Groundbreaking Insights Into SIRT1 / NRF2 ‐Mediated Ferroptosis Inhibition by Resveratrol in Parkinson's Disease Models

In the network pharmacology analysis, potential therapeutic targets for RSV in the context of PD and ferroptosis were identified through a multi‐database approach. RSV treatment targets were obtained from the HERB database (http://herb.ac.cn/↗), PD‐related targets from the GeneCards database with a correlation score > 1 (http://www.genecards.org/↗), and ferroptosis‐related targets from the FerrDb database (http://www.zhounan.org/ferrdb/current/↗). Overlapping targets were subsequently determined using a Venn diagram to pinpoint potential targets of RSV for PD treatment. To explore protein interactions, both direct and indirect, the STRING database (https://string‐db.org/↗) was used to analyze these overlapping targets, with species limited to Homo sapiens and default settings applied. Cytoscape 3.9.0 was employed to create a protein–protein interaction (PPI) network diagram, and the top 10 core targets were selected based on degree centrality values using Network Analyzer. Further functional and pathway enrichment analyses were performed using R software, focusing on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. GO analysis covered three categories: biological process (BP), cellular component (CC), and molecular function (MF), providing insight into the functional roles of the target proteins involved in the pharmacological intervention of PD.

The viability of PC12 cells was assessed using the MTT assay. Briefly, PC12 cells (1.5 × 10⁴) were seeded into 96‐well plates and pretreated with varying concentrations of RSV (1–50 μM) for 1 h prior to 6‐OHDA exposure (24 h) to assess neuroprotective effects. MTT (10 μM) was then added to each well, and the cells were incubated for 2 h. Absorbance was measured at 490 nm using a microplate reader (BioTek, VT, USA).

Intracellular reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were determined using DCFH‐DA (5 μM) and LiperFluo (10 μM) probes, respectively. PC12 cells in 6‐well plates were treated for 24 h, stained with probes (37°C, 1 h), and visualized using an inverted fluorescence microscope.

Levels of MDA and GSH were measured using specific assay kits. After treatment, brain tissues from mice were homogenized and sonicated in lysis buffer, followed by centrifugation at 12,000 rpm at 4°C for 30 min. The supernatants were analyzed for MDA and GSH activities using a multimode plate reader.

Mitochondrial membrane potential (MMP) was evaluated using the JC‐1 fluorescent probe. PC12 cells (3.5 × 10⁴) were seeded in 6‐well plates overnight, treated for 24 h, and then incubated with JC‐1 according to the manufacturer's protocol. Images were captured using an inverted fluorescence microscope. Healthy mitochondria with high membrane potential emitted red fluorescence, while green fluorescence indicated a decrease in membrane potential. The change in MMP was detected by measuring the fluorescence shift from red to green.

Mitochondrial ROS levels were assessed using MitoSOX Red. Briefly, PC12 cells were seeded in 6‐well plates, treated, and incubated with 10 μM MitoSOX Red at 37°C for 30 min. After washing with PBS, fluorescence was analyzed using laser scanning confocal microscopy.

The morphological structure of mitochondria, including mitochondrial size, shape, and cristae structure, was directly observed by using a JEM‐1400‐FLASH transmission electron microscope (TEM). Briefly, PC12 cells were collected and fixed with 2% glutaraldehyde for 2 h, followed by 1% osmium tetroxide fixation. The cells were dehydrated in a graded ethanol series, embedded in resin and imaged by TEM.

For the lifespan analysis assay, synchronized L1 larvae (N2) were allowed to grow on NGM plates seeded with E. coli OP50 for 1 day at 22°C, reaching the L2 stage to assess lifespan. The nematodes were collected by centrifugation at 3000 rpm for 1 min and then divided into 6‐OHDA (50 mM) lesion groups and control groups (20 mM ascorbic acid) for 1 h. Nematodes in the control and PD groups were transferred to plates seeded with inactive OP50, while those in the RSV group were transferred to plates seeded with 20 mM RSV and inactive OP50 after 6‐OHDA treatment. Synchronized late L4 larvae or young adults were transferred to fresh plates with or without RSV every other day (n = 50). The survival of the nematodes was monitored daily throughout the experiment. The survival rate on each plate was recorded until all the nematodes died. The lifespan experiments were repeated independently at least three times. Statistical analyses were performed using Kaplan–Meier lifespan analysis, and P‐values were calculated using the log‐rank test.

Quantitative analysis of DA neurodegeneration and mitochondrial damage was performed in transgenic BZ555 and PD4251 strains, respectively, under the treatment conditions described above. After 48 h of treatment, the worms were washed three times with M9 buffer and fixed onto an agar pad on a glass slide. They were then straightened using 100 mM sodium azide before being sealed with a coverslip. Fluorescence microscopy was used to assess DA neurodegeneration and mitochondrial damage in each group.

Quantitative analysis of Fe²⁺ and ROS levels was performed in N2 nematodes, with the same treatment and groups mentioned above. After 48 h of treatment, the nematodes were exposed to bacterial solutions containing 150 μM ROS probe (DCFH‐DA) or 10 μM Fe²⁺ probe (FerroOrange) for 2 h, following a 1‐h starvation period. ROS and Fe²⁺ levels were analyzed using a fluorescence microscope.

Seventy mice were randomly assigned to control, PD, RSV10, RSV20, RSV30, ferrostatin‐1 (Fer‐1) and levodopa (Lev) groups (10 per group) (Figure 6A). A separate cohort of 50 mice was divided into control, PD, RSV, EX527, and ML385 groups (10 per group) (Figure 8A). Mice in the PD, RSV, Fer‐1, Lev, EX527, and ML385 groups received intraperitoneal injections of MPTP (30 mg/kg/day) for 5 consecutive days (days 1–5) [11]. From day 6 to day 15, the RSV group was administered RSV (10, 20 or 30 mg/kg/day) intraperitoneally, the Fer‐1 group was administered Fer‐1 (3 mg/kg/day) intraperitoneally, the Lev group was administered Lev (3 mg/kg/day) intraperitoneally. while the EX527 (5 mg/kg/day) and ML385 (30 mg/kg/day) groups were treated with the respective compounds 1 h prior to RSV (30 mg/kg/day) administration. The control group received an equivalent volume of 0.9% saline.

Mouse brain tissues or cells were collected and homogenized, and the supernatants were obtained by centrifugation. Total iron levels were quantified using an iron assay kit, and the supernatant was analyzed for iron content using an ultraviolet spectrophotometer at 593 nm. For iron staining, Prussian blue staining was performed on 5 μm paraffin sections of three brain tissues. The sections were deparaffinized and rehydrated in distilled water, followed by sequential incubation with Perls' working solution, incubation solution, and enhanced working solution. After three washes with PBS, the sections were counterstained, dehydrated through a graded ethanol series, cleared in xylene, and sealed with resin.

All data were analyzed using GraphPad Prism 8.0 software (San Diego, CA, USA). Data are expressed as the mean ± standard deviation (SD). The Shapiro–Wilk test was used for the normality test. Nonparametric tests were employed for data that did not follow a normal distribution. Comparison of means among groups was performed using the one‐way ANOVA followed by Tukey's post hoc test. A value of p < 0.05 was considered statistically significant.

RSV is a plant‐derived polyphenolic compound with natural chelating activity, attributed to the catechol moieties in its structure, which contain three potential metal‐binding sites. Given the critical role of iron in ferroptosis, the iron‐chelating capacity of RSV was systematically evaluated by titrating RSV with trivalent iron (Fe³⁺) in a neutral buffer for UV–vis spectrophotometric analysis. With the addition of Fe³⁺, the characteristic absorbance of free RSV at 312 nm decreased, and the color of the Fe³⁺ solution became lighter, suggesting that RSV may chelate iron ions (Figure 2A). We further assessed the antioxidant capacity of RSV using the well‐established 1,1‐diphenyl‐2‐picrylhydrazide (DPPH) assays and 3,3′,5,5′‐tetramethylbenzidine (TMB)‐H₂O₂ system. Results demonstrated that RSV efficiently scavenged DPPH• radicals and H₂O₂ in a dose‐dependent manner, respectively (Figure 2B,C). These results indicate the potential of RSV in iron chelation and antioxidant activity.

Subsequently, the impact of RSV on neuronal survival under iron overload was assessed. The MTT assay was conducted to determine the optimal concentration of RSV for the experiment. High RSV concentrations (≥ 30 μM) were found to induce cytotoxicity. Consequently, 10 μM of RSV was selected as the optimal concentration for subsequent experiments (Figure S1). PC12 cells were treated with ferric ammonium citrate (FAC) to establish an in vitro model of iron overload‐induced neuronal injury, resulting in a rapid decline in cell viability to approximately 61.8% (Figure S2). RSV treatment significantly increased cell viability, and the cell survival rate was 98.6%, which was close to the control level. (Figure 2D). These results suggest that RSV may have protective effects against ferroptosis.

To further investigate RSV's anti‐ferroptotic effects, PC12 cells were treated with two classical ferroptosis inducers, Erastin and RSL3. As shown in Figure 2E,F, RSV pretreatment effectively attenuated cytotoxicity induced by both compounds. Comparative analysis with the ferroptosis inhibitor ferrostatin‐1 (Fer‐1) revealed that RSV exhibited superior cytoprotective effects in a dose‐dependent manner. These results support the hypothesis that RSV can protect PC12 cells from Erastin‐ and RSL3‐induced ferroptosis.

Given its potential as an iron chelator and ROS scavenger, as well as its ability to protect against Erastin‐ and RSL3‐induced ferroptosis in PC12 cells, RSV was hypothesized to alleviate cytotoxicity in a PD model. Initially, PC12 cells were exposed to varying doses and durations of 6‐OHDA to establish an in vitro PD model [12]. The results revealed a dose‐dependent cytotoxic effect of 6‐OHDA, with concentrations ranging from 50 to 400 μM (Figure S3). At 200 μM, cell viability dropped to approximately 50% (50 ± 2.42, p < 0.0001). Consequently, 200 μM of 6‐OHDA was selected for subsequent experiments. PC12 cells were then treated with or without Fer‐1 (1 μM, 1 h), followed by 6‐OHDA. Fer‐1 treatment effectively mitigated 6‐OHDA‐induced cell death (Figure S4), consistent with previous studies suggesting that 6‐OHDA induces ferroptosis in PC12 cells [13]. To determine whether RSV could protect against 6‐OHDA‐induced ferroptosis, PC12 cells were treated with RSV (1–50 μM) prior to 6‐OHDA exposure. RSV at 15 μM provided the most significant protective effect, with cell viability reaching 90.22% ± 5.08% (Figure 2G). Calcein‐AM/propidium iodide (PI) cell staining was used to identify live and dead PC12 cells exposed to 6‐OHDA with or without RSV. Both the photographs and quantitative examination revealed that after 6‐OHDA stimulation, there was a prominent number of dead cells, whereas RSV treatment caused a noticeable increase in the number of viable cells (p < 0.05; Figure 2H and Figure S5). These results suggest that RSV may inhibit 6‐OHDA‐induced ferroptosis.

Excess intracellular iron contributes directly to ferroptosis. To assess the effect of RSV on iron levels in PD cell models, Prussian blue staining was performed to detect iron‐positive cells, and total iron content was quantified. Prussian blue staining revealed a 3‐fold increase in iron‐positive cells in the PD group versus controls (p < 0.01), which was reduced by 50% following RSV treatment (p < 0.05; Figure 3A,B). Consistent with these findings, quantitative analysis demonstrated significantly elevated total iron content in PD cells (p < 0.01), with RSV treatment effectively normalizing iron levels (p < 0.05) (Figure 3C). Further, Calcein‐AM dye was used to assess the intracellular labile iron pool (LIP) [14]. Fluorescence imaging revealed quenched green fluorescence, indicating a marked increase in the LIP, suggesting that 6‐OHDA pretreatment augmented the intracellular LIP (p < 0.01). Upon treatment with RSV, the fluorescence intensity increased (p < 0.05; Figure 3D,E), indicating a continuous reduction in the intracellular LIP. These results collectively support that RSV's cellular actions involve iron chelation.

LPO, a pivotal process in ferroptosis, leads to excessive ROS generation. To assess the effect of RSV on ROS levels, ROS content was measured in each group. Quantitative analysis revealed that 6‐OHDA treatment induced a dramatic elevation in intracellular ROS levels compared to normal controls (p < 0.0001), while RSV treatment significantly reduced ROS levels in the PD model group (p < 0.0001; Figure 3F,G). This suggests that RSV can attenuate ROS accumulation, a central product of LPO, in PD cell models. Using a LPO fluorescent probe, we further confirmed that RSV significantly suppressed 6‐OHDA‐induced lipid peroxidation (p < 0.01; Figure 3H,I).

To further evaluate LPO, we quantified the levels of GSH and MDA, two critical biomarkers of oxidative stress. GSH mitigates oxidative stress in ferroptotic cells, while MDA is a byproduct of free radicals and LPO. Our findings revealed a marked depletion of GSH in PD model cells relative to normal controls (p < 0.01), which was substantially reversed by RSV administration (p < 0.01; Figure 3J). In contrast, we observed a pronounced accumulation of MDA in PD cells relative to normal controls (p < 0.0001), but RSV treatment significantly attenuated MDA production (p < 0.001; Figure 3K). In addition, we observed a significant increase in 4‐HNE in PD cells compared to normal controls (p < 0.0001), but RSV treatment remarkably reduced the production of 4‐HNE (p < 0.0001; Figure S6).

Mitochondria are primary sources of ROS and are closely linked to ferroptosis. To systematically evaluate mitochondrial functional alterations induced by 6‐OHDA exposure and the potential protective effects of RSV, we quantitatively assessed mitochondrial ROS (mROS) production using MitoSOX Red fluorescence staining. Comparative analysis revealed a marked increase in mROS in the PD group compared to the NC group (p < 0.0001). However, RSV treatment significantly attenuated mROS levels, highlighting its capacity to scavenge mROS and mitigate oxidative stress‐induced damage (p < 0.0001; Figure 4A,B).

The JC‐1 probe, a widely used fluorescent marker for detecting mitochondrial membrane potential (Δψm), was employed to assess Δψm alterations, a key indicator of ferroptosis. The probe accumulates in mitochondria in a Δψm‐dependent manner, with changes in staining reflecting mitochondrial polarization. The ratio of green to red fluorescence was calculated to quantify mitochondrial depolarization. Compared to the NC group, the ratio of green to red fluorescence in the PD group was significantly increased (p < 0.01). RSV treatment significantly reduced this ratio (p < 0.01), suggesting that RSV alleviated mitochondrial depolarization in the PD model (Figure 4C,D).

The primary morphological characteristics of mitochondria undergoing ferroptosis include increased mitochondrial membrane density, reduced volume, and the loss of inner cristae structure. To evaluate the impact of RSV on these morphological changes, transmission electron microscopy was used to examine mitochondrial ultrastructure in each group. The results revealed that, compared to the NC group, the PD group exhibited typical ferroptotic mitochondrial alterations, such as increased membrane density, decreased volume, and the disappearance of inner cristae. Notably, RSV treatment significantly ameliorated these morphological changes (p < 0.01; Figure 4E,F). Taken together, these results suggest that RSV improves mitochondrial function and mitigates the detrimental effects of 6‐OHDA treatment.

Numerous studies have established a strong correlation between iron accumulation, ROS production, and oxidative stress, all of which contribute to mitochondrial damage and ultimately lead to the death of DA neurons. To further investigate the neuroprotective potential of RSV across different species, a Caenorhabditis elegans (C. elegans) model was employed to observe the effects on iron levels, ROS production, and DA neuron morphology.

The 6‐OHDA‐pretreated wild‐type C. elegans (Bristol N2 strain, did not have any markers) was initially utilized to assess Fe²⁺ and ROS levels [15, 16]. As depicted in Figure 5A,B, red FerroOrange (a detection probe for Fe²⁺) fluorescence was prominently observed in the nematodes of the PD group (p < 0.001). In contrast, RSV treatment resulted in a noticeable reduction in red fluorescence intensity, indicating a marked decrease in Fe²⁺ production compared to the PD group (p < 0.01). This suggests that RSV effectively attenuates iron accumulation in the model. Subsequently, the 6‐OHDA‐pretreated Bristol N2 strain model was used to evaluate ROS levels. The DCFH‐DA assay revealed a relative increase in intracellular ROS levels in the PD group compared with that in the control group (p < 0.0001). However, RSV treatment significantly reduced the green fluorescence intensity (p < 0.0001; Figure 5C,D), further validating its role in reducing Fe²⁺ levels and eliminating ROS.

Mitochondrial damage was assessed using the PD4251 transgenic nematode model, which expresses GFP in body wall muscle mitochondria [17]. As illustrated in Figure 5E,F, the fluorescence intensity was significantly reduced in the PD group (p < 0.001), indicating mitochondrial damage. Remarkably, RSV treatment effectively restored fluorescence levels (p < 0.01), suggesting a protective role in mitigating 6‐OHDA‐induced mitochondrial dysfunction.

To further evaluate the neuroprotective effects of RSV, we employed the BZ555 nematode strain, a PD model featuring GFP‐labeled DA neurons (DAns) [18]. 6‐OHDA treatment significantly reduced the number of DAns in the head region (p < 0.01), indicating substantial neuronal damage. Notably, RSV treatment restored DAn integrity (p < 0.05; Figure 5G,H), supporting its potential to protect DA neurons from degeneration. Finally, we assessed the impact of RSV on lifespan using 6‐OHDA‐treated Bristol N2 nematodes. The median survival time (MST) of the 6‐OHDA‐treated nematodes was markedly reduced (7 days) compared to controls (16 days). Strikingly, RSV treatment extended the MST to 11 days (Figure 5I), further corroborating its neuroprotective efficacy in this PD model.

To evaluate whether RSV alleviates motor deficits in a mouse model of PD, various behavioral assays, including forced swimming, open field, and pole climbing tests, were conducted. In the open field test, RSV‐treated groups (20 and 30 mg/kg/day) exhibited significant improvements in locomotor activity, with both total movement distance and average speed showing marked increases compared to the PD model group (Figure 6B–D). Similar enhancements were observed in Fer‐1 and Lev treatment groups. The pole climbing test revealed substantial motor coordination improvements across all treatment groups. RSV administration (20 and 30 mg/kg) reduced climbing time by 23.8% and 34.9%, respectively, comparable to the effects of Fer‐1 (31.2% reduction) and Lev (24.8% reduction) (Figure 6E). Consistent with these findings, the forced swimming test indicated significantly reduced immobility scores in treated groups (RSV20:2.17 ± 0.058, RSV30: 2.60 ± 0.10, Fer‐1: 2.23 ± 0.58, and Lev:2.30 ± 0.17) versus the PD model group (1.93 ± 0.58) (Figure 6F). Collectively, these behavioral assessments demonstrate that both RSV doses (20 and 30 mg/kg/day) effectively ameliorate MPTP‐induced motor impairments in mice, with efficacy comparable to that of Fer‐1 and Lev treatments.

A key pathological hallmark of PD is the degeneration of DA neurons. Tyrosine hydroxylase (TH), an enzyme critical to DA synthesis, serves as a marker for DA neuron damage [19]. To assess the extent of DA neuron loss in the SN, the number of TH‐positive neurons and the expression of TH protein were evaluated. The results demonstrated that RSV treatment (20 mg/kg/day) significantly restored the number of TH‐positive neurons compared to the PD model group (p < 0.01; Figure 6G,H). Furthermore, Western blot analysis revealed a marked upregulation of TH protein expression in all treatment groups relative to the PD model group (p < 0.05; Figure 6I,J). These findings suggest that RSV effectively attenuates MPTP‐induced neurodegeneration of DA neurons in the SN, with a neuroprotective efficacy comparable to that of Fer‐1 and Lev treatments.

The Xc‐system‐GSH‐GPX4 axis is a well‐known regulator of ferroptosis, with GPX4 playing a pivotal role in clearing lipid peroxides, thus acting as a key negative regulator of ferroptosis [20]. Ferritinophagy, regulated by FTH1, also inhibits ferroptosis [21]. The expression levels of GPX4 and FTH1 in the SN region of the mice were examined. The results revealed that both RSV doses (20 and 30 mg/kg/day) as well as Fer‐1 treatment significantly enhanced the expression levels of GPX4 and FTH1 compared to the PD model group (p < 0.05, p < 0.05; Figure 6K–M), suggesting that RSV may protect against ferroptosis through this axis.

Prussian blue staining was used to examine iron deposition in the SN region. The results demonstrated significantly elevated iron deposition in the SN of PD mice compared to the NC group. Notably, both RSV treatment (20 and 30 mg/kg/day) and Fer‐1 administration effectively attenuated this iron accumulation, as evidenced by reduced iron‐positive cell counts (Figure 6N). These results suggest that RSV possesses iron‐chelating properties comparable to Fer‐1, potentially contributing to its neuroprotective effects in this PD model.

To identify potential therapeutic targets of RSV for PD and ferroptosis, 225 RSV‐related targets, 484 ferroptosis‐related targets, and 8633 PD‐related targets were retrieved from existing datasets. The Venn diagram revealed 46 overlapping targets among RSV, ferroptosis, and PD, which were considered potential therapeutic targets (Figure 7A). These targets were further analyzed using the STRING database to evaluate protein–protein interactions, and the resulting data were visualized using Cytoscape 3.9.0 (Figure 7B). The top ten core targets, identified based on their degree values, included SIRT1, TP53, HIF1A, PPARG, MTOR, JUN, IL6, IL1B, STAT3, and NFE2L2 (Figure 7C).

To explore the biological processes and signaling pathways associated with the overlapping targets, GO and KEGG enrichment analyses were performed. As shown in Figure S7A, the BP analysis revealed that the target genes were primarily enriched in processes such as the positive regulation of RNA polymerase II, positive regulation of gene expression, cellular response to oxidative stress, and negative regulation of ferroptosis. In the CC analysis (Figure S7B), enrichment was observed in the nucleus, cytosol, cytoplasm, nucleoplasm, and mitochondrion. In terms of MF (Figure 7D), protein binding, identical protein binding, and enzyme binding were identified as key molecular activities. KEGG analysis (Figure 7E) highlighted the involvement of the FoxO signaling pathway and cellular senescence in the regulation of these targets. Notably, silent information regulator 1 (SIRT1) emerged as a central signal in these pathways, prompting further focus on its role in RSV's therapeutic effects on PD.

To validate the network pharmacology findings regarding the interaction between key targets and RSV, molecular docking was performed to investigate the binding affinity of RSV with SIRT1 and NRF2, two critical proteins identified in the analysis. The docking results demonstrated robust and stable binding interactions between RSV and both target proteins. Specifically, RSV formed multiple hydrogen bonds with SIRT1 at critical residues, including ILE403, GLU408, and ASN409, exhibiting a binding affinity of −6.3 kcal/mol (Figure 7F and Table S1). Similarly, RSV exhibited strong binding with NRF2, engaging residues GLU518 and ASP526, and yielding a favorable binding energy of −7.1 kcal/mol (Figure 7G and Table S2). These interactions confirm the potential of RSV to bind to these key proteins.

To further validate the role of the SIRT1‐NRF2 pathway in RSV's figure using WB analysis. The results revealed a significant upregulation of both SIRT1 and NRF2 in the RSV‐treated group compared to the PD model group (Figure 7H–J). To further validate our molecular docking predictions, we conducted in vitro experiments, which confirmed that RSV treatment markedly enhanced the protein expression of both SIRT1 and NRF2 (p < 0.05, p < 0.01; Figure 7K–M). These results are consistent with the results from the network pharmacology analysis and molecular docking.

To investigate whether the effects of RSV on motor deficits in the PD mouse model are linked to the SIRT1‐NRF2 signaling pathway, this study employed pharmacological inhibition of SIRT1 and NRF2 using EX527 and ML385, respectively [22]. Motor function was evaluated through open field, pole climbing, and forced swimming experiments. The open field experiment revealed that inhibition of SIRT1 significantly reduced the total distance and average speed of mouse movement compared to the RSV treatment group (p < 0.01; Figure 8B–D). The same phenomenon was detected after inhibition of NRF2 compared to the RSV treatment group (p < 0.001). Furthermore, the pole climbing time was notably increased in the inhibitor‐treated groups compared to the RSV‐treated mice (p < 0.05; Figure 8E,F). Similarly, in the forced swimming test, swim scores were significantly higher in the SIRT1 and NRF2 inhibitor groups than in the RSV‐treated group (p < 0.05, p < 0.05; Figure 8G). These results suggest that blocking SIRT1 and NRF2 effectively eliminated RSV's beneficial effects on MPTP‐induced motor deficits.

To further explore whether the effect of RSV on ferroptosis is mediated by the SIRT1/NRF2 pathway, key biomarkers involved in ferroptosis were assessed. The results demonstrated that inhibition of SIRT1 or NRF2 blocked the increase in GSH levels induced by RSV in PD mice (p < 0.05, p < 0.05; Figure 8H). The inhibitory effects of EX527 and ML385 on RSV were also evident in the decreased levels of MDA and total iron (p < 0.001, p < 0.001; Figure 8I,J). Moreover, SIRT1 and NRF2 inhibitors blocked the decrease in 4‐HNE levels induced by RSV in PD mice (p < 0.05, p < 0.05; Figure S8). Additionally, the expression of key proteins associated with the SIRT1–NRF2 pathway was examined. The results showed that EX527 treatment led to decreased expression of SIRT1, NRF2, FTH1, and GPX4 compared to the RSV group. In contrast, ML385 treatment resulted in reduced levels of NRF2, FTH1, and GPX4, but had no significant effect on SIRT1 expression compared to the RSV group (Figure 8K–O and Figure S9).