Oral iron sulfide prevents acute alcohol intoxication by initiating the endogenous multienzymatic antioxidant defense system

Abstract

Acute alcohol intoxication could cause multiorgan damage, including nervous, digestive, and cardiovascular systems, and in particular, irreversible damage to the brain and liver. Emerging studies have revealed that the endogenous multienzymatic antioxidant defense system (MEAODS) plays a central role in preventing oxidative stress and other toxicological compounds produced by alcohol. However, few available drugs could quickly regulate MEAODS. Herein, we report a nanosized iron sulfide (nFeS) that can rapidly release polysulfide species in gastric juice. The released hydrogen polysulfide activates the Keap1/Nrf2 pathway via S-persulfidation of cysteine residues in Keap1, which promotes the expression of antioxidant enzymes and glutathione synthesis–related enzymes, thus potentiating MEAODS. Results indicate that the activated MEAODS not only alleviates oxidative stress and inflammation in the brain and liver but also mitigates movement dysfunction after only 2.5 hours of oral nFeS treatment. Collectively, this study provides a MEAODS-regulated strategy with nFeS and may aid the prevention of acute alcoholic injury.

Oral iron sulfide activates the endogenous multienzymatic antioxidant defense system to reduce excessive drinking–induced injury.

INTRODUCTION

Acute alcohol intoxication not only triggers violence and criminal activities, which threatens the security and harmony of society, but also brings about a large variety of tissue damages severely impacting the nervous system, digestive, and cardiovascular systems (1). Therein, brain and liver tissues undergo the most severe damage, as neurons in the brain are not regenerative and the liver is the primary site of alcohol metabolism. Acute alcohol intoxication–induced brain and liver injury has become a global public health problem, which annually results in several million deaths worldwide according to the World Health Organization report (2). After drinking, alcohol is rapidly absorbed into the blood circulation and accumulated in the brain, which directly destroys neurological function and brings about abnormal behavior and consciousness (3). Mounting experimental and clinical evidence indicates that the alcohol metabolites, acetaldehyde and reactive oxygen species (ROS), are largely responsible for the toxicity of alcohol (4, 5). On the one hand, excessive production of ROS leads to redox disequilibrium and oxidative damage once the scavenging capacity of the antioxidant system is exceeded. Highly reactive free radicals could not only cause permanent damage to lipids, proteins, and nucleic acids but also destroy mitochondrial function, leading to the inflammatory response and the activation of apoptosis. On the other hand, acetaldehyde, to a certain extent, mediates the effect of alcohol on neurotransmitters in the brain (6). Moreover, acetaldehyde could directly induce mitochondrial dysfunction and cause antioxidant depletion, which synergizes the ROS-mediated tissue injury (7, 8). Thus, it is imperative to develop a strategy to counteract the produced ROS and acetaldehyde after excessive drinking.

Although increasing studies have explored natural antioxidants, such as cysteine, catechol, and ascorbic acid, to reduce oxidative stress and acetaldehyde in alcoholic injury, these antioxidants failed to exert satisfactory therapeutic effects because of rapid consumption by excessive ROS. In recent years, booming research has developed multifarious nanozymes for scavenging miscellaneous free radicals (9–11). Nevertheless, these exogenous nanozymes face the challenges of biocompatibility and physiologic barriers. Furthermore, in consideration of frequent drinking in daily life, oral administration is the practical and feasible way to improve compliance. However, these above natural antioxidants and well-designed nanozymes have poor stability in gastric acid and low bioavailability. There is a well-established endogenous multienzyme antioxidant defense system (MEAODS) in the human body, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase–1 (HO-1), NAD(P)H:NQO1 (quinone acceptor oxidoreductase 1), etc., which plays an important role in maintaining redox balance (12, 13). However, this system struggles to cope with the marked elevation of ROS and acetaldehyde levels in acute alcohol intoxication. Thus, enhancing MEAODS to eliminate ROS- and acetaldehyde-mediated injury has great potential in alcoholic injury therapy.

It is widely recognized that the transcription factor, nuclear factor erythroid 2–related factor (Nrf2), is a master regulator of cellular stress response, providing cytoprotection against cellular insults and disrupted redox balance (14–16). Note that the Kelch-like ECH-associated protein 1 (Keap1)/Nrf2–mediated signaling pathway could regulate the expression of ROS-involved genes, producing a variety of antioxidant enzymes and phase 2 detoxifying enzymes. These endogenous enzymes could specifically and efficiently scavenge various free radicals and intoxicants. For instance, one CAT is capable of catalyzing the decomposition of 6,000,000 hydrogen peroxide (H₂O₂) per minute (17). Therefore, activation of the Keap1/Nrf2 pathway becomes a promising therapeutic strategy to stimulate MEAODS for counteracting ROS- and acetaldehyde-mediated damage.

Under normally physiological conditions, Nrf2 is bound to Keap1 in the cytoplasm, which falls into an inactive and easily degradable state (18). Two strategies are capable of disrupting the Keap1-Nrf2 interaction, including (i) competitively binding with Keap1 via noncovalent interaction and (ii) modifying the cysteine sulfhydryl groups on Keap1 (19). Thereinto, the S-persulfidation (─SH → ─SSH) of cysteine residues on Keap1 would change the protein conformation, further inducing the release of Nrf2 and translocation into the nucleus where it activates downstream gene expression. Notably, reactive sulfide sulfur, such as persulfide and polysulfide (H₂S_n), has the unique ability of reversibly binding with other sulfur atoms and completing the S-persulfidation modification of cysteine residues (20, 21). However, the commonly used polysulfide donors, such as Na₂S₂ and Na₂S₃, will be decomposed into H₂S in gastric acid and lose the ability of S-persulfidation during oral administration.

Our previous work successfully synthesized nanosized iron sulfide (nFeS), which could release Fe²⁺, H₂S₂, and H₂S₃ in aqueous solution with prolonged incubation (22, 23). Moreover, we reported that S₃²⁻ had the capability to achieve S-persulfidation of cysteine residues in glucokinase (24). Although these studies were all explored for antibacterial application, we synchronously stated that nFeS and its decomposition products were both biocompatible. Inspired by that, we herein explored whether nFeS could alleviate alcohol-induced brain and liver injury. Notably, results indicated that nFeS could release H₂S_n in gastric fluid. On the basis of the intrinsic lipophilicity, H₂S_n could enter the liver and brain tissues through gastrointestinal absorption. As expected, H₂S_n interacted with the cysteine residue of Keap1, thus changing the conformation of Keap1 and resulting in Nrf2 translocation into the nucleus. Consequently, the activation of Nrf2 induces the expression of antioxidant enzymes and detoxifying enzymes, thus potentiating MEAODS. Facing subsequent high-dose injection of alcohol, the activated antioxidant enzymes effectively eliminated ROS and acetaldehyde in time so as to reduce oxidative stress and the pathological cascade process and lastly alleviate alcoholic injury (Fig. 1).

Fig. 1. Scheme illustration of the therapeutic process of nFeS.

Alcohol metabolism boosted the content of ROS and acetaldehyde, causing inflammatory response and tissue injury in both the brain and liver. Oral administration of nFeS before drinking releases H₂S_n in gastric fluid, which activates the Keap1/Nrf2 pathway by the S-persulfidation effect and enhances the expression of antioxidant enzymes and GSH synthesis–related enzymes. The improved MEAODS will defend the alcohol metabolism–caused damage, therefore protecting the brain and liver. J. Mu from Aier Eye Institute provided technical support in drawing the schematic illustrations.

RESULTS

Preparation and characterization of nFeS

We previously reported the preparation of nFeS via a solvothermal method (22). Briefly, nFeS was formed under high temperature and high pressure using Fe³⁺ as the iron source as well as cysteine as the sulfur source and coordinating ligand (Fig. 2A). Scanning electron microscopy (SEM) and transmission electron microscopy images revealed that the as-prepared nFeS presented sheet-like and hexagonal nanostructures (Fig. 2B and fig. S1). The length of the nFeS nanosheet was around 1 μm, and the thickness was 20 to 30 nm. The corresponding energy-dispersive x-ray spectroscopy mapping demonstrated that nFeS mainly consisted of iron and sulfide elements, which presented uniform distribution (Fig. 2B).

Fig. 2. Synthesis, characterization, and decomposed products of nFeS.

(A) Scheme illustration of the preparation process of nFeS. h, hours. (B) Transmission electron microscopy image and the corresponding element mapping of nFeS. Scale bars, 500 nm. (C) Fe and S content in 1 mg of nFeS detected by inductively coupled plasma optical emission spectrometry. (D) Mass change of nFeS in H₂O and SGF. (E) SEM images of SGF-treated nFeS after different processing times. Scale bars, 20 μm. The upper right corner is the enlarged images. Scale bars, 200 nm. (F) Release of polysulfides within 20 min detected by the SSP4 probe. a.u., arbitrary units. (G) Composition of polysulfides analyzed by LC-MS/MS. (H) Quantitative detection of hydrogen sulfide (H₂S) released from nFeS within 20 min. (I) Quantitative detection of Fe²⁺ released from nFeS within 20 min. (J) Scheme illustration of the decomposition products of nFeS in SGF. (K) Polysulfides detected by the SSP4 probe in Na₂S, NaSH, Na₂S₂, and Na₂S₃ in SGF. DI, deionized.

To further clarify the components of nFeS, we applied inductively coupled plasma optical emission spectrometry to quantitatively determine the element ratio. As shown in Fig. 2C, there are about 8.3 μmol of Fe and 5.1 μmol of S in 1 mg of nFeS. The ratio between iron and sulfur in nFeS was about 1.6. The x-ray diffraction pattern showed that nFeS matched well with gregite (Fe₃S₄, 16-0713) and pyrrhotite (Fe₇S₈, 24-0220), confirming the coexistence of two phases of nFeS (fig. S2). Furthermore, x-ray photoelectron spectroscopy was used to analyze the surface chemical composition and elemental state (fig. S3). Results further confirmed that nFeS mainly contains iron and sulfur elements. The high-resolution Fe 2p spectra and S 2p spectra of nFeS further showed the formation of Fe─S bonds. Together, these results confirmed the formation of nFeS.

The polysulfide released from nFeS achieves S-persulfidation on Keap1

Given that nFeS was taken via oral administration, the active components released in the gastrointestinal tract will be effective instead of iron sulfide. Although the decoction strategy could cause the release of H₂S_n from nFeS, the decomposition products of that in gastric acid remain unclear (23). Therefore, we next investigated the degradation performance in simulated gastric fluid (SGF) and the possible functional components. As shown, nFeS underwent rapid degradation in SGF. Compared to that in H₂O, nFeS lost more mass in SGF, proving that SGF accelerated the degradation of nFeS (Fig. 2D). SEM images confirmed the mass loss. Moreover, the surface of the dense structure of nFeS appeared to be concave and the structure gradually collapsed with the prolongation of incubation time (Fig. 2E). These predicted that nFeS can decompose to release products in gastric fluid. Then, the possible functional components in decomposition products of nFeS were identified. First, the supernatant of SGF-treated nFeS was clear and odorless, preliminarily proving that the H₂S gas and S precipitate are minimal or absent. Upon incubating with sulfane sulfur probe 4 (SSP4), the supernatant produced enhanced fluorescence emission, indicating the existence of polysulfide in the decomposition products of nFeS (Fig. 2F). Note that the fluorescence signal gradually increased within 20 min, which implied the rapid and continuous release of polysulfide. The liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was further carried out to evaluate the state of polysulfide (Fig. 2G). According to these results, we identified that the released polysulfide was mainly persulfide (H₂S₂) and trisulfide (H₂S₃). Besides, the H₂S assay kit was applied to determine the proportion of H₂S in the sulfurated products. Figure 2H shows that the released H₂S was less than 10 μM, which accounts for a small part of sulfurated products. Moreover, the state of iron in the supernatant also deserved attention. The iron assay kit was used to detect the state and content of iron. As shown in Fig. 2I, Fe²⁺ was the main state and increased over time within 20 min, while the content of Fe³⁺ was minimal. The above results indicated that the decomposition products of nFeS in gastric acid mainly included hydrogen polysulfide (H₂S_n) and Fe²⁺ (Fig. 2J).

Our previous research reported that H₂S_n was released slowly from nFeS in aqueous solution (22). However, SGF hastened the decomposition of nFeS, leading to a greater release of H₂S_n compared to that observed in aqueous solution (Fig. 2D). This implied that H₂S_n could be rapidly released from nFeS in the presence of gastric acid after oral administration, which is crucial for the rapid onset of therapeutic efficacy. This enhanced release is likely attributable to abundant hydrogen ions in the acidic environment that are necessary for the generation of H₂S_n. Furthermore, we have investigated the impact of the crystalline structure on the release of H₂S_n (fig. S4). In contrast to FeS and FeS₂, our nFeS, predominantly composed of Fe₃S₄ and Fe₇S₈, features longer iron-sulfur bonds, which are more susceptible to cleavage, facilitating sulfur release (24). Therefore, the as-prepared nFeS is deemed appropriate for oral administration and rapid onset.

The property of nFeS that releases H₂S_n in gastric acid is unique. The commonly used polysulfide species (Na₂S₂ and Na₂S₃) and sulfide species (Na₂S and NaHS) are acid labile, which rapidly decomposes into S and H₂S in SGF. This has been confirmed by the phenomenon that these polysulfide species and sulfide species produced white suspension and malodorous gas at once upon adding into SGF (fig. S5). The decreased fluorescence signal of SSP4 also indicated that these sulfur species were decomposed (Fig. 2K).

To ascertain whether H₂S_n released from nFeS in gastric acid could be absorbed into the bloodstream, we measured the polysulfide levels in the blood of mice after oral gavage administration of nFeS over various time intervals. Our findings revealed that H₂S_n was rapidly absorbed into the bloodstream, achieving peak concentration at 60 min (fig. S6). The elimination half-life of H₂S_n was determined to be 69.31 min. Notably, H₂S_n maintained a high concentration for up to 6 hours, suggesting a minimum effective duration of 6 hours. Moreover, H₂S_n was found to rapidly penetrate the brain and liver tissues within 30 min, reaching maximum concentration at 3 hours, which is conducive to the rapid manifestation of its therapeutic effects (fig. S7). It is known that Fe²⁺ is transported into enterocytes by divalent metal cation transporter 1 and then stored as ferritin (25). The process is limited by high storage, which in turn regulates the uptake of iron. Accordingly, a few dosages of nFeS will not change the iron content in the body. Therefore, the active component might mainly be H₂S_n. To eliminate interference from Fe²⁺ in the in vitro experiment, we used EDTA to remove Fe²⁺ in the nFeS decomposition product.

nFeS protects cells from alcohol-induced injury by activating the Keap1/Nrf2 pathway

We next investigated whether the S-persulfidation of cysteine residues of Keap1 promoted the nuclear translocation of Nrf2 and protected cells from oxidative injury. Given that nFeS and its decomposition products will be used in biomedical applications, the cytotoxicity must be considered. The results exhibited that the decomposition products of nFeS showed negligible cytotoxicity below a concentration of 1000 μg/ml, which guaranteed the in vitro and in vivo application (fig. S8). To evaluate the intracellular effect of H₂S_n, we first confirmed the efficient cellular internalization (Fig. 3A). Fluorescence images and quantitative results showed an enhanced signal in the H₂S_n-treated group, implying rapid cellular uptake (Fig. 3B and fig. S9). The membrane permeability of H₂S_n may be similar to that of H₂S, which experiences essentially no barrier to permeation on the basis of its lipophilicity (26).

Fig. 3. In vitro protection effect of nFeS decomposition products.

(A) Illustration of cellular internalization of H₂S_n and triggered activation of the SSP4 probe. (B) Fluorescence imaging of intracellular H₂S_n by the SSP4 probe (green) after the treatment by the supernatant from decomposition products of nFeS in SGF. Scale bar, 100 μm. Ctrl, control. (C and D) MS/MS spectra of the β-(4-hydroxyphenyl)ethyl iodoacetamide–labeled Cys151-containing peptide (KCVLHVMNGAVMYQIDSVVR) from Keap1, with the treatment of H₂S (C) or H₂S_n (D). (E) Immunofluorescence staining of Nrf2 in SH-SY5Y cells after different treatments. Red fluorescence represents anti-Nrf2, and blue fluorescence represents 4′,6-diamidino-2-phenylindole. Scale bar, 20 μm. (F) Western blot analysis of cytoplasmic Nrf2 and nuclear Nrf2 in SH-SY5Y cells before and after nFeS treatment. (G) Luciferase activity in ARE-Luc-EF1α-mCherry SH-SY5Y cells with or without H₂S_n treatment. (H) Western blotting for CAT and GCLC in SH-SY5Y cells in different groups. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (I and J) Qualitative analysis of expression levels of CAT (I) and GCLC (J) in different groups in (H). (K and L) Representative fluorescence images (K) and qualitative analysis (L) of intracellular ROS levels in different groups stained with DCFH-DA (green). 0.25 and 0.5 represent degradation products of nFeS (0.25 and 0.5 mg/ml, respectively). Scale bar, 200 μm. (M) Scheme illustration of the process in which released H₂S_n activates the Keap1/Nrf2 pathway by the S-persulfidation effect, induces the nuclear translocation of Nrf2, and promotes the expression of antioxidant enzymes, thus potentiating MEAODS and reducing oxidative stress. Data are presented as means ± SD (n = 3). Statistical significance was determined using [(G), (I), and (J)] two-tailed unpaired Student’s t test and (L) one-way ANOVA followed by Sidak’s multiple comparison test. *P < 0.05 and **P < 0.01 versus the alcohol group. ^#P < 0.05 and ^###P < 0.001 versus the control group.

To explore whether the released H₂S_n causes S-persulfidation on Keap1 protein, we characterized the formation of Keap1 persulfide by LC-MS/MS. In H₂S_n-treated samples, the extracted ion chromatogram signals clearly showed a substantial level of persulfide (─SSH) adduct on the peptide GLVLIAFSQYLQQCPFDEHVK (Fig. 3, C and D). Specifically, ─SH in cysteine C151 was changed to ─SSH. The results definitely demonstrated that H₂S_n completed the S-persulfidation of Keap1. Therefore, our results showed that the released H₂S_n from nFeS could cause the S-persulfidation of Keap1.

Then, we provided definitive identification that H₂S_n was capable of breaking the binding between Keap1 and Nrf2 and promoting the nuclear translocation of Nrf2. Immunofluorescence images revealed that the released H₂S_n-treated group showed a brighter fluorescence signal in the nucleus than the sham group, suggesting that H₂S_n promoted Nrf2 separation and nuclear accumulation in SH-SY5Y cells (Fig. 3E). This result was further verified by Western blot analysis. It is obvious that Nrf2 is mainly located in the cytoplasm in control cells, while that is translocated into the nucleus after H₂S_n treatment (Fig. 3F). The antioxidant response element (ARE) is a specific DNA sequence present in the promoter regions of Nrf2-target genes. It is well established that intranuclear Nrf2 binds to the ARE, thereby inducing the expression of antioxidant enzymes (27–30). Thus, we next performed an ARE-driven luciferase (ARE-luc) assay, as previously described, to validate the activity of Nrf2. Results showed that the released H₂S_n enhanced the luciferase activity, indicating that Nrf2 could bind to ARE and activate the transcription of downstream genes (Fig. 3G). Moreover, Western blot experiment was conducted to detect the expression of antioxidant enzymes. H₂S_n treatment markedly increased the expression of CAT and GCLC, implying the activation of MEAODS (Fig. 3, H to J).

Although our previous work has stated that the presence of polysulfide species would cause the oxidation of glutathione (GSH) and therefore exhaust reducing substances inside the bacteria, the GSH depletion was also found to become invalid in mammalian cells such as RAW 264.7 cells, human umbilical vein endothelial cells, hepatocyte line (L02), and vaginal cells (VK2) (23). The elevated GSH/GSSH (glutathione persulfide) ratio in RAW 264.7 cells implies the different action mechanism and pharmacological effects in comparison with bacteria. The contradictory result prompted us to speculate that H₂S_n activates intracellular MEAODS and therefore potentiates ROS resistance. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was used to detect the intracellular ROS level. Upon stimulation by alcohol, SH-SY5Y cells emerged bright green fluorescence, which illuminates the successful production of ROS. Notably, the fluorescence signal significantly decreased with preincubating with H₂S_n (Fig. 3, K and L). Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to assess the cytoprotection effect of H₂S_n. Results revealed that alcohol treatment decreased SH-SY5Y cell viability, while this damage was absolutely recovered by preincubating with H₂S_n (fig. S10). These results demonstrated that H₂S_n treatment strengthened the cellular antioxidative ability and thus increased cell viability. Together, the released H₂S_n activated the Keap1/Nrf2 pathway to strengthen MEAODS, thus effectively alleviated the alcohol-induced damage in vitro, presenting potential therapeutic effects on both the liver and brain (Fig. 3M).

Oral nFeS presents a neuroprotective effect by potentiating endogenous MEAODS in a mouse model of acute alcohol intoxication

Encouraged by the superior cytoprotection effect of nFeS, animal experiments were performed to investigate the in vivo therapeutic effect. The in vivo toxicity of nFeS was initially evaluated. The body weight, food intake, water intake, and organ wet–to–body weight ratios were observed in healthy C57BL/6J mice after administration of high dosages of nFeS (~800 times of therapeutic dose). Results indicated that no obvious negative effect was observed on the four indicators during 14 days (fig. S11). Moreover, considering that H₂S_n is absorbed into the blood circulation and tissues, we performed blood routine examination and blood biochemistry tests on day 7 after oral nFeS treatment. All indicators showed that oral administration of nFeS did not cause adverse effects (figs. S12 and S13). Meanwhile, histological examination of the brain, heart, liver, spleen, lung, and kidney tissues was conducted to affirm the high biocompatibility of nFeS (fig. S14). The above results indicate that there is no toxicity to the body and organs after oral administration of high doses of nFeS. Then, we evaluated the minimum effective dose of nFeS required to elicit a therapeutic response in acute alcohol intoxication mice. Obviously, a significant therapeutic effect became discernible at an administered dose of 2.5 mg/kg (fig. S15). Consequently, this dose was selected for administration in subsequent experiments.

We subsequently explored whether nFeS activated the Keap1/Nrf2 pathway and induced the expression of antioxidant enzymes in the brain. Immunofluorescence images revealed that the nFeS-treated group showed a brighter fluorescence signal in the nucleus than the sham group (Fig. 4A). Statistical results also exhibited higher colocalization of red fluorescence with a blue nucleus in the nFeS group, suggesting that nFeS promoted Nrf2 nuclear accumulation in the cerebral cortex (Fig. 4B). To further verify the nuclear translocation of Nrf2, we applied Western blot experiment to quantify the expression of Nrf2 in the cytoplasm and nucleus. Compared with the sham group, the nFeS group presented decreased expression in the cytoplasm and increased expression in the nucleus, confirming the nuclear translocation of Nrf2 (Fig. 4, C and D). As stated above, Nrf2 could bind to ARE and thus activate the transcription of downstream cytoprotective genes. Therefore, we detected the transcription level of several crucial antioxidant enzymes. As shown in Fig. 4E, with the pretreatment of nFeS, the transcription levels of Gr, Gclc, Sod, and Cat all increased, suggesting higher ROS resistance than the sham group. Western blot results further demonstrated the increased expression of these antioxidant enzymes at the protein level (Fig. 4F and fig. S16). GCLC is the rate-limiting enzyme in the synthesis of GSH, and GR mainly reduces glutathione disulfide (GSSG) to GSH (31, 32). nFeS pretreatment boosted the expression of GR and GCLC in comparison with the sham group. Therefore, even if alcohol administration could deplete GSH and decrease the ratio of GSH/GSSG, the nFeS pretreatment was able to withstand this depletion (Fig. 4G and fig. S17). The enhanced GSH is also important for removing ROS.

Fig. 4. Therapeutic mechanism and neuroprotective effects of oral nFeS.

(A) Immunofluorescence staining of Nrf2 (red) in the cortex in different groups. The yellow line is used for intensity analysis. Scale bars, 10 μm. (B) Plots of pixel intensity along the yellow line in (A). (C) Western blot analysis of cytoplasmic Nrf2 and nuclear Nrf2 in the cortex with or without nFeS treatment. (D) Qualitative analysis of expression levels of cytoplasmic Nrf2 and nuclear Nrf2 in (C). (E) RT-qPCR quantitative analysis of Gr, Gclc, Sod1, and Cat levels in the cortex with or without nFeS treatment. (F) Western blotting for GR, GCLC, SOD1, and CAT in the cortex with or without nFeS treatment. (G) GSH/GSSG ratio in brain tissue of alcohol-treated mice with or without nFeS treatment. (H) Acetaldehyde (Ace) content in brain tissue of alcohol-treated mice with or without nFeS treatment. (I) MDA content and (J) H₂O₂ level in brain tissue of alcohol-treated mice with or without nFeS treatment. (K and L) RT-qPCR quantitative analysis of Tnfα (J) and Inos (K) in brain tissue of alcohol-treated mice with or without nFeS treatment. (M) Western blot analysis of TNF-α and iNOS (inducible nitric oxide synthase) in different groups. (N) Representative images of Nissl staining of brain tissues in different groups. Scale bars, 25 μm. Data are presented as means ± SD (n = 3). Statistical significance was determined using [(D) and (E)] two-tailed unpaired Student’s t test and [(G) to (L)] one-way ANOVA followed by Sidak’s multiple comparison test. *P < 0.05 and **P < 0.01 versus the alcohol group. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus the sham group.

It is well documented that acetaldehyde and ROS produced by alcohol metabolism are largely responsible for the virulence (33). Thus, we first detected the acetaldehyde in the brain through 2,4-dinitrophenylhydrazine (DNPH) precolumn derivation high-performance liquid chromatography. Results indicated that nFeS pretreatment significantly decreased the acetaldehyde caused by alcohol metabolism, which may be due to elevated GSH (Fig. 4H and fig. S18). The oxidative stress level was assessed by testing malonaldehyde (MDA) and H₂O₂ content (Fig. 4, I and J). MDA is the decomposition product of oxidative fatty acid that represents the lipid peroxidation level. H₂O₂, as the main ROS, directly reflects the oxidative stress level. Results showed that both MDA and H₂O₂ content increased after alcohol exposure. Obviously, nFeS treatment not only lightened lipid peroxidation but also attenuated H₂O₂ content, preventing brain tissue from oxidative damage. To evaluate the inflammation state that likewise aids the development of tissue injury, we used reverse transcription quantitative polymerase chain reaction (RT-qPCR) to detect the inflammatory cytokines including Tnfα and Inos. These cytokines were significantly up-regulated in the alcohol-treated group but down-regulated in the nFeS-administered group (Fig. 4, K and L). Western blot results verified the increased inflammation in the alcohol group and the alleviated inflammation in the nFeS-treated group (Fig. 4M and fig. S19). We next inspected whether the ROS decrease could alleviate neuron injury by Nissl staining. The Nissl body in neurons is a vital part for protein synthesis, which could reflect the status of neurons. In the normal group, Nissl bodies were large and numerous, indicating that nerve cells had a strong function of synthesizing proteins. On the contrary, the number of Nissl bodies decreased when exposed to alcohol, which revealed the damage on neurons by alcohol (Fig. 4N). Oral nFeS enhanced the number of Nissl bodies. The Nissl staining result indicated that nFeS was conducive to guarding the function of nerve cells. The above results stated that nFeS pretreatment successfully activated the Keap1/Nrf2 pathway, promoted the expression of endogenous antioxidant enzymes, and potentiated MEAODS, thus alleviating the alcohol-caused oxidative stress, inflammatory response, and neuronal damage.

The duration of the therapeutic efficacy of nFeS is noteworthy. Although H₂S_n can only maintain a high concentration in the bloodstream for 6 hours, the produced antioxidant enzymes are able to extend the effective period. This protracted action potentially confers an extended effective duration to nFeS. Our findings demonstrated a marked reduction in the expression of SOD1 at 24 hours after administration (fig. S20). This observation suggests that the effective therapeutic window of nFeS spans at least 12 hours. This provides sufficient time for preadministration of nFeS before drinking.

Oral nFeS mitigates the motor dysfunction induced by acute alcohol intoxication

The behavior tests were performed to evaluate whether nFeS ameliorated alcohol metabolism–caused central nervous system depression. The timeline in Fig. 5A showed the arrangement of several representative behavior tests. Footprint analysis was used to assess the locomotor states (Fig. 5B). The footprints of the hindpaws almost overlapped with those of the forepaws during walking for the sham group, which showed good coordination. The alcohol exposure resulted in nonoverlapping of forepaw and hindpaw footprints and the oblique trajectory (Fig. 5C). In the alcohol group, both the relative position and stride width significantly increased in comparison with those of the sham group. The nFeS pretreatment significantly reduced the relative position and stride width, which indicated that the nFeS treatment resulted in enhanced coordination between the forepaw and the hindpaw (Fig. 5, D and E). Furthermore, a rotarod test was carried out to estimate the motor function performance (Fig. 5F). Figure 5G shows that alcohol exposure decreased latency to fall, resulting in obvious motor dysfunction. With the pretreatment of nFeS, mice could prolong the time in the rod. To further investigate the mental state before lethargy, we subsequently performed a righting reflex test (Fig. 5H). As shown in Fig. 5I, mice in the sham group swiftly recovered to normal position within a few seconds, while alcohol treatment extended the time needed. But the pretreatment of nFeS significantly mitigated the alcohol-caused sluggishness, suggesting that the inhibitory effect on the central nervous system was possibly weakened. Overall, the nFeS pretreatment significantly relieved the degree of intoxication.

Fig. 5. Behavioral evaluation of oral nFeS treatment.

(A) Schematic of the experimental timeline. All groups were trained for 3 days before drinking. ig, intragastrically; ip, intraperitoneally. (B) Illustration of footprint test. (C) Representative footprint and quantitative analysis of (D) relative position and (E) stride width in different groups (n = 10). (F) Illustration of rotarod test. Created using BioRender.com. (G) Rotarod analysis of motor function with or without nFeS treatment (n = 10). (H) Illustration of the righting reflex test. (I) Time for righting reflex after alcohol exposure with or without nFeS treatment (n = 10). (J) Time for righting reflex and (K) latency to fall in rotarod analysis after alcohol exposure with the treatment of different concentrations of NaSH (n = 5). (L) Time for righting reflex and (M) latency to fall in rotarod analysis after alcohol exposure with the treatment of Na₂S₂ or Na₂S₃ (n = 5). Data are presented as means ± SD (one-way ANOVA and Sidak’s multiple comparison tests; **P < 0.01 and ***P < 0.001 versus the alcohol group; ^###P < 0.001 and ^####P < 0.0001 versus the sham group; ns means no significance).

Believing that the decomposition products of nFeS in gastric acid contain tiny amounts of hydrogen sulfide, the effect of hydrogen sulfide is indistinct. To ensure the behavior improvement results from H₂S_n rather than hydrogen sulfide, we selected NaSH as the donor for oral administration. NaSH is unstable in gastric acid and rapidly decomposes into hydrogen sulfide. We assessed the therapeutic effect of hydrogen sulfide by righting reflex test and rotarod test (Fig. 5, J and K). Obviously, the pretreatment of NaSH failed to elevate the motor performance regardless of the concentration of NaSH. To further explore whether the commonly used polysulfide species could alleviate alcohol-caused behavior deficiency, the mice were orally administered Na₂S₂ and Na₂S₃ before the alcohol exposure. No obvious therapeutic effect was observed in the Na₂S₂-pretreated group and the Na₂S₃-pretreated group (Fig. 5, L and M). As stated above, the result may be due to the decomposition of polysulfide species. Overall, nFeS has unique properties that other commonly used polysulfide species do not own, which could effectively mitigate alcoholic brain injury.

Oral nFeS simultaneously protects the liver from alcoholic injury

The liver is the major organ for alcohol metabolism, accounting for 90 to 98% of alcohol intake. Alcohol is first oxidized into acetaldehyde via alcohol dehydrogenase, cytochrome P450 2E1 (CYP2E1), and catalase (Fig. 6A). The produced acetaldehyde and oxidative stress play a critical role in the pathogenesis of acute alcohol intake–induced hepatic injury. Therefore, the hepatoprotective effects are a vital indicator for evaluating medicine potency. To investigate whether oral nFeS in the liver shares the same mechanism with neuroprotection, we observed the nuclear translocation of Nrf2 in liver tissue. As shown in Fig. 6 (B and C), the red fluorescence–labeled Nrf2 was located in the nucleus, indicating that the nFeS treatment successfully induced the separation of Keap1 and Nrf2 and caused Nrf2 accumulation in the nucleus. The expression of Nrf2 in the cytoplasm and nucleus also confirmed the nuclear translocation of Nrf2 in liver tissue (Fig. 6D). Moreover, we found that the transcription level of Ho1 and Gr was increased (Fig. 6, E and F). The detection in protein levels also showed higher expression of both enzymes in the nFeS group than in the sham group (Fig. 6, G to I). We inferred that the enhanced MEAODS would mediate the hepatoprotective effects. This contention was supported by monitoring the GSH/GSSH ratio and acetaldehyde content. As a result, the ratio of GSH/GSSG was significantly elevated in the nFeS-treated group and also reversed the depletion of reduced GSH caused by acetaldehyde and ROS (Fig. 6J). Figure 6K shows that acute alcoholism resulted in higher acetaldehyde accumulation than the sham group, which was significantly reduced by the pretreatment of nFeS. This may be ascribed to the high GR expression and GSH/GSSG ratio.

Fig. 6. Hepatoprotective effects of oral nFeS in alcohol-treated mice.

(A) Scheme illustration of the metabolic path of alcohol in the liver. NAD⁺, nicotinamide adenine dinucleotide; NADH, reduced form of NAD⁺, NADP⁺, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP⁺. (B) Immunofluorescence staining of Nrf2 (red) in the liver with or without nFeS treatment. Scale bars, 10 μm. (C) Plots of pixel intensity along the yellow line in (B). (D) Western blot analysis of cytoplasmic Nrf2 and nuclear Nrf2 in the liver before and after nFeS treatment. (E and F) RT-qPCR quantitative analysis of (E) Gr and (F) Ho1 levels in the liver with or without nFeS treatment. (G) Western blotting for HO-1 and GR in different groups. (H and I) Qualitative analysis of expression levels of HO-1 (H) and GR (I) in different groups in (G). (J) GSH/GSSG ratio in the liver. (K) Acetaldehyde content in the liver. (L and M) RT-qPCR quantitative analysis of (L) Tnfα and (M) Il1β in the liver with or without nFeS treatment. (N) Western blot analysis of TNF-α and IL-1β in different groups. (O and P) Qualitative analysis of expression levels of TNF-α (O) and IL-1β (P) in (N). (Q) ALT and (R) AST levels in the blood from different groups. (S) Representative images of TUNEL staining (green) of liver tissues in different groups. Scale bars, 100 μm. (T) Hematoxylin and eosin staining of liver tissues in alcohol-exposed mice with or without nFeS treatment. Scale bars, 100 μm. Scale bars in the enlarged image, 10 μm. Data are presented as means ± SD (n = 3). Statistical significance was determined using [(E), (F), (H), and (I)] two-tailed unpaired Student’s t test and [(J) to (R)] one-way ANOVA followed by Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus the alcohol group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001 versus the sham group.

To ascertain the therapeutic effect of nFeS, we further assessed the inflammation response in the liver via inflammatory cytokine monitoring and pathological analyses. RT-qPCR analysis indicated that nFeS pretreatment down-regulated the expression of cytokines including Il-1β and Tnfα, suggesting the inhibitory effect of nFeS on inflammation (Fig. 6, L and M). The same tendency was observed at the protein level by Western blot test (Fig. 6, N to P). Accumulating evidence demonstrates that the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) could sensitively point out the injury degree, which thus works as an evaluation index of liver function level in the clinic (34). Once hepatic cells suffer injury, the destroyed cell membrane will enhance cell permeability, leading to the infiltration of ALT and AST into the blood. As expected, nFeS treatment reversed the damage induced by alcohol (Fig. 6, Q and R).

It is well documented that alcohol-induced oxidative stress could bring about cell apoptosis (35). TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) assay was performed to inspect the apoptosis rate. The representative images and statistical result demonstrated alcohol-induced excessive apoptosis, while that was depressed in the presence of nFeS treatment (Fig. 6S and fig. S21). Then, hematoxylin and eosin staining showed that alcohol resulted in severe inflammatory cell infiltration. Comparatively, no obvious inflammatory features were observed in the nFeS-treated group. Not only that, hepatic cells exhibited obvious swelling but an indistinct boundary along with blood clot in the alcohol-treated group. On the contrary, the injury was mitigated in the nFeS-treated group (Fig. 6T).

Collectively, acute alcohol intoxication could induce acetaldehyde accumulation and oxidative stress in the liver, which is attenuated by the nFeS-mediated protective effect. This hepatoprotective effect originates from the fact that H₂S_n released from nFeS activates Nrf2, triggers the expression of downstream antioxidant enzymes, and thus potentiates MEAODS, which is similar to that in the brain.

DISCUSSION

Alcohol could be metabolized to acetaldehyde through three pathways, which include the ADH (alcohol dehydrogenase) ethanol oxidation system in the cytoplasm, the microsomal ethanol-oxidizing system in the endoplasmic reticulum, and the catalase system in peroxisome (36, 37). However, because of the low expression of ALDH (aldehyde dehydrogenase), it needs a long time to convert acetaldehyde to nontoxic acetic acid, which causes excessive accumulation of acetaldehyde. Acetaldehyde could change the mitochondrial structure and result in mitochondrial dysfunction, thus reducing adenosine 5′-triphosphate synthesis and increasing ROS production by inhibiting the respiratory chain (8). Besides, the metabolic process of alcohol directly enhances free radicals’ production and induces oxidative stress. Accumulating evidence indicates that ROS and acetaldehyde mediate the alcohol-induced injury.

Acute alcohol intoxication invariably induces multiple-organ injury of the nervous, digestive, and cardiovascular systems or even causes death. Single-organ treatment strategies are not feasible for acute alcoholism. Among the organs, brain and liver tissues were the most severely damaged because of the fact that the brain tissue is extremely sensitive to ROS and acetaldehyde, and the liver is the main organ for alcohol metabolism (38, 39). These characteristics prompt us to focus on the protection of both the liver and brain. Although nanozyme-mediated catalytic therapy has made great progress in ROS-related disease recently, oral nanozymes are acid labile and faced with a physiologic barrier, such as a dense mucus barrier and the blood-brain barrier. It is difficult for nanozymes to intervene in oxidative stress in systemic tissues. The body itself has a variety of antioxidant enzymes comprising MEAODS, which become a powerful weapon against damage from acetaldehyde and ROS. Pre-regulating the MEAODS before drinking has tremendous potential. It is like a vaccine that stimulates the body to produce antibodies in response to the invading antigen, and our pre-regulation strategy beforehand potentiates the expression of antioxidant enzymes and enhances the content of GSH, which subsequently contributes to blocking the ROS- and acetaldehyde-mediated pathological injury in acute alcohol intoxication. The pre-regulation strategy relies on the H₂S_n released from as-prepared nFeS. H₂S_n achieves S-persulfidation of cysteine C151 residues in Keap1, thus promoting the nuclear accumulation of Nrf2. This activates the transcription of numerous cytoprotective genes and therefore stimulates MEAODS, maintaining cellular homeostasis. Notably, nFeS could simultaneously regulate MEAODS in both the brain and liver, which lets us speculate that nFeS could potentiate MEAODS throughout the body and enable multiorgan therapy at the same time. In conclusion, oral nFeS successfully potentiated MEAODS, thus decreasing alcohol-induced ROS and acetaldehyde, alleviating inflammatory response and tissue injury, and simultaneously reversing motor deficits. This work clearly manifests that the nFeS-based pre-regulation strategy is featured as an important therapeutic candidate for preventing alcoholic injury.

MATERIALS AND METHODS

Preparation of nFeS nanoparticles

nFeS synthesis was conducted with the typical solvothermal method. Briefly, 0.82 g of FeCl₃•6H₂O was dissolved in 40 ml of ethylene glycol. Once the solution was clear, 3.6 g of NaOAc was added with continuous and vigorous stirring for 30 min. The mixture was sonicated for 10 min, transferred to a 50 ml Teflon-lined stainless-steel autoclave, and reacted at 200°C for 12 hours. After the reaction was completed, the autoclave was cooled to room temperature. The products were washed three times with alcohol and dried at 60°C for 3 hours. The final products were sealed in a tube and placed in a desiccator for long-term storage.

Determination of decomposition products released from nFeS

nFeS with a concentration of 2 mg/ml was prepared with SGF, placed in a constant temperature incubator at 37°C, and centrifuged for 0, 4, 8, 12, 16, and 20 min to collect the supernatant. NaOH (0.5 M) was added to neutralize the pH of the supernatant to 7. Then, 100 μl of the supernatant from different times was added into a 96-well plate, and 0.1 μl of 10 mM SSP4 probe solution was added to the above well. The reaction was performed at room temperature for 30 min. Last, the fluorescence intensity was measured with a microplate reader (excitation, 482 nm; emission, 515 nm). The contents of H₂S and Fe were determined by the H₂S assay kit and serum iron assay kit, respectively.

Detection of polysulfide species by LC-MS

First, nFeS with a concentration of 2 mg/ml was prepared with SGF, placed in a constant temperature incubator at 37°C, and centrifuged for 20 min to collect the supernatant. The obtained supernatant was detected by LC-MS.

Determination of decomposition products released from sulfide and polysulfide

Na₂S (60 μM), NaSH (60 μM), Na₂S₂ (30 μM), and Na₂S₃ (20 μM) solutions were prepared with SGF. These solutions were placed in a constant temperature incubator at 37°C. After that, samples were taken at 0 and 20 min and the supernatant was obtained by centrifugation. NaOH (0.5 M) was added to neutralize the pH to 7. Next, 100 μl of collected supernatant and 1 μl of 10 mM SSP4 probe solution were added to a 96-well plate and incubated at 37°C for 30 min for complete reaction. Last, the fluorescence intensity was measured with a microplate reader (excitation, 482 nm; emission, 515 nm). At the same time, 60 μM Na₂S, 60 μM NaSH, 30 μM Na₂S₂, and 20 μM Na₂S₃ aqueous solutions were incubated with the SSP4 probe according to the above method.

Absorption of H₂S_n in the bloodstream, brain, and liver

First, nFeS was administered via oral gavage at a dosage of 1 g/kg body weight. At various time points after administration (0, 1, 2, 5, 10, 20, and 30 min and 1, 2, 3, 4, 6, 8, 12, and 24 hours), 10 μl of blood was extracted from the tail vein of each mouse and introduced into a centrifuge tube that had been precoated with 5 μl of sodium citrate as an anticoagulant. Subsequently, 490 μl of phosphate-buffered saline (PBS) was added, and the mixture was thoroughly homogenized. The samples were then centrifuged at 2000 rpm for 3 min. Thereafter, 400 μl of the supernatant was aspirated and transferred to a new centrifuge tube. To this, 20 μM SSP4 probe was added, and the fluorescence intensity was measured using a microplate reader with excitation at 488 nm and emission at 525 nm.

Likewise, another three mice were administered via oral gavage at a dosage rate of 1 g/kg body weight. At various time points after administration (0, 0.5, 1, 3, 6, 12, and 24 hours), the mice were euthanized. Then, the liver and brain tissues were collected and rinsed, followed by addition of 1 ml of PBS. After mincing, the samples were centrifuged at 10,000 rpm for 5 min. Thereafter, 800 μl of the supernatant was transferred to a fresh centrifugation tube, and 10 μM SSP4 probe was introduced. The fluorescence intensity was measured using a microplate reader. The fluorescence intensity in the tissue is the subtracted background fluorescence.

Cell culture

SH-SY5Y cells were cultured by using Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37°C.

Intracellular H₂S_n detection

The SSP4 probe was applied to detect the intracellular accumulation of H₂S_n. First, SH-SY5Y cells were seeded into 24-well plates with 1 × 10⁵ cells per well and cultured for 24 hours. Then, the nFeS decomposition products were added for incubation. After a certain time, the cells were washed thrice with PBS and added to 10 μl of 10 mM SSP4 probe solution for 30 min. Last, the fluorescence images were obtained by a fluorescence microscope. ImageJ software was used to count the fluorescence intensity.

S-persulfidation of Keap1 detected by LC-MS/MS

Keap1 protein (5 μg) was incubated with 25 μl of nFeS decomposition products [released from nFeS (2 mg/ml) during 20 min]. After 1 hour, the protein was collected and lyophilized for LC-MS/MS. Keap1 protein (5 μg) incubated with 25 μl of H₂O was selected as the control. The peptide mixture obtained from enzymatic hydrolysis is first analyzed by LC-MS/MS. The chromatographic column was a reversed-phase C₁₈ column (75 μm by 20 cm, 3 μm). Acetonitrile and an aqueous solution (0 min, 4:96; 8 min, 8:92; 58 min, 22:78; 70 min, 32:68; 71 min, 90:10; 78 min, 90:10; v/v) containing 0.1% formic acid were used as the mobile phase. The flow rate was 300 nl/min. Then, the peptide was searched and identified in the target protein database using the SEQUEST HT search engine of Thermo Proteome Discoverer (2.4.1.15).

Intracellular expression of Nrf2 and antioxidant enzymes

SH-SY5Y cells were seeded into six-well plates with 5 × 10⁵ cells per well and cultured for 24 hours. Subsequently, SH-SY5Y cells were treated with nFeS decomposition products for 2 hours. The cytoplasmic proteins and nuclear proteins were extracted by the Cellular Nuclear Protein Extraction Kit (EX1470, Beijing Solarbio Science & Technology Co., Ltd.). Briefly, 200 μl of extract A was added to the cell precipitate, containing approximately 2 × 10⁶ cells, and the mixture was thoroughly vortexed. After incubation for 30 min, the cells were centrifuged at 4°C and 2000g for 5 min. The cytoplasmic proteins were obtained by collecting the supernatant. The precipitate was then washed with PBS. Extract B (100 μl) was introduced and mixed by vortexing. After 30 min, the mixture was centrifuged at 4°C and 12,000g for 10 min. The supernatant was collected to acquire nuclear proteins. Both extracting solutions were supplemented with 1% protease inhibitor and 1% phosphatase inhibitor. For extraction of whole proteins, 150 μl of radioimmunoprecipitation assay lysis buffer, supplemented with protease inhibitors, was added to the wells 6 hours after nFeS treatment. The adherent cells were gently detached using a cell scraper. The lysate was vortexed three times over a 30-min period to ensure thorough cell lysis. Subsequently, the lysate was centrifuged at 13,400 rpm to pellet the cellular debris. The supernatant, containing the solubilized proteins, was then carefully collected to obtain whole proteins. All proteins were quantified using a bicinchoninic acid protein assay kit. The protein samples were next mixed with loading buffer and heated at 95°C for 10 min to denature the proteins. These prepared samples were subsequently utilized for Western blot analysis.

Cell viability study

Cell viability was investigated with MTT assay. For SH-SY5Y cells, the cells were seeded into 96-well plates with 1 × 10⁵ cells per well and cultured for 24 hours. nFeS with various concentrations of 1000, 500, 250, 125, 62.5, and 0 μg/ml in SGF was prepared. The collected liquid supernatant was added into the same volume of 2× Dulbecco’s modified Eagle’s medium/F12 complete medium for cell incubation. After being incubating for 24 hours, the cells were washed with PBS and added to 100 μl of fresh medium [containing MTT (0.5 mg/ml)]. After incubation for another 4 hours, the cells were washed with PBS and added to 150 μl of dimethyl sulfoxide to dissolve the formed formazan. The absorbance at 570 nm was recorded after shaking at 100 rpm for 10 min, and the viability of cells without any treatments was used as a control.

To assess the therapeutic effect of nFeS on alcohol-induced cell damage, MTT assay was applied to evaluate the cell viability. The experimental procedure was similar to the above steps, except that SH-SY5Y cells were treated with nFeS decomposition products for 2 hours before incubating with 500 mM alcohol for 15 min.

Intracellular ROS scavenging by nFeS decomposition products

According to previous reports (40, 41), the intracellular ROS was induced by alcohol. Fluorescence imaging was used to detect intracellular ROS levels by DCFH-DA, which was nonfluorescent but generated strong fluorescence upon encountering intracellular ROS. The fluorescence signal was positively associated with the amount of the ROS. In detail, cells were preincubated with nFeS decomposition products for 2 hours and next incubated with 500 mM alcohol. After the removal of medium, the cells were treated with 10 μM DCFH-DA for another 1 hour and lastly analyzed by a fluorescence microscope.

Animal experiments

All animal studies were conducted under the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals, and the overall project protocols were approved by the Animal Ethics Committee of Nankai University (approval ID: 2021-SYDWLL-000457).

Biosafety assessment of nFeS

Mice were randomly divided into control and nFeS groups, with nine females and nine males in each group. Mice in the nFeS group received nFeS (2 g/kg) by gavage. The control group received the same dose of deionized water. Food consumption, water consumption, and body weight of the mice were recorded daily. On day 7 after oral nFeS treatment, blood was collected from three mice in each group for blood routine examination and blood biochemistry tests. On days 1, 7, and 14 after nFeS administration, one male mouse and one female mouse from each group were collected for pathological analysis (heart, liver, spleen, lung, kidney, and brain). On day 14, all mice were euthanized and the heart, liver, spleen, lung, kidney, brain, small intestine, and didymus/ovaries were weighed.

Acute alcohol intoxication mice establishment and nFeS treatment

Ten-week-old male C57BL/6J mice were randomly divided into three groups as follows: (i) sham, (ii) alcohol, and (iii) nFeS + alcohol groups. An acute alcohol intoxication model was induced by intraperitoneal injection of alcohol at a dosage of 2.2 g/kg (25%, v/v). In the nFeS treatment group, the mice were administered nFeS (2.5 mg/kg) via oral gavage 2 hours before the administration of alcohol. Behavioral assessments were conducted 20 min after ethanol administration in mice. Subsequently, immunofluorescence analysis, protein extraction, and the evaluation of intracellular biochemical parameters were carried out 4 hours after ethanol injection.

Immunofluorescence

nFeS was administered to the mice following the procedure previously described, and an acute alcohol intoxication model was established. Six hours subsequent to the administration, the mice were euthanized (n = 3 per group). The brain and liver tissues were separated after saline perfusion and fixed in 4% paraformaldehyde. After dehydration through gradient sucrose, the brain tissue was embedded in Tissue OCT-freeze medium and further sliced. The slices were permeabilized with 0.1% Triton for 1 hour, blocked with normal goat serum, and then incubated with anti-Nrf2 primary antibody (ab137550, Abcam) for 1 hour. After washing thrice with Tris-buffered saline with Tween 20, the slices were incubated with TRITC-conjugated goat secondary antibody for 1 hour. Last, the slices were washed thrice with Tris-buffered saline with Tween 20, stained with 4′,6-diamidino-2-phenylindole before sealing the surface, and observed on the an upright fluorescence microscope.

Assessment of antioxidant genes expression

The cortex was separated and homogenized in 1 ml of TRIzol at 24 hours after injury (n = 3 per group). RNA was transferred to the aqueous phase through chloroform, precipitated with isopropanol, and lastly separated by centrifugation. The obtained RNA was quantified by a NanoDrop 8000. A certain amount of RNA was used to invert into cDNA and further quantified by RT-qPCR. β-Actin functioned as an endogenous housekeeping gene to normalize the corresponding mRNA. The mRNA expression level was calculated on the basis of the comparative C_t method (2^−ΔΔCt). All of primers were designed by Primer-BLAST (National Center for Biotechnology Information) and are listed in table S1.

Protein expression in tissue

To assess the protein expression, the Tissue Nuclear Protein Extraction Kit (EX1480, Beijing Solarbio Science & Technology Co., Ltd.) was used to extract proteins in the brain and liver (n = 3 per group). Briefly, 20 mg of brain or liver tissue was added to 300 μl of extract A and homogenized using a tissue homogenizer until no visible solids remain. Subsequently, the homogenate should be shaken at 4°C for 30 min. After this, the mixture was centrifuged at 4°C at 2000g for 5 min. The supernatant was then collected, providing the cytoplasmic proteins. Next, 100 μl of extract B was added to the precipitate and vortexed for 15 s. The mixture was shaken at 4°C for 30 min until the precipitate was no longer visible. After centrifugation at 4°C at 12,000g for 10 min, the supernatant was collected to yield the nuclear proteins. Total protein extraction was performed using radioimmunoprecipitation assay lysis buffer. Protein concentrations were determined using the bicinchoninic acid protein assay kit. The extracts were supplemented with both protease and phosphatase inhibitors.

Assessment of GSH/GSSG

Cortex tissues (70 mg) were collected and homogenized in 1.5 ml of cell lysates (n = 3 per group). The homogenate was left on ice for 15 min and then centrifuged 12,000g at 4°C for 10 min. The supernatant was taken for subsequent determination. The GSH/GSSG ratio was determined by the GSH and GSSG assay kit.

Assessment of acetaldehyde content

The acetaldehyde content was detected through by DNPH precolumn derivation high-performance liquid chromatography. The samples were obtained with DNPH for 60 min in a 40°C water bath, extracted with dichloromethane, and determined at a wavelength of 365 nm using acetonitrile:water (58:42, v/v) as the mobile phase. The flow rate was 1.0 ml/min. Paclitaxel was selected as the internal standard.

Assessment of inflammation

Total RNA extraction and relative expression were conducted according to the above methods. All of primers were designed by Primer-BLAST (National Center for Biotechnology Information) and are listed in table S1.

Assessment of H₂O₂ and lipid peroxidation

The brain and liver tissues were separated, weighted, and homogenized in lysis buffer (3 ml) at 24 hours after injury (n = 3 per group). The supernatant was collected by centrifugation at 5000g for 10 min and used for further quantification. The levels of H₂O₂ and lipid peroxidation were quantified through the Hydrogen Peroxide Assay Kit and Lipid Peroxidation MDA Assay Kit, respectively.

Footprint test

The footprint test was performed as described elsewhere (42). Mice were trained to walk in a corridor 3 days before experiment (n = 10 per group). The length was 60 cm, and the width was 7 cm. The forefeet and hindfeet of the mice were painted with nontoxic and different color ink. The footprint pattern was analyzed for on the white paper. The stride width was measured as the perpendicular distance between the left and right footprints of a given step. The relative position was measured as the average distance of the forebase and hindbase of a given step.

Rotarod test

To assess motion function after acute alcohol damage, the rotarod test was conducted (43, 44). In detail, mice were placed onto the rotated cylinders, and the rotation speed of those uniformly increased from 0 to 25 rpm within 5 min. Mice were trained for 5 days before modeling acute alcohol intoxication mice to achieve the same baseline. After alcohol treatment, the time of fall from the rotating rod was recorded (n = 10 per group).

Righting reflex test

Normally, animals could maintain a standing posture. If an animal is pushed or flipped, it can quickly turn right and return to upright position. This reflex is called the righting reflex. The righting reflex disappearance experiment is an important method to test whether mice are drunk. After alcohol treatment, the mice were placed in a supine position with their backs facing down in a V-shaped groove for observation (n = 10 per group). The degree of intoxication was determined by counting the time the mice were righted.

Statistical analysis

Results were analyzed by using GraphPad Prism software. Differences between two groups were assessed using two-tailed unpaired t tests. For multiple comparisons, statistical significance was analyzed using one-way analysis of variance (ANOVA), followed by Sidak’s multiple comparison tests, which was used when comparing all the conditions. The level of statistical significance was set at P < 0.05. *P < 0.05 was considered significant, and **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered highly significant. All data were expressed as means ± SD, unless otherwise indicated.

Supplementary Materials

This PDF file includes:

Supplementary Text

Figs. S1 to S21

Table S1