into iron sulfides (CFeSNs, DFeSNs, GFeSNs, and TFeSNs) with lowly toxic ferric ions.The as-synthesized GFeSNs showed the strongest ability to suppress superoxide anions and release hydrogen polysulfides and were therefore used for further study.Given their synergistic mode of action, GFeSNs exhibited broad-spectrum ROS scavenging activities, including against hydroxyl radical (•OH), superoxide anion (O 2

•−
), and hydrogen peroxide (H 2 O 2 ), and protected cells from oxidative damage in vitro.GFeSNs showed high biocompatibility and strong ROS scavenging ability at a working concentration of 5 μg/ mL in vitro and 500 μg/kg for AKI in vivo.To the best of our knowledge, this dose is much lower than that of any other iron sulfide compounds reported for the treatment of bacterial infections or tumors [49][50][51].This study provides a new approach for preparing nano-sized mackinawite nanozymes with robust ROS scavenging capability.These as-prepared novel mackinawite nanozymes show considerable application potential as efficient antioxidant agents to meet the clinical needs of AKI and other ROSrelated therapy.

Synthesis of GFeSNs
FeCl 3 (5 mmol) was dissolved in EG (40 mL) with magnetic stirring for 15 min, after which NaAc•3H 2 O (40 mmol) and GSH (4 mmol) were slowly added with continuous stirring until the solution became transparent.The solution was then transferred to a polytetrafluoroethylene reactor and reacted at 200 ℃ for 12 h.The reactant was then washed with ethanol absolute and ultrapure water three times each.Finally, the product was freeze-dried overnight and stored at 4 ℃ until further use.

H 2 O 2 scavenging activity of GFeSNs
The H 2 O 2 scavenging effect of GFeSNs at different concentrations (2.5-80 μg/mL) was examined using a hydrogen peroxide detection kit (Nanjing Jiancheng Bioengineering Institute, China).In brief, GFeSNs were incubated at a range of concentrations (2.5-80 μg/mL) with 500 mM H 2 O 2 for 24 h at 37 °C, respectively, after which the samples were reacted with ammonium molybdate and measured at 405 nm.

•OH scavenging activity of GFeSNs
The •OH generated by the Fenton reaction reacts with salicylic acid to generate 2,3-dihydroxybenzoic acid with special absorption at 510 nm, with the reaction formula: Here, 9 mM salicylic acid and different concentrations of GFeSNs (2.5-80 μg/ mL) were successively mixed, followed by the addition of an appropriate amount of deionized water and 8.8 mM H 2 O 2 .Absorbance was measured at 510 nm after incubation in a water bath at 37 °C for 15 min.

O 2 •− scavenging activity of GFeSNs
The GFeSN scavenging activity of O 2 •− was assessed using a superoxide anion assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions.Different concentrations of GFeSNs (2.5-80 μg/mL) were added to the working solution.After standing for 30 min, absorbance was measured at 560 nm using a multiple plate reader.EPR spectroscopy further confirmed the superoxide dismutase (SOD)-like activity of GFeSNs.Briefly, superoxide anion radicals were produced by illuminating riboflavin solution, with 2 mM riboflavin, 100 mM DMPO, and different concentrations of GFeSNs (0, 10, and 20 μg/mL) added to the buffer and EPR signals recorded immediately.

Cell culture
Normal rat kidney-52E (NRK-52E) cells and human embryonic kidney 293 (HEK293) cells were purchased from the American Type Culture Collection (ATCC).The cells were cultured in DMEM (10% FBS and 1% penicillin/streptomycin) at 37 °C in an incubator under a humidified atmosphere of 5% CO 2 .

Cell viability assay
GFeSN cytotoxicity was determined by the cell counting kit-8 assay in vitro.The HEK293 and NRK-52E cells were seeded into 96-well cell culture plates at a density of 10 4 cells/well, then incubated at 37 °C in an incubator with 5% CO 2 overnight.After incubation with different concentrations of GFeSNs (0, 2.5, 5, 10, and 20 µg/mL) for 24 h, the cell culture medium was removed and 100 µL of CCK-8 solution was added to the plate for 2.5 h incubation at 37 °C.Absorbance was measured at 450 nm using a microplate reader and cell viability was then calculated.

Mitochondrial membrane potential (MMP) assay
A loss of MMP assay was carried out using a JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′tetraethylimidacarbocyanine) mitochondrial membrane potential assay kit (Beyotime Biotechnology, Shanghai, China).NRK-52E cells were seeded into a 6-well plate at a density of 2 × 10 5 cells/well and cultured at 37 °C under 5% CO 2 overnight.The cells were pretreated with PBS or GFeSNs (5 or 10 μg/mL) for 2 h, then incubated with H 2 O 2 (300 μM) for a further 24 h at 37 °C under 5% CO 2 .Finally, the cells were washed with PBS before the addition of JC-1, followed by 20 min of incubation at 37 °C.After washing with the cell culture medium, the cells were imaged using a fluorescence microscope.

In vitro ROS scavenging of GFeSNs
To investigate the ROS scavenging ability of GFeSNs, NRK-52E cells were seeded into 96-and 24-well plates at densities of 10 4 cells/well and 10 5 cells/well, respectively.After 24 h of incubation, different concentrations of GFeSNs (0-10 μg/mL) were added to each well and incubated for 30 min.Cells seeded in the 96-well plates were treated with 300 μM or 500 μM H 2 O 2 and further incubated at 37 °C for 24 h.After that, the cell culture medium was removed and 100 µL of CCK-8 solution was added to the plate and incubated for 2.5 h at 37 °C.Absorbance was measured at 450 nm using a microplate reader.Wells without the addition of H 2 O 2 were regarded as the negative control.
DCFH-DA was used to detect intracellular ROS levels.After pretreatment with different concentrations of GFeSNs (0-10 μg/mL) for 30 min, cells seeded in 6-well plates were further incubated with 300 μM H 2 O 2 at 37 °C for 24 h.Cells were gently washed three times with serum-free medium, then incubated with 10 μM DCFH-DA in serum-free medium in the dark at 37 °C for 30 min.The cells were then washed thrice with serum-free medium and imaged using a fluorescence microscope.

In vivo biocompatibility evaluation of GFeSNs
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Yangzhou University and approved by the Animal Ethics Committee of Medical College of Yangzhou University.Female BALB/c mice (aged 4-6 weeks, obtained from Comparative Medicine Centre of Yangzhou University) were intravenously administered a single dose of 500 μg/kg GFeSNs.Mice injected with PBS were used as the control group.At 1 day post injection, blood samples were collected for complete blood panel analysis and serum biochemical tests.At 7th and 30th day post injection, the mice were sacrificed to harvest major organs (heart, liver, spleen, lung, and kidney) for hematoxylin and eosin (H&E) staining and histological analysis.
Partial renal tissues from all groups were harvested and fixed with paraformaldehyde, then sectioned and stained with H&E.Other kidney samples from all groups were frozen and subsequently sectioned into tissue slices.The obtained sections were stained by 4′,6-diamidino-2-phenylindole (DAPI), dihydroethidium (DHE), or terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL).

Synthesis and characterization
GFeSNs, CFeSNs, DFeSNs, and TFeSNs were synthesized using the solvothermal method detailed in our previous study (Fig. 1a) [52].As shown in the scanning electron microscopy (SEM) images (Fig. 1b-e), CFeSNs and TFeSNs displayed a mixture of spheres and sheets, while GFeSNs and DFeSNs exhibited free-standing nanosheets with a diameter of several hundred nanometers.It is found that the products with different morphologies are closely related to the different sulfur precursors introduced in the synthesis.In the previous literatures of our research group, there have been preliminary reports on the synthesis of iron sulfide nanoparticles with L-Cysteine and DATS as sulfur precursor [52,53].With the increase of sulfur precursor input, the proportion of spherical nanoparticles in the product decreased.Meanwhile only certain amount of GSH can be used as sulfur precursor to produce pure mackinawite FeS.Therefore, we concluded that the different sulfur sources are closely related to the composition and morphology of the iron sulfide products.According to the results, we speculate that sulfur sources with sulfhydryl and carbonyl group are the key raw material for the synthesis of pure mackinawite FeS by hydrothermal method.First, we evaluated the O 2 •− clearance efficiency of the four iron sulfide nanoparticles.As shown in Fig. 1f, GFeSNs exhibited the strongest O 2 •− scavenging ability compared with the other iron sulfide nanoparticles.Accumulating evidence suggests that hydrogen polysulfide-generating prodrugs can increase intracellular hydrogen polysulfide levels to regulate redox balance in cells, including protection of cells against harmful ROS levels and enhancing host antioxidant capacity [54,55].The hydrogen polysulfidedonating abilities of the four iron sulfide nanoparticles were detected using sulfane sulfur probe 4 (SSP4, 3′,6′-Di(O-thiosalicyl) fluorescein) at a concentration of 10 μg/mL.GFeSNs exhibited the strongest hydrogen polysulfide-producing ability among the four nanoparticles (Fig. 1g).
After incubation with SSP4 for 30 min, the fluorescence spectrum intensity of GFeSNs in PBS buffer were gradually enhanced with the increase of concentration (Fig. 1h).We also assessed hydrogen polysulfide release based on the ability of hydrogen polysulfides to rapidly react with L-cysteine to generate H 2 S.
GFeSNs reacting with L-cysteine showed the strongest fluorescence intensity among the iron sulfide nanoparticles.Incubation with either 10 mM phosphate buffer (pH 7.4) or L-cysteine alone generated limited fluorescence intensity (Fig. 1i).The release trend of hydrogen polysulfide from GFeSNs was then monitored.As shown in Additional file 1: Fig. S1, hydrogen polysulfide was released steadily and continuously over time in 12 h.To better understand the properties of GFeSNs, we monitored the release trend of iron ions in PBS by using the Iron Content Assay Kit.We found that the concentration of released iron ions in PBS rose rapidly in the first 2 h, and then continued to rise slowly until reaching the maximum concentration in 12 h (Additional file 1: Fig. S2), which was consistent with the release trend of hydrogen polysulfide.
X-ray diffraction (XRD) pattern confirmed that GFeSNs were in pure phases of mackinawite FeS (PDF no.15-0037) (Fig. 2a).The height of GFeSNs obtained from atomic force microscopy (AFM) measurement was approximately 55-62 nm (Additional file 1: Fig. S3).Elemental analysis by energy dispersive X-ray spectroscopy (EDS) revealed that the GFeSNs consisted of iron (Fe) and sulfur (S) (Fig. 2b).The high-resolution transmission electron microscope (HRTEM) image and selected area electron diffraction (SAED) analysis indicated that GFeSNs were flake-shaped with well-defined crystallinity, which exhibited clear lattice fringes with a d-spacing of 0.29 nm, corresponding to (101) crystal planes of mackinawite FeS (Fig. 2c).The STEM-energy dispersive X-ray spectroscopy (STEM-EDX) elemental mapping images of Fe-K, Fe-L, and S (Fig. 2d-g) clearly revealed the homogeneous distribution of Fe and S elements in GFeSNs.Furthermore, X-ray photoelectron spectroscopy (XPS) was used to analyze the surface chemical composition of the nanozymes (Fig. 2h, i) [56].In the high-resolution spectra of Fe 2p 3/2 , the peak located at 709.6 eV corresponded to Fe(II)-S, and the peak at 711 eV was attributed to Fe(III)-S bonds, which was always observed at , respectively.The optimized bulk structure of GFeSNs were computer-simulated and displayed in Fig. 2j.

ROS scavenging activities of GFeSNs
Given the major role of ROS in AKI prevention and treatment, representative ROS, such as , and H 2 O 2 , were used to systematically evaluate the antioxidant abilities of GFeSNs [57].As seen in Fig. 3, GFeSNs showed high concentration-dependent ROS scavenging activity.GFeSNs significantly removed •OH by inhibiting salicylic acid oxidation (Fig. 3a, b).Notably, 60% of •OH was scavenged at a GFeSN concentration of 2.5 μg/mL.Methylene blue (MB) was used to detect the •OH radicals generated by the Fenton reaction.MB was degraded to a colorless state in the presence of •OH.However, MB remained blue under the protection of GFeSNs, indicating that the nanoparticles possessed strong •OH radical scavenging ability (Additional file 1: Fig. S4).The antioxidant capacity of GFeSNs against DPPH and ABTS radicals was evaluated.As shown in Fig. 3c, d, the absorbance of DPPH at 517 nm markedly decreased after mixing with various concentrations of GFeSNs (2.5, 5, 20, 40, and 80 μg/ mL).Similarly, as shown in Fig. 3e, f, > 70% of ABTS was scavenged by GFeSNs at a concentration of 40 μg/mL.Thus, GFeSNs demonstrated strong radical scavenging properties.GFeSNs further showed strong antioxidant activity against O 2 •− and H 2 O 2 , scavenging more than 62% of O 2 •− (Fig. 3g) and 76% of H 2 O 2 (Fig. 3h) at a concentration of 80 µg/mL.In comparison, GSH exhibited negligible antioxidant activity in vitro (Additional file 1: Fig. S5).We also measured the O 2 •− radical scavenging ability of GFeSNs in aqueous solution after 24 and 48 h, with results showing that GFeSNs still removed more •− (Additional file 1: Fig. S6).Given its longer half-life than O 2 •− and •OH, H 2 O 2 is considered one of the most important ROS and exhibits the highest intracellular concentration.As such, we next investigated the concentration-dependent catalase (CAT)-like activity of GFeSNs (Additional file 1: Fig. S7), which was further assessed by monitoring dissolved O 2 .A gradual increase in O 2 concentration was observed in the presence of GFeSNs (Fig. 3i).Consistent with this, the formation of many O 2 bubbles were photographed after the addition of GFeSNs to 1000 mM H 2 O 2 solution in 0.1 M NaAc (pH = 7) at 37 ℃ (Fig. 3j).We further investigated the multiple enzyme-mimicking properties of GFeSNs under different pH conditions.GFeSNs exerted SOD-like and CAT-like activity in pH-independent manners.The results revealed that GFeSNs possess SOD-like activity under both neutral and acidic conditions but with much better performance at neutral conditions.Similarly, under acidic conditions (pH = 6.5/5.5), the decomposition efficiency of H 2 O 2 catalyzed by GFeSNs was significantly reduced.Intriguingly, the POD-like activity of GFeSNs was detected under different pH conditions.The results revealed that GFeSNs possess weak POD activity, which could hardly convert H 2 O 2 into toxic free radicals even in the acidic environment (Additional file 1: •− decreased significantly upon the addition of 10 and 20 μg/mL GFeSNs (Fig. 3k, l).At present, the hydrogen polysulfide donors reported in the literatures are mainly small molecular compounds, with complex synthesis steps, poor stability and rapid release of hydrogen polysulfide in solution [46,58,59].GFeSNs is a new type of polysulfide donor, which is easy to be synthesized in large quantity, easy to be preserved and well tolerated.More strikingly, GFeSNs can release hydrogen polysulfide steadily and continuously over time during degradation and exert antioxidant enzyme activity.These results indicate that GFeSNs are strong ROS scavenging agents.

GFeSN-mediated protection of H 2 O 2 -stimulated cells
The HEK293 and NRK-52E cells were used to detect the cytoprotective effects of GFeSNs under ROS damage in vitro.First, the CCK-8 assay results showed that GFeSNs were nontoxic to both cell lines at concentrations ranging from 2.5 to 20 μg/mL over 24 h.Surprisingly, GFeSNs promoted cell proliferation at concentrations of 10 and 20 μg/mL, which may be because hydrogen polysulfides can promote cell proliferation (Fig. 4a, b).Subsequently, NRK-52E cells were used to evaluate the cytoprotective properties of GFeSNs against ROS damage in vitro.As shown in Fig. 4C, compared to those without pretreatment, the viability of NRK-52E cells pretreated with 5 and 10 μg/mL GFeSNs for 1 h after treatment with 300 or 500 μM H 2 O 2 significantly increased.Under 10 μg/mL GFeSN pretreatment, approximately 92% and 82% of cells were alive in the presence of 300 and 500 μM H 2 O 2 , respectively.The antioxidant performance of GFeSNs was further studied using the DCFH-DA probe.As seen in Fig. 4d.ROS levels in the H 2 O 2 group showed the highest signals.In contrast, when cells were pretreated with GFeSNs, intracellular ROS levels were significantly suppressed, We then examined the effect of GFeSNs on H 2 O 2 -induced cell death/apoptosis using Calcein AM/ PI dual staining.As shown in Fig. 4e, the stained cells with Calcein AM/PI were found an increase in red fluorescence intensity after H 2 O 2 treatment, and the addition of GFeSNs significantly reduced the ratio of dead cells induced by H 2 O 2 treatment, indicating the cytoprotective properties of GFeSNs against ROS at the cellular level.

GFeSNs mediated ROS scavenging and anti-inflammation in vitro
MMP is a key indicator of mitochondrial function.Excessive ROS can cause MMP loss, resulting in mitochondrial damage and fragmentation [25].Here, changes in MMP were observed by JC-1 staining, determined by the transition between JC-1 aggregates (red fluorescence) and JC-1 monomers (green fluorescence) [60].Results showed that green fluorescence signal intensity increased sharply in cells treated with H 2 O 2 , but decreased significantly in cells pretreated with GFeSNs, suggesting that GFeSNs can maintain mitochondrial function by inhibiting ROS-induced damage (Fig. 5a).We further characterized the entry of hydrogen polysulfide species into NRK-52E cells using the SSP4 probe.As shown in Fig. 5b, significant green fluorescence signals were observed in cells treated with GFeSNs compared to NRK-52E cells alone, indicating that GFeSNs releasing hydrogen polysulfide species entered the cells to alleviate intracellular oxidative stress.To better understand the ROS scavenging performance of GFeSNs at the cellular level, various biochemical measures of intracellular redox homeostasis were determined.
Several inflammatory cytokines involved in AKI progression were first examined, including TNF-α, IL-6 and IL-1β.As illustrated in Fig. 5c-e, H 2 O 2 increased TNFα, IL-6, and IL-1β levels in NRK-52E cells, while GFeSN treatment effectively decreased their expression.We then evaluated the level of lipid peroxidation and intracellular antioxidant status by assessing the level of intracellular MDA and GSH.Results showed that MDA levels increased and GSH levels decreased in the H 2 O 2 group, while the PBS-and GFeSNs-treated groups showed no significant differences (Fig. 5f, g).These results strongly suggest that GFeSNs may exert synergistic anti-inflammatory and ROS scavenging effects to restore cellular redox balance.

In vitro and in vivo biocompatibility of GFeSNs
SEM images were taken of GFeSNs placed in PBS solution for different times, and we found that GFeSNs degraded to small-sized lamellae after 24 h, indicating good degradability (Additional file 1: Fig. S9).As good red blood cell compatibility indicates high safety of intravenous administration [61], we studied hemolysis performance of GFeSN.Results showed that even at a high concentration, the hemolysis rate of GFeSNs was less than 5%, indicating high biocompatibility (Additional file 1: Fig. S10).Thus, GFeSNs were administered intravenously at a high dose of 1 mg/kg and in vivo toxicity was studied.H&E staining showed no obvious changes in the heart, liver, spleen, lung, and kidney at different time points (Additional file 1: Fig. S11).In addition, serum biochemical indicators including aspartate aminotransferase (AST), alanine aminotransferase (ALT) and blood routine indices in GFeSN-treated mice showed no obvious differences between PBS and GFeSN treated groups (Additional file 1: Fig. S12).

In vivo therapeutic outcome of GFeSNs on AKI mice
Given the excellent anti-inflammatory and ROS scavenging abilities of GFeSNs, we speculated that the nanoparticles may be capable of protecting renal health from ROS damage, thereby alleviating AKI.As GFeSNs possessed a typical thin sheet-like morphology of hundreds of nanometers, the high planar/thickness ratio may facilitate accumulation in the kidney, thereby enabling efficient AKI therapy [62].Furthermore, the hydrogen polysulfides generated by GFeSNs may be easily taken up by cells to play a synergistic role in maintaining cellular redox balance.
As illustrated in Fig. 6a, the AKI model was established by intramuscular injection of 50% glycerol into 15 h-dehydrated mice, which induced rhabdomyolysis and oxidative stress-related renal dysfunction.GFeSNs (500 μg/kg) were injected intravenously at 2 h post glycerol injection to verify their potential feasibility as antioxidants in the treatment of AKI.For comparison, NAC (20 mg/kg) was utilized as a positive control.The pictures of the corresponding kidneys were shown in Fig. 6b.The kidney of PBS-treated AKI mice showed a lighter color compared to PBS-and GFeSN-treated mice, while the kidney of GFeSN-treated AKI mice improved markedly.To further evaluate the therapeutic effect of GFeSNs, we measured the levels of two clinical renal excretion indicators (serum CRE and BUN) in different groups.AKI mice treated with PBS alone showed significantly high levels of CRE and BUN, while GFeSN-treated AKI mice showed significantly reduced levels of CRE and BUN.NAC treatment in AKI mice did not produce an obvious therapeutic effect (Fig. 6c, d).In addition, all AKI mice treated with PBS died within 6 days, whereas administration of GFeSNs significantly prolonged survival time beyond 14 days (Fig. 6e).Next, we evaluated the plasma concentration of GFeSNs after intravenous injection at 1.5 and 24 h by measuring the iron ion concentration.Results showed no significant difference between the PBS-and GFeSN-treated groups, indicating that GFeSNs exhibited therapeutic effects against AKI at very low concentrations (Fig. 6f ).In addition, the AKI mice treated with GFeSNs showed sustained weight gain to normal levels after slight weight loss in the first few days, whereas AKI mice treated with PBS underwent sustained weight loss until death (Fig. 6g-j).

Therapeutic mechanisms of GFeSNs on AKI
Given the encouraging ROS scavenging capacity and biocompatibility of GFeSNs in vitro and in vivo, we investigated whether the ability of GFeSNs to treat AKI is correlated with ROS levels and antioxidant activities in renal tissues after treatment in AKI mice.We first stained kidney slices with DHE to detect superoxide generation.Compared with AKI mice, the superoxide levels in GFeSN-treated AKI mice were significantly reduced to normal mouse levels (Fig. 7a).In addition, we examined the renal expression levels of two important biomarkers of kidney injury, i.e., HO-1 and KIM-1.As shown in Fig. 7b, c, the levels of KIM-1 and HO-1 were significantly lower in GFeSN-treated AKI mice compared to AKI mice, approaching levels observed in normal mice.
We then assessed antioxidant activity in renal tissues of AKI mice (Fig. 7d-f ).MDA levels in the GFeSN-treated mice were consistent with those in healthy mice, whereas MDA levels were increased in the PBS-treated AKI mice.Furthermore, SOD and GSH levels in the GFeSN-treated AKI mice were restored to those similar in normal mice, while SOD and GSH levels in the AKI mice were significantly reduced.The protective effects of GFeSNs in AKI mice were also examined using LDH release assay (Fig. 7g).Our data showed that GFeSNs almost completely abolished LDH release in AKI mice to the same level as healthy mice, suggesting that GFeSNs can protect renal cells in AKI mice.To better understand the mechanism by which GFeSNs combat AKI, we evaluated the renal inflammatory response based on TNF-α, IL-6, and IL-1β levels using ELISA.As shown in Fig. 7h-j, GFeSN treatment reduced the release of proinflammatory factors TNF-α, IL-6, and IL-1β in AKI mice, indicating that GFeSNs can protect kidney tissues from oxidative stress by inhibiting the immune response during AKI.
Furthermore, the therapeutic effects of GFeSNs on AKI-induced apoptosis in the renal cortex were evaluated by TUNEL staining.As shown in Fig. 8a, massive apoptosis in the renal cortex of AKI mice was observed with loss of tubular structure.In contrast, no significant apoptosis was detected in the GFeSN-treated group, demonstrating the anti-apoptotic properties of GFeSNs against AKI, consistent with the renal function and blood analysis results.In addition, H&E staining of kidney tissues showed severe renal tubular injury with damaged tubules in AKI mice (Fig. 8b), whereas significantly reduced kidney damage was observed in the GFeSNtreated AKI mice.These results clearly demonstrate the outstanding therapeutic performance of GFeSNs against AKI and their potential as candidates for the treatment of ROS-related diseases.
To reveal the underlying therapeutic mechanisms of GFeSNs against AKI, we conducted RNA-seq profiling based on the GFeSN-and PBS-treated AKI mice.Principal component analysis (PCA) revealed substantially different transcriptomic profiles between the GFeSN-and PBS-treated AKI mouse kidneys (Fig. 8c).Given these differences, Deseq2, edgeR, and limma analyses were applied to identify significantly differentially expressed genes (DEGs), identifying 745, 630, and 766, respectively.The Venn diagram showed that a total of 408 DEGs were identified from the three methods (Fig. 8d).Volcano plots identified 766 significant DEGs, including 447 upregulated and 319 down-regulated DEGs (Fig. 8e).Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and gene set enrichment analysis (GSEA) based on integrated differential expression and pathway analysis were performed to determine the effects of GFeSN-induced transcriptomic changes on biological functions and pathways (Fig. 8f, g).During the analysis, inflammation response exhibited significantly value.The significances were ranked by adjusted p-values using the limma algorithm and the top 10 enrichment (Fig. 8f ) and 2 representative figures (Fig. 8g) were visualized.Consistent with the biochemical analyses, GFeSN-treated AKI mice kidneys exhibited the enrichment of inflammatory pathways including IL-6/JAK/STAT3 pathway, interferon (IFN)-γ and TNF-α signaling via NF-κB, demonstrating that the level of inflammation in the kidney of AKI mice was substantially decreased by GFeSN treatment, providing an improved environment for AKI alleviation.

Conclusions
Here, we developed a novel mackinawite nanozyme synthesized from GSH and ferric ions using a hydrothermal method, which exhibited broad-spectrum ROS-eliminating activity against oxidative stress.In vitro experiments demonstrated that GFeSNs promoted cell proliferation and effectively protected cells from H 2 O 2 -induced oxidative stress.Our study suggested that the conversion of active sulfides into mackinawite nanozymes is an effective approach to enhance the ROS scavenging activity of sulfur-containing compounds.In addition, GFeSNs exhibited multiple enzyme-like activities and hydrogen polysulfide-releasing properties, which were strongly correlated with the capacity to scavenge superoxide anions.Therefore, the hydrothermal method may be an effective approach to convert sulfur and iron into metastable mackinawite nanozymes with high ROS scavenging ability.In addition, with high biocompatibility and robust ROS scavenging activity, GFeSNs exhibited superior therapeutic efficiency towards in vivo ROS-induced AKI.Taken together, these advantageous features suggest that GFeSNs may be promising therapeutic agents for the treatment of AKI and ROS scavengers in biomedical treatment and research.

Fig. 2
Fig. 2 Characterization of GFeSNs.a XRD pattern of GFeSNs.b EDS analysis of GFeSNs.c High-resolution transmission electron microscope (HRTEM) image and selected-area electron diffraction (SAED) pattern of GFeSNs.Scale bar = 200 nm.d HAADF-STEM image of GFeSNs.Fe-K (e), Fe-L (f), and S (g) elemental mapping images of GFeSNs.Scale bar = 100 μm.Fe 2p (h) and S 2p (i) regions of XPS spectra of GFeSNs.j A optimized schematic atom structure represents crystal phases of GFeSNs

Fig
Fig. S8).Electron spin resonance (ESR) spectra were used to detect the •OH generated by the Fenton reaction and O 2 •− generated from riboflavin exposed to light, using DMPO as the trapping agent of free radicals.Notably, ESR signals of •OH and O 2•− decreased significantly upon the addition of 10 and 20 μg/mL GFeSNs (Fig.3k, l).At present, the hydrogen polysulfide donors reported in the literatures are mainly small molecular compounds, with complex synthesis steps, poor stability and rapid release of hydrogen polysulfide in solution[46,58,59].GFeSNs is a new type of polysulfide donor, which is easy to be synthesized in large quantity, easy to be preserved and well tolerated.More strikingly, GFeSNs can release hydrogen polysulfide steadily and continuously over time during degradation and exert antioxidant enzyme activity.These results indicate that GFeSNs are strong ROS scavenging agents.

Fig. 8
Fig. 8 Therapeutic mechanisms of GFeSNs on AKI. a Fluorescence images of TUNEL-stained kidney sections from each group.b H&E staining of kidney tissues from each group.c PCA was performed on DEGs from the kidneys of GFeSN-and PBS-treated AKI mice (n = 5).Each data point corresponds to PCA of each sample.d Venn diagram of transcriptomic profiles of PBS-and GFeSN-treated AKI mice.e Volcano plots showing up-regulated and down-regulated genes by GFeSNs.f GSEA was performed to analyze enriched signaling pathways.Top 20 most significantly enriched pathways are shown.g GSEA identified two representative signaling pathways regulated by GFeSN treatment