Esculetin inhibits ferroptosis after ICH by promoting NUDT1-mediated m7G methylation modification of GPX4

Abstract

Intracerebral hemorrhage (ICH) is the most morbid of all stroke types with a high mortality and significant disability burden. Inhibiting ferroptosis after ICH will effectively treat ICH and improve prognosis. Esculetin is the main active ingredient of the Chinese medicine Cortex Fraxini, which has neuroprotective effects. 7-methylguanosine (m7G) methylation is a common RNA modification that regulates mRNA stability and translation, playing an important role in neural development and function, as well as in ferroptosis-related pathways. In this study, we used hemin-induced PC12 cells to investigate the impact of esculetin on ferroptosis and the involvement of m7G modification. Cell viability was measured by cell counting kit-8. Cell death was evaluated by measuring the levels of LDH release and PI-positive cells. Ferroptosis was assessed by measuring the levels of Fe²⁺, glutathione, lipid reactive oxygen species, and malondialdehyde. The underlying mechanism was investigated by quantitative real-time PCR, methylated RNA immunoprecipitation (MeRIP), and RIP. Results suggested that esculetin promoted cell viability, inhibited ferroptosis, and increased NUDT1-mediated m7G levels in hemin-induced PC12 cells. Besides, NUDT1 interacted with GPX4 and enhanced the mRNA stability of GPX4. Silencing of GPX4 abolished the inhibitory influences of NUDT1 overexpressing on ferroptosis in hemin-induced PC12 cells. The ICH mouse model revealed that esculetin treatment effectively reduced neurological deficits and inhibited ferroptosis. In conclusion, esculetin treatment inhibits ICH-induced ferroptosis by promoting NUDT1-mediated m7G methylation modification and enhancing GPX4 stability. This study contributes to understanding the mechanisms by which esculetin may mitigate ICH damage and may provide a new potential therapeutic target for ICH.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16869-7.

Introduction

Stroke is a common cerebrovascular disease, including ischemic stroke and hemorrhagic stroke¹. It is the second leading cause of death and the third leading cause of disability in humans^2,3. Hemorrhagic stroke accounts for 35% of strokes, mainly including subarachnoid hemorrhage and intracerebral hemorrhage (ICH). The incidence of ICH has increased year by year and shows a trend towards affecting younger populations. Therefore, it is crucial to study the pathological mechanism of degeneration, death, and repair of nerve cells after ICH and explore active and effective intervention methods to improve the prognosis of ICH. Studies have shown that inhibiting ferroptosis is one of the effective means to treat ICH^4,5, but its mechanism has not been fully clarified.

Esculetin, a naturally occurring 6,7-dihydroxy derivative of coumarin, is the main active ingredient of the traditional Chinese medicine Cortex Fraxini⁶. Studies have shown that esculetin has a wide range of pharmacological activities^7,8, including antibacterial, antioxidant, anti-inflammatory, anti-apoptotic, anticancer, neuroprotective, and cardiovascular protective activities. It has a good therapeutic effect on cancer, diabetes, cardiovascular disease, nervous system disease, and non-alcoholic fatty liver disease⁹. However, the role of esculetin in ICH has not been studied, and the related mechanism is unknown.

More than 170 RNA modifications have been identified, and RNA methylation is one of the most common and well-studied post-transcriptional regulation methods¹⁰. Common RNA methylation modifications include N6-methyladenosine (m6A), 2-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), 5-methylcytosine (m5C), and 7-methylguanosine (m7G)¹¹. It is worth noting that m7G modification plays an important role in the central nervous system. Dysfunction of m7G methylation contributes to the development of neurological diseases, including Alzheimer’s disease, down syndrome, ischemic stroke, multiple sclerosis, and glioma¹². However, there is limited research on regulating m7G modification and its effect on ICH. In addition, RNA methylation plays an important regulatory role in ferroptosis, and targeting epigenetic mechanisms that regulate ferroptosis could treat the disease¹³.

Nudix (nucleoside diphosphate-l inked moiety X)-type motif 1 (NUDT1)[¹⁴], also known as mutT human homolog 1 (MTH1), is considered to be a protective agent against nucleotide oxidation with a wide range of substrate recognition capabilities¹⁵. NUDT1 has attracted much attention not only in anti-cancer treatment, but also in the nervous Fe²⁺ detection kitsystem, for example, it prevents neurotoxicity, protects the brain from oxidative stress¹⁶, and effectively inhibits the progression of Alzheimer’s disease¹⁷. More importantly, NUDT1 is a key m7G-related regulator¹⁸. Therefore, we speculate that NUDT1-mediated m7G modification is involved in ICH injury, but the specific mechanism is still unclear.

Herein, we investigated the therapeutic potential of esculetin in hemin-induced ferroptosis using PC12 cells and an ICH mouse model. We focused on elucidating the role of m7G methylation mediated by NUDT1, a key m7G-related enzyme, in regulating the ferroptosis suppressor GPX4. Our findings demonstrate that esculetin attenuates ferroptosis by enhancing NUDT1-dependent m7G modification of GPX4 mRNA. These results not only reveal a novel mechanism by which esculetin confers neuroprotection but also highlight the therapeutic potential of targeting RNA methylation in ICH and other ferroptosis-related disorders.

Methods

Cell culture and treatment

The pheochromocytoma (PC12) cells were purchased from the Chinese Academy of Science (Shanghai, China). PC12 cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco BRL) and 1% penicillin-streptomycin (Gibco BRL) at 37℃ with 5% CO₂. PC12 cells were exposed to 40 µM hemin (Sigma-Aldrich, St. Louis, MO, USA) for 12 h to simulate the ICH in vitro. Various concentrations (0, 5, 10, and 20 µM) of esculetin (Sigma-Aldrich) were added and incubated with the cells for 12 h, and the optimal concentration was determined by detecting the cell viability using CCK-8 assay and the LDH release using LDH kit. Subsequently, PC12 cells were divided into four groups: control, hemin, hemin + Fer-1, and hemin + esculetin group. The cells were pretreated with 20 µM esculetin for 12 h or 5 µM ferroptosis inhibitor Fer-1 for 1 h (Sigma-Aldrich) and then exposed to hemin for another 12 h.

Cell transfection

Short hairpin (sh) RNA targeting NUDT1 (shNUDT1#1/2/3), shRNA negative control (shNC), shRNA targeting GPX4 (shGPX4#1/2/3), NUDT1 overexpressing plasmids, and empty vector were provided by GenePharma Company (Shanghai, China). These plasmids were transfected into PC12 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and the cells were harvested after 48 h of transfection.

shNUDT1#1: forward: 5′-GGAAGAAAGUGGUCUGAGAGU-3′; reverse: 5′-ACTCTCAGACCACTTTCTTCC-3′.

shNUDT1#2: forward: 5′-CCAAUAUGUUUCAAUUCUUCA-3′; reverse: 5′-TGAAGAATTGAAACATATTGG-3′.

shNUDT1#3: forward: 5′-GAGUGGAUACACUGCACAAGG-3′; reverse: 5′-CCTTGTGCAGTGTATCCACTC-3′.

shGPX4#1: forward: 5′-TACTAAAGTGCCGGGATTAAA-3′; reverse: 5′-TTTAATCCCGGCACTTTAGTA-3′.

shGPX4#2: forward: 5′-GGCCTGGAAGTGGAGTTAATA-3′; reverse: 5′-TATTAACTCCACTTCCAGGCC-3′.

shGPX4#3: forward: 5′-AGGCAGGGTCTCACCATATAT-3′; reverse: 5′-ATATATGGTGAGACCCTGCCT-3′.

Cell viability

Cell viability was assessed using a cell-counting kit-8 (CCK-8; Beyotime, Shanghai, China). PC12 cells were seeded to 96-well plates at the density of 1 × 10⁴ cells/well and incubated for 24 h. Then, it was replaced with a serum-free medium containing 10% CCK-8 reagent and continued incubation for 2 h at 37℃. The absorbance at 450 nm in each period was measured by a microplate reader (BMG Labtech, Offenburg, Germany).

Measurement of lactate dehydrogenase (LDH) release

LDH release of PC12 cells in different groups was measured using an LDH assay kit (Beyotime Biotechnology, Shanghai, China) followed by the manufacturer’s instructions. The cells were inoculated in 96-well plates (1 × 10⁴ cells/well). Cells were grouped and the experimental group added 20 µM esculetin and continued culture. After 12 h, the cell culture plates were centrifuged at 400 g for 5 min. Then, 120 µl of the supernatant from each well was added to a new 96-well plate for sample determination. LDH working liquid (60 µl) was added to each well, mixed well, and incubated at room temperature for 30 min away from light. The absorbance was measured at 490 nm.

Measurement of Fe²⁺, malondialdehyde (MDA), and glutathione (GSH) levels

The levels of Fe²⁺, MDA, and GSH in different groups were measured to assess the level of ferroptosis. The concentrations of Fe²⁺, MDA, and GSH in PC12 cell lysate were assessed using a Fe²⁺ detection kit (Solarbio, Beijing, China), an MDA detection kit (Sigma-Aldrich), and a GSH detection kit (Solarbio) according to the manufacturer’s instructions.

Measurement of lipid reactive oxygen species (ROS)

Cells were seeded in a 6-well plate and were cultured in a serum-free medium containing 5 µM BODIPY C11 (D3861, Invitrogen, Waltham, MA, USA) for 30 min protected from light for staining. Next, the cells were washed with PBS three times to remove excess BODIPY C11. The fluorescence microscope was used to measure the fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The green fluorescence intensity that indicates the amount of lipid ROS was calculated using ImageJ software.

Propidium iodide (PI) staining

PI staining was used to determine PC12 cell death. After transfection, cells were collected and incubated with 10 µL PI (Vazyme Biotech, Jiangsu, China) reagent for 10 min. Images of multiple cell fields were taken using a fluorescence microscope immediately after staining and calculating the percentage (%) of PI-positive cells.

Quantitative real-time PCR (qPCR)

Total RNA was separated and extracted from cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions, and RNA concentration was measured. The reverse transcription kit (Takara, Tokyo, Japan) reversely transcribed 1 µg of total RNA into cDNA. Primers were synthesized by Sangon Biotech (Shanghai, China). The mRNA levels of GPX4 and m7G-related genes were measured by qPCR in a 20 µl reaction system containing specific primers and SYBR Green Master Mix (Takara) according to the manufacturer’s protocols on the LightCycler96 Real-Time PCR detection system (Roche, IN, USA). A melting curve of each amplicon was determined to verify its specificity. The comparative 2^−ΔΔCt method was used to analyze the relative mRNA expression in each group.

RNA m1A, m5C, m6A, and m7G quantification

Total RNA was extracted using the TRIzol reagent (Invitrogen) and was bound to strip wells using an RNA high-binding solution. The m6A RNA methylation assay kit (Colorimetric) (ab185912, Abcam, Cambridge, MA, UK) and global RNA methylation assay kit (5 Methyl Cytosine, Fluorometric) (ab233492) were used to measure the relative m6A and m5C content following the recommended procedures. To measure m1A or m7G RNA methylation levels, the capture antibody in the kit was replaced with m1A (ab208196) or m7G antibodies (ab300740). The other procedures were the same as m6A level detection. The absorbance was measured at 450 nm.

Methylated RNA Immunoprecipitation (MeRIP)

The m7G level of ferroptosis-related genes was measured using a GenSeq^® m7G MeRIP kit (Cloudseq, Shanghai, China). Total RNA was extracted from 2 × 10⁷ cells and RNA fragmented to a size of ~ 200 nt. The magnetic beads were incubated with 2 µl m7G or IgG antibody for 1 h on a rotator and washed with IP buffer. Next, 200 µl of nuclease-free water containing fragmented RNA was added to the prepared beads for 1 h incubation on a rotator at 4℃ and washed with IP buffer. The enriched RNA was purified according to the instructions and the expression of genes was evaluated by qPCR.

RNA Immunoprecipitation (RIP)

RIP assays were conducted using an EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Shanghai, China). PC12 cells were washed twice in PBS and then centrifuged at 1,000 × g for 5 min. Next, the cells were lysed in 1 mL of RIP lysis buffer on ice for 10 min. Next, the cell lysates were incubated with magnetic beads conjugated with anti-NUDT1 antibody (ab197028, Abcam, CA, USA), or anti-IgG (#2729, CST, Danvers, MA, USA) at 4℃ overnight. After purification, RNA was extracted from the mixture and GPX4 expression was analyzed by qPCR.

Luciferase reporter assay

The wild-type (WT) GPX4 sequence and the mutant-type (MUT) GPX4 sequence were separately inserted into the pmirGLO reporter plasmid (Promega, Madison, WI, USA). With the knockdown of NUDT1, the firefly activity of the abovementioned reporter plasmids was measured following the protocols of the Dual-Luciferase reporter kit (Promega) by normalizing to the internal control Renilla luciferase activity.

Detection of RNA stability

GPX4 mRNA stability was measured using qPCR after PC12 cells were treated with 5 µg/mL actinomycin D (Merck, Darmstadt, Germany) for 0, 4, 8, and 12 h.

Western blot analysis

Cells or tissues were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors (Roche, Basel, Switzerland), and protein concentrations were determined using a BCA assay (Beyotime). Equal amounts of protein (20 µg) were resolved by 10% SDS-PAGE, transferred to PVDF membranes (Millipore), and blocked with 5% non-fat milk. Membranes were incubated overnight at 4 °C with the following primary antibodies from Abcam (Cambridge, MA, USA): ACSL4 (ab155282), SLC7A11 (ab307601), NUDT1 (ab200832), GPX4 (ab125066), and GAPDH (ab8245). After incubation with HRP-conjugated secondary antibodies (Abcam, 1:5000), signals were detected using ECL substrate (Bio-Rad, Hercules, CA, USA).

ICH mouse model

Male C57BL/6 mice (8–10 weeks old, purchased from Charles River Laboratories, Wilmington, MA, USA) were housed under standard conditions (12-h light/dark cycle, 22 ± 1 °C, 50 ± 5% humidity) with free access to food and water. After one week of acclimatization, mice were randomly divided into three groups (n = 5/group): (1) Sham group (craniotomy without collagenase injection), (2) ICH model group (collagenase-induced ICH + vehicle treatment), and (3) ICH + Esculetin group (collagenase-induced ICH + Esculetin treatment). For ICH induction, mice were anesthetized with isoflurane (3% induction, 1.5% maintenance, Sigma-Aldrich) and positioned in a stereotaxic frame. A midline scalp incision was made, and a 1-mm burr hole was drilled at 0.2 mm anterior and 2.3 mm lateral to the bregma. A 30-gauge needle was inserted 3.5 mm deep into the right striatum, and 0.5 µL of bacterial collagenase VII-S (0.075 U in 0.5 µL saline; Sigma-Aldrich) was infused at 0.1 µL/min using a microinjection pump. The needle was left in place for 5 min post-injection to prevent backflow. Esculetin was administered orally at 20 mg/kg once daily for 7 consecutive days prior to surgery, with the final dose given on the day of the procedure. Sham and ICH model mice received equal volumes of vehicle (5% DMSO in saline) at the same time points. Post-surgery, mice were monitored for recovery and provided with analgesia (buprenorphine, 0.05 mg/kg, s.c.). Neurological function was assessed at 24 h post-ICH and mice were euthanized with 5% isoflurane at 72 h post-ICH.

Neurological behavioral assessments

A systematic evaluation of neurological function in mice was performed using the modified Garcia score test, forelimb placement test, and corner turn test, as previously described in the literature¹⁹.

The modified Garcia scoring system employs a 21-point scale comprising seven neurological assessment items, each graded from 0 to 3 (0 indicating complete functional deficit, 3 indicating normal function). The evaluated parameters include spontaneous activity, axial sensation, vibrissae proprioception, limb symmetry, lateral turning, forelimb walking, and climbing ability.

The forelimb placement test was used to assess sensorimotor integration. The number of successful left forelimb placements onto the table in response to whisker stimulation was recorded, and the success rate (%) was calculated over 10 consecutive trials.

The corner turn test was conducted to evaluate motor lateralization. A 30° angled enclosure was constructed using two vertical boards. Mice were placed facing the corner and allowed to move forward freely, choosing to exit by turning either left or right. The test was repeated 10 times, and the percentage of left turns was recorded.

Statistical analysis

Statistical analyses were conducted with GraphPad Prism 7 software (GraphPad, La Jolla, CA, USA). All values are expressed as the mean ± SD. Student’s t-test was employed to analyze the differences between the two groups. Differences among multiple groups were analyzed by one-way ANOVA or two-way ANOVA followed by Tukey’s post hoc test. P values < 0.05 were considered to be statistically significant.

Results

Esculetin induces cell viability and reduces the LDH release in hemin-induced PC12 cells

As shown in Fig. 1A, the chemical structural formula of esculetin was obtained. To investigate how esculetin affects PC12 cell behaviors, we established an in vitro model using hemin to treat PC12 cells, and increasing concentrations of esculetin (0, 5, 10, 20 µM) were used to treat the cells. The CCK-8 data showed that the viability of hemin-induced PC12 cells was efficiently and gradually enhanced while cell viability in the control group was not changed (Fig. 1B). Then, we observed that esculetin substantially reduced the LDH release of hemin-induced PC12 cells in a dose-dependent manner, with 20 µM showing the maximal reduction, without affecting LDH release in control cells (Fig. 1C). These data suggested that hemin inhibits cell viability and promotes LDH release, while esculetin treatment inhibits the effects of hemin. Since 20 µM esculetin optimally enhanced cell viability and minimized LDH release, we selected this concentration for all subsequent experiments.

Fig. 1.

Esculetin induces cell viability and reduces the LDH release in hemin-induced PC12 cells. (A) The chemical structural formula of esculetin. (B) Hemin and various concentrations of esculetin were used to treat PC12 cells for cell viability detection by CCK-8 assays. (C) LDH release was measured using an LDH release assay kit after the treatment of hemin and esculetin. (n = 3 independent biological replicates/group). All data are expressed as the means ± SD. *p < 0.05 and ***p < 0.001.

Esculetin alleviates ferroptosis in hemin-induced PC12 cells

To investigate whether hemin-induced PC12 cell death occurred via ferroptosis, we treated cells with the ferroptosis inhibitor Fer-1 (5 µM) or esculetin (20 µM) prior to hemin exposure. Compared with the hemin group, we demonstrated that the Fe²⁺, lipid ROS, and MDA levels were significantly decreased after the treatment of Fer-1 or esculetin (Fig. 2A-C). Besides, Both esculetin and Fer-1 treatment increased the level of GSH in hemin-induced PC12 cells (Fig. 2D). Later, we examined cell death by PI staining and found that either esculetin or Fer-1 treatment suppressed the hemin-induced increase in PI-positive cells (Fig. 2E and F). In addition, Western blot analysis revealed that treatment with either esculetin or Fer-1 downregulated ACSL4 expression while upregulating SLC7A11 expression (Fig. 2G). Collectively, the data robustly confirmed that hemin triggers ferroptosis in PC12 cells, and both esculetin and Fer-1 exert protective effects by inhibiting ferroptosis.

Fig. 2.

Esculetin alleviates ferroptosis in hemin-induced PC12 cells. (A) The Fe²⁺ level was measured using a ferrous ion content assay kit. (B) The lipid ROS level was measured by C11-BODIPY. (C) The MDA level was measured using a lipid peroxidation MDA assay kit. (D) The GSH level was measured using a GSH content assay kit. (E,F) Representative fluorescence images and quantification of PI-stained cells in PC12 cells. (G) The expression of ACSL4 and SLC7A11 was detected by western blot. (n = 3 independent biological replicates/group). All data are expressed as the means ± SD. **p < 0.01 and ***p < 0.001.

Esculetin increases the m7G methylation level in hemin-induced PC12 cells

To investigate the specific mechanism of RNA methylation in alleviating ferroptosis by esculetin, we measured different RNA methylation levels in PC12 cells. As is shown in Fig. 3A, the m5C, m6A, and m1A levels had no significant difference after the treatment of esculetin, while only the m7G level was decreased remarkably in the hemin cell model and therewith increased by esculetin treatment. Next, we measured the expression of several m7G-related enzymes in different groups, and the results of the heatmap showed that NUDT1 expression was most significantly increased after the treatment of esculetin compared to the hemin group (Fig. 3B). Therefore, we demonstrated that esculetin increases the m7G methylation level in hemin-induced PC12 cells, which is caused by the high expression of NUDT1.

Fig. 3.

Esculetin increases the m7G methylation level in hemin-induced PC12 cells. (A) Different RNA methylation levels in PC12 cells were measured by quantitative assays. (B) The expression of several methylases was measured by qPCR. (n = 3 independent biological replicates/group). All data are expressed as the means ± SD. **p < 0.01 and ***p < 0.001.

Silencing of NUDT1 abolishes the inhibitory effect of Esculetin on ferroptosis in hemin-induced PC12 cells

Subsequently, this study continued to investigate the effect of NUDT1 on hemin-induced PC12 cells. For functional detection, PC12 cells were transfected with three distinct shRNA sequences targeting the NUDT1 gene, all of which successfully knocked down NUDT1 expression, as confirmed by both qPCR and Western blot analyses (Fig. 4A,B). The cell viability of hemin-stimulated PC12 cells increased by esculetin treatment was recovered by silencing of NUDT1 (Fig. 4C). Later, the LDH release, Fe²⁺, lipid ROS, and MDA levels were significantly decreased after the treatment of esculetin, while silencing of NUDT1 reversed the inhibiting effects induced by esculetin treatment (Fig. 4D-G). Meanwhile, the GSH level was increased by esculetin treatment, whereas NUDT1 knockdown restored this induction (Fig. 4H). Synchronously, silencing of NUDT1 abolished the inhibitory influences of esculetin on the PI-positive cells in hemin-induced PC12 cells (Fig. 4I). Additionally, the esculetin-induced downregulation of ACSL4 and upregulation of SLC7A11 were reversed by NUDT1 knockdown (Fig. 4J). These data indicated that silencing of NUDT1 abolishes the protective effects of esculetin on ferroptosis of hemin-induced PC12 cells.

Fig. 4.

Silencing of NUDT1 abolishes the inhibitory effect of esculetin on ferroptosis in hemin-induced PC12 cells. (A,B) The mRNA level and protein expression of NUDT1 after the shNUDT1#1/2/3 transfecting into PC12 cells was measured by qPCR and western blot, respectively. (C) The cell viability detection by CCK-8 assays. (D) LDH release was measured using an LDH release assay kit. (E) The Fe²⁺ level was measured using a ferrous ion content assay kit. (F) The lipid ROS level was measured by C11-BODIPY. (G) The MDA level was measured using a lipid peroxidation MDA assay kit. (H) The GSH level was measured using a GSH content assay kit. (I) Representative fluorescence images and quantification of PI-stained cells in PC12 cells. (J) The expression of ACSL4 and SLC7A11 was detected by western blot. (n = 3 independent biological replicates/group). All data are expressed as the means ± SD. **p < 0.01 and ***p < 0.001.

NUDT1 enhances RNA stability of the ferroptosis-related gene GPX4

Given the above results, the relationship between NUDT1 and ferroptosis can be known. Next, we further investigated which target gene regulated by NUDT1 exerts its m7G methylation modification function. Firstly, we assessed the m7G level on several ferroptosis-related genes in PC12 cells and found only the level of m7G on the GPX4 gene was significantly inhibited by NUDT1 knockdown (Fig. 5A). Subsequently, we predicted using the m7GHub database that GPX4 contains only a single m7G modification site (Fig. 5B). Further, the interaction between GPX4 and NUDT1 was examined by RIP (Fig. 5C). Luciferase assay examined the luciferase activity in mut or wt GPX4 cells. Silencing of NUDT1 resulted in significantly lower luciferase activity than that of the shNC group in GPX4-wt cells. However, there was no difference in GPX4-mut cells (Fig. 5D). Compared to GPX4-mut cells, NUDT1 knockdown accelerated GPX4 mRNA degradation in GPX4-wt cells (Fig. 5E). In addition, the knockdown of NUDT1 decreased the expression of GPX4 (Fig. 5F). Based on the above results, we concluded that NUDT1 enhanced the stability of GPX4 mRNA by facilitating m7G modification of GPX4, and then we chose GPX4 as a candidate of NUDT1 in the regulation of ferroptosis for further studies.

Fig. 5.

NUDT1 promotes RNA stability of the ferroptosis-related gene GPX4. (A) The regulatory effect of NUDT1 on the m7G level of ferroptosis-related genes was measured by Me-RIP. (B) The m7G modification sites of GPX4 were predicted using the m7GHub database. (C) The binding between NUDT1 and GPX4 was measured by RIP. (D) Luciferase assay was conducted to examine the binding relationship between NUDT1 and GPX4. (E) The stability of GPX4 mRNA was measured by qPCR after PC12 cells were treated with actinomycin D at 0, 4, 8, and 12 h. (F) The expression of GPX4 was detected by western blot. (n = 3 independent biological replicates/group). All data are expressed as the means ± SD. **p < 0.01 and ***p < 0.001.

NUDT1 inhibits ferroptosis of hemin-induced PC12 cells through GPX4

Ultimately, the role of GPX4 on ferroptosis in hemin-induced PC12 cells was verified through rescue experiments. The NUDT1 expression was increased after NUDT1-overexpressing vector transfection, and GPX4 expression was decreased after three distinct shGPX4 sequences transfection (Fig. 6A-D). The cell viability was enhanced by NUDT1 overexpressing, whereas GPX4 knockdown recovered this enhancement (Fig. 6E). Subsequently, the silencing of GPX4 recovered the suppressing effect on the LDH release, Fe²⁺, lipid ROS, and MDA levels and the promoting effect on the GSH level induced by NUDT1 overexpressing (Fig. 6F-J). Besides, the knockdown of GPX4 abolished the inhibitory influences of NUDT1 overexpressing on the PI-positive cells in hemin-induced PC12 cells (Fig. 6K). GPX4 silencing reversed the NUDT1 overexpression-induced downregulation of ACSL4 and upregulation of SLC7A11 (Fig. 6L). Taken together, we certified that NUDT1 inhibits ferroptosis of hemin-induced PC12 cells via enhancing GPX4.

Fig. 6.

NUDT1 inhibits ferroptosis of hemin-induced PC12 cells through GPX4. (A,B) The mRNA level and protein expression of NUDT1 after the NUDT1 overexpression plasmid transfecting into PC12 cells was measured by qPCR and western blot, respectively. (C,D) The mRNA level and protein expression of GPX4 after the shGPX4#1/2/3 transfecting into PC12 cells was measured by qPCR and western blot, respectively. (E) The cell viability was detected by CCK-8 assays. (F) LDH release was measured using an LDH release assay kit. (G) The Fe²⁺ level was measured using a ferrous ion content assay kit. (H) The lipid ROS level was measured by C11-BODIPY. (I) The MDA level was measured using a lipid peroxidation MDA assay kit. (J) The GSH level was measured using a GSH content assay kit. (K) Representative fluorescence images and quantification of PI-stained cells in PC12 cells. (L) The expression of ACSL4 and SLC7A11 was detected by western blot. (n = 3 independent biological replicates/group). All data are expressed as the means ± SD. **p < 0.01 and ***p < 0.001.

Esculetin ameliorates ICH-induced neurobehavioral deficits and ferroptosis

To evaluate the therapeutic potential of esculetin in ICH, we established a murine ICH model and assessed its effects on neurological function, gene expression, and ferroptosis markers. The Modified Garcia score revealed severe neurological deficits in ICH mice compared to the Sham group, whereas esculetin-treated mice exhibited significant functional recovery, suggesting its potential to ameliorate ICH-induced neurological impairment (Fig. 7A). ICH mice exhibited significant impairments in left forelimb placement and left-turning behaviors, indicating marked motor dysfunction, which were effectively ameliorated by esculetin treatment (Fig. 7B and C). Notably, ICH induced pronounced downregulation of NUDT1 and GPX4 mRNA expression, both of which were partially restored following esculetin administration (Fig. 7D and E). Esculetin significantly reduced Fe²⁺, ROS, and MDA levels while partially recovering GSH levels in ICH mice (Fig. 7F-I). Furthermore, esculetin treatment markedly decreased ACSL4 expression and increased SLC7A11 expression (Fig. 7J). These data demonstrated that esculetin mitigates ICH-induced neurological and motor deficits via the anti-ferroptosis pathway.

Fig. 7.

Esculetin ameliorates ICH-induced neurobehavioral deficits and ferroptosis. (A) The neurological function of mice was evaluated by the modified Garcia score. (B,C) The motor function of mice was evaluated by the left forelimb placement test and the corner turn test. (D,E) The mRNA level of NUDT1 and GPX4 was measured by qPCR. (F) The Fe²⁺ level was measured using a ferrous ion content assay kit. (G) The lipid ROS level was measured by C11-BODIPY. (H) The MDA level was measured using a lipid peroxidation MDA assay kit. (I) The GSH level was measured using a GSH content assay kit. (J) The expression of ACSL4 and SLC7A11 was detected by western blot. (n = 5 independent biological replicates/group in animal experiments ). All data are expressed as the means ± SD. ***p < 0.001.

Discussion

There is increasing evidence that ferroptosis plays a dominant role in the pathogenesis of ICH injury, and inhibiting ferroptosis can significantly improve the prognosis of ICH^20,21. Esculetin has various pharmacological activities, especially neuroprotective effects. A study shows that esculetin prevents oxidative stress and neuronal death induced by amyloid protein in SH-SY5Y cells by activating Nrf2 and increasing GSH²². Esculetin can save cultured primary neurons from N-Methyl-D-Aspartate-induced toxicity in cerebral ischemia/reperfusion injury²³. However, how esculetin plays a protective role in ICH needs further study. Hemin affects cell survival, metabolism, and function, and is widely used as an in vitro ICH model due to its ability to simulate secondary brain injury, including blood-brain barrier disruption and neuroinflammation²⁴. PC12 cells have been widely used as models for neural differentiation²⁵. In this study, we stimulated PC12 cells with hemin to establish an ICH model, treated the cells with different concentrations of esculetin, and selected the most effective concentration of esculetin. Then, we found that esculetin promoted hemin-induced PC12 cell viability, inhibited the level of ferroptosis-related indicators (LDH release, Fe²⁺, lipid ROS, and MDA levels), and increased the level of GSH, suggesting that esculetin inhibits ferroptosis.

At present, studies on RNA methylation modifications mainly focus on the regulation of ischemic stroke^26,27, but there are few studies on their role in ICH. A study has shown that differentially expressed m6A methylation-modified genes affect the immune microenvironment of acute ICH²⁸. Silencing of METTL3 inhibits hemin-induced pyroptosis of HT-22 cells by inhibiting m6A methylation of NEK7, thereby relieving ICH-induced nerve damage²⁹. Here, we examined several RNA modifications and found that only the m7G modification level was decreased in hemin-induced PC12 cells, which was increased after esculetin treatment. Previous studies focusing on the pathological role of m7G modification disorders have identified the role of METTL1/WDR4 complex-mediated m7G methylation in central nervous system development and the pathogenesis of nervous system diseases³⁰. Therefore, we speculated that m7G modification might be involved in esculetin inhibition of ferroptosis in ICH. Then, we screened the genes involved in m7G regulation and found that esculetin significantly increased NUDT1 expression in hemin-induced cells.

NUDT1 is an m7G-associated risk gene predicted in a variety of diseases, such as clear cell renal cell carcinoma³¹ and head and neck squamous cell carcinoma³², and may serve as a potential diagnostic biomarker for ischemic stroke¹⁸. In addition, NUDT1 effectively inhibits the accumulation of 8-oxoguanine in cells, protecting microglia³³ and dopamine neurons³⁴ from oxidative damage by nucleic acids, suggesting the function of NUDT1 on nerve cells. In this study, we conducted the function experiment to explore the cell biological function of NUDT1 and found that silencing of NUDT1 abolished the inhibitory effect of esculetin on ferroptosis in hemin-induced PC12 cells. These findings are the first to confirm the mechanism by which NUDT1 is involved in regulating esculetin to inhibit hemin-induced ferroptosis and is related to m7G methylation modification.

We then further explored the molecular mechanism of NUDT1 regulating ferroptosis and found the significant inhibition of m7G levels on the GPX4 gene by NUDT1 knockdown. GPX4 moonlights as a structural protein and antioxidase that powerfully inhibits lipid oxidation³⁵. Loss of GPX4 induces apoptosis, necroptosis, and pyroptosis, which mediates or accelerates developmental defects, tissue damage, and aseptic inflammation³⁶. GPX4 is thought to be a key regulator of ferroptosis which blocks it by eliminating phospholipid hydroperoxides³⁷. It is worth noting that the up-regulated GPX4 expression can inhibit ferroptosis of nerve cells and alleviate brain injury after ICH³⁸. In our study, we found that NUDT1 interacted with GPX4 and enhanced the mRNA stability of GPX4. Moreover, the rescue experiments further solidify this mechanism by showing that GPX4 knockdown reverses all protective effects of NUDT1 overexpression, confirming GPX4 as the critical downstream effector in this pathway.

Our in vivo studies using an ICH mouse model significantly bolster the translational potential of this study. The consistent upregulation of NUDT1 and GPX4, along with reduced ferroptotic markers and improved neurological outcomes, confirms the conserved action of this pathway across experimental systems. Importantly, esculetin treatment not only protected against cellular damage but also restored motor function, highlighting its translational value for neurological recovery.

However, the clinical translation of esculetin for ICH treatment faces several challenges that need to be addressed. The pharmacokinetic and pharmacodynamic properties of esculetin, including its blood-brain barrier permeability, bioavailability, metabolism, and optimal dosing regimen, remain to be fully characterized. Comprehensive toxicological assessments are also necessary to evaluate potential hepatotoxicity or off-target effects, especially for long-term administration. Furthermore, the exact molecular mechanism by which esculetin upregulates NUDT1 expression remains unclear and warrants further investigation. Given the complexity of human ICH pathophysiology involving multiple cell death pathways, future studies should explore whether esculetin’s efficacy could be enhanced through combination therapies with iron chelators or anti-inflammatory agents. These investigations will be crucial for determining esculetin’s potential as a clinically viable therapeutic option for ICH.

On the whole, we prove the molecular mechanism of the alleviation of ICH injury mediated by esculetin. Esculetin upregulates NUDT1 expression to facilitate the m7G methylation modification of GPX4 and increases the mRNA stability of GPX4, thereby promoting PC12 cell viability and inhibiting cell ferroptosis. These findings establish esculetin as a promising candidate for targeting ferroptosis in ICH treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. J G drafted the work and revised it critically for important intellectual content; Z Z and L Lwere responsible for the acquisition, analysis and interpretation of data for the work; Z M and T C made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Hunan Province (Grant No. 2025JJ80950).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of The First Affiliated Hospital of Hunan University of Chinese Medicine. All animal experiments were complied with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.