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Brain-targeted ursolic acid nanoparticles for anti-ferroptosis therapy in subarachnoid hemorrhage

Journal of Nanobiotechnology volume 22, Article number: 641 (2024) Cite this article

Abstract

Background

Subarachnoid hemorrhage (SAH) is a life -threatening cerebrovascular disease, where early brain injury (EBI) stands as a primary contributor to mortality and unfavorable patient outcomes. Neuronal ferroptosis emerges as a key pathological mechanism underlying EBI in SAH. Targeting ferroptosis for therapeutic intervention in SAH holds significant promise as a treatment strategy.

Methods

SAH model was induced via intravascular puncture and quantitatively assessed the presence of neuronal ferroptosis in the early phase of SAH using FJC staining, Prussian blue staining, as well as malondialdehyde (MDA) and glutathione (GSH) measurements. Hyaluronic acid-coated ursolic acid nanoparticles (HA-PEG-UA NPs) were prepared using the solvent evaporation method. We investigated the in vivo distribution of HA-PEG-UA NPs in SAH model through IVIS and fluorescence observation, and examined their impact on short-term neurological function and cortical neurological injury. Finally, we assessed the effect of UA on the Nrf-2/SLC7A11/GPX4 axis via Western Blot analysis.

Results

We successfully developed self-assembled UA NPs with hyaluronic acid to target the increased CD44 expression in the SAH-afflicted brain. The resulting HA-PEG-UA NPs facilitated delivery and enrichment of UA within the SAH-affected region. The targeted delivery of UA to the SAH region can effectively inhibit neuronal ferroptosis, improve neurological deficits, and prognosis in mice. Its mechanism of action is associated with the activation of the Nrf-2/SLC7A11/GPX4 signaling pathway.

Conclusions

Brain-targeted HA-PEG-UA NPs was successfully developed and hold the potential to enhance SAH prognosis by limiting neuronal ferroptosis via modulation of the Nrf-2/SLC7A11/GPX4 signal.

Graphical Abstract

Introduction

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease, contributing to 5% of all strokes [1]. Within three months of onset, approximately 35% of SAH patients die, and over 50% of survivors experience neurological impairments [2]. Despite substantial advancements in SAH treatment, patient prognoses remain unsatisfactory.

Early brain injury (EBI) refers to secondary brain damage occurring within 72 h of SAH onset, widely acknowledged as the primary contributor to both mortality and poor patient prognosis. However, the precise underlying pathological mechanisms of EBI remain elusive [3]. Ferroptosis is an iron-dependent programmed cell death triggered by the accumulation of lipid peroxides (LPO), resulting in the eventual disruption of the plasma membrane [4]. Recent research indicates that ferroptosis may play a vital role in the pathological process following SAH, making it one of the important factors exacerbating EBI after SAH [5]. The processes such as hypoxia, mitochondrial dysfunction, hemoglobin autooxidation, and iron dependent Fenton reaction after SAH result in the excessive production of reactive oxygen species (ROS). When antioxidant defense is exceeded, this leads to oxidation of unsaturated fatty acids in the cell membrane, forming lipid peroxides, ultimately causing ferroptosis and irreversible brain damage [6, 7]. The SLC7A11-GPX4 axis plays a crucial role in the cellular defense against ferroptosis. Glutathione peroxidase 4 (GPX4) is vital for resisting lipid peroxidation, reducing lipid peroxides (LPO) in the plasma membrane to non-toxic fatty alcohols. GPX4 functionality depends on the availability of reduced glutathione (GSH) [8, 9]. Intracellular GSH synthesis is regulated by the a cystine/glutamate antiporter system (system xc-), with SLC7A11 being its primary subunit. SLC7A11 facilitates GSH synthesis by exchanging extracellular glutamic acid for cystine [10, 11].

Nrf2 is a principal transcription factor intracellularly responsible for maintaining redox homeostasis and countering oxidative stress responses [12, 13]. Concurrently, Nrf2 has been shown to exert anti-inflammatory and antioxidant effects by activating the expression of target genes such as antioxidant enzymes and phase II detoxification enzymes, positioning it as a crucial regulator of lipid peroxidation and ferroptosis [14]. Recent studies have demonstrated that in cerebral ischemia injuries, targeted activation of Nrf2 and its downstream SLC7A11 can inhibit neuronal ferroptosis, offering neuroprotection [15, 16]. In murine SAH models, the activation of Nrf2 has been found to suppress oxidative stress and ameliorate early brain injury (EBI) following SAH [17]. Nrf2 is negatively regulated by Keap1 in the cytoplasm. Keap1, functioning as an adaptor for the Cul3-dependent E3 ubiquitin ligase complex (Keap1-Cul3-Rbx1), regulates Nrf2. Keap1 consists of five functional domains: NTR, BTB, IVR, DGR, and CTR, the latter two collectively known as the DC domain [18]. Nrf2 is composed of six functional domains (Neh1 ~ Neh6), among which the ETGE sequence in the Neh2 domain binds to the DC domain of Keap1, mediating the Keap1-associated ubiquitination degradation of Nrf2 [19].

Triterpenoids represent a substantial category of plant secondary metabolites, exhibiting antitumor, anti-inflammatory, and antioxidant biological activities, and hold significant potential for pharmaceutical development [20]. Dinkova-Kostova and colleagues engineered a series of derivatives using the pentacyclic triterpenoid oleanolic acid as a pioneer compound, disclosing Bardoxolone as the most efficacious Nrf2 activator known to date [21]. Ursolic acid (UA) belongs to the pentacyclic triterpenoid compound family, which is widely distributed in various plants, offering potent antioxidant and anti-inflammatory effects [22, 23]. Zhang et al. reported that UA mediates an anti-inflammatory response in a murine model of multiple sclerosis, mitigating neuronal damage [24]. In scenarios of cerebral ischemia and SAH, UA has also been shown to confer neuroprotection by suppressing oxidative stress [25, 26]. Moreover, prior studies have elucidated that UA can modulate the activity of diverse transcription factors, including Nrf2, NF-κB, and Skn-1, with Nrf2 being primarily associated with UA's antioxidant function [27,28,29]. In this study, we found that UA can significantly enhance the expression of Nrf2 in the context of SAH.

However, akin to most natural bioactive molecules, UA faces challenges such as low water solubility, limited bioavailability, reduced tissue targeting, and restricted blood–brain barrier (BBB) penetration, significantly constraining it in vivo applications [30, 31]. In previous studies, we prepared self-assembled ursolic acid nanoparticles (UA NPs) and explored their application in glioma [32]. We found that it could cross the BBB and reach brain tissue.

In this study, based on the microenvironment characteristics of SAH, we developed hyaluronic acid -coated UA nanoparticles (HA-PEG-UA NPs) to further improve the brain delivery efficiency and in vivo therapeutic effect of UA nanoparticles. We further investigated the impact of UA on neuronal ferroptosis in SAH and elucidated the mechanism by which it regulates ferroptosis through the modulation of Nrf2.

Methods

Materials

Ursolic acid was purchased from Nanjing Spring and Autumn Biological Products Co., Ltd [purity (98%), China]. Coumarin 6 and Rhodamine B200 was purchased from Shanghai yuanye Bio-Technology Co., Ltd. PEG-DSPE (2 k) and HA-PEG-DSPE(2k) was purchased from Qiyue Technology Co., Ltd. ML385 and Cycloheximide (Chx) was purchased from MedChemExpress Co., Ltd. The antibodies included the following: anti-NeuN [26975-1-AP, Proteintech, China], anti-SLC7A11/xcT [GB115276-100, Servicebio, China], anti- Glutathione Peroxidase 4 [GB113091, Servicebio, China], anti-Nrf-2 [12721S, Cell Signaling Technology (CST), USA], anti-Keap1 [8047S, CST, USA] and anti-beta Actin [GB15001, Servicebio, China].

Cell culture

The mouse hippocampal (HT22) cell line originally from ATCC were cultured at 37 °C and 5% CO2, in high-glucose DMEM Medium [Gibco, USA] supplemented with 10% fetal bovine serum [Gibco, USA], 100 U/mL penicillin, and 100 μg/mL streptomycin. We added 30 μM hemin to HT22 cells to simulate the biological process of neuronal cells being stimulated by blood after SAH. In the cell culture, 5 μM of ML385 and 1 μM of Chx were added to evaluate the impact of UA on the ferroptosis pathway.

CCK-8

According to the protocol [C0037,Beyotime Ltd., China], the Cell Counting Kit-8 (CCK8) assay was utilized to evaluate the impact of UA on the proliferation of HT22 cells. The HT22 cells were seeded into a 96-well plate at a density of 10,000 cells per well and cultured for 24 h. After the cells had adhered, the culture medium was replaced with DMEM containing UA at concentrations of 0, 1, 4, 6, 8, 10, 12, and 16 μg/ml. Following a 24-h treatment, 10 μL of CCK8 solution was added to each well and incubated at 37 °C for 1 h. The absorbance was then measured at 450 nm using a microplate reader to assess cell viability.

Nuclear protein separation

The cell pellet was obtained and 10 μl of cytoplasmic protein extraction reagent B were added. After mixing, the mixture was centrifuged at 4 °C and 16,000 g for 5 min. The supernatant was completely removed, and 50 µl of nuclear protein extraction reagent, supplemented with PMSF, were added. The mixture was then vigorously vortexed at maximum speed, followed by centrifugation at 4 °C and 16,000 g for 10 min. The supernatant was immediately transferred to a pre-chilled plastic tube, which contained the extracted nuclear proteins [P0028, Beyotime Ltd., China].

Western blot

The HT22 cells or brain tissues were lysed on ice for approximately 30 min using a modified RIPA buffer [G2002, Servicebio, China). The lysates were then centrifuged at 12,000 rpm for 15 min. The protein concentration of the samples was measured using the BCA protein assay method [Thermo Fisher, USA]. Subsequently, the lysates were heated at 95 °C for 5 min and mixed with 5X SDS PAGE loading buffer [G2075, Servicebio, China). Equal amounts of protein were loaded onto an 8–12% SDS-PAGE gel and separated. The proteins were then transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk for 1 h, followed by incubation with primary and secondary antibodies for 1 h each at 4 °C.

SAH model

The SAH model is established through perforation in the vascular. As reported [33], anesthesia C57BL/6 J mice are exposed to the common carotid artery and its branch. Then, we ligate and cut open the external carotid artery, insert a sharp 4–0 nylon suture from the external carotid artery, walk along the internal carotid artery into the brain, enter the bifurcation of the anterior and middle cerebral arteries, and continue to puncture for 1 mm until the artery is punctured. The mice in the sham group accepted the same procedure, except for removing the stitching without a puncture.

CBF

To monitor the changes in cerebral blood flow (CBF) before and after SAH, mice were anesthetized and transferred to a stereotaxic frame. Following a midline incision to expose the skull, the mice were positioned under a laser speckle contrast imaging system [RFLSI ZW, RWD Technology Co., Ltd, China) and illuminated with a 784 nm laser (60 mW) on the surface of the skull. Blood flow was recorded for 10 s. The region of interest was focused on the site of injury, and data analysis was conducted using specialized software [RWD Life Science).

SAH grade

As reported [34], the basal cistern is divided into six components, and each part is scored according to the amount of bleeding, with a maximum of 3 points and a minimum of 0 points. Finally, the score is the sum of the scores of the six parts. Mice with mild SAH (SAH grading score < 8) were excluded from the current study.

Experimental design

As shown in Fig. 1, mice were randomly allocated to four separate experiments. Mice that died within 24 h or those with mild SAH (SAH grading score < 8) were excluded from the current study.

Fig. 1
figure 1

Experimental designs of the study

Full size image

Experiment 1

To determine the ferroptosis during EBI in SAH, 31 mice were assigned to 2 groups: sham and SAH. TUNEL, FJC, Nissl, Prussian blue staining, GSH, MDA, LPO were assessed 24 h after SAH (n >  = 3). And Western blotting was used to detect the expression of proteins related to the ferroptosis pathway in the ipsilateral temporal cortex of mice at 1, 3, and 7 days (n = 3).

Experiment 2

To evaluate the effect of HA-PEG-UA NPs on short-term neurological function and Ferroptosis, 50 mice were assigned to 6 groups: sham (PBS, iv.), SAH (PBS, iv.), SAH + UA (0.1 mg/g dissolved in PEG400, iv.), SAH + UA NPs (0.1 mg/g dissolved in PBS, iv.), SAH + PEG-UA NPs (0.1 mg/g calculated by UA, iv.) and SAH + HA-PEG-UA NPs (0.1 mg/g calculated by UA, iv.). 24 h following SAH, SAH grade, modified Garcia, and Neurological score tests were evaluated 24 h after SAH. Western blotting was performed on samples from the ipsilateral temporal cortex (n = 3). Levels of GSH, MDA, LPO, and iron were also assessed 24 h after SAH (n >  = 7).

Experiment 3

To evaluate the impact of Nrf2 on short-term neurological deficits and ferroptosis induced by HA-PEG-UA NPs, 31 mice were assigned to 4 groups: SAH (PBS, iv.), SAH + ML385 (30 μg/g icv.), SAH + HA-PEG-UA NPs (0.1 mg/g calculated by UA, iv.) and SAH + ML385 + HA-PEG-UA NPs (30 μg/g ML385 icv., 0.1 mg/g HA-PEG-UA NPs calculated by UA, iv.). 24 h following SAH, Western blotting was performed on samples from the ipsilateral temporal cortex (n = 3). Levels of GSH, MDA, LPO, and iron were also assessed 24 h post-SAH (n >  = 7).

Experiment 4

To evaluate the effect of HA-PEG-UA NPs on short-term neurological function and Ferroptosis, 42 mice were assigned to 6 groups: sham (PBS, iv.), SAH (PBS, iv.), SAH + UA (0.1 mg/g dissolved in PEG400, iv.), SAH + UA NPs (0.1 mg/g dissolved in PBS, iv.), SAH + PEG-UA NPs (0.1 mg/g calculated by UA, iv.) and SAH + HA-PEG-UA NPs (0.1 mg/g calculated by UA, iv.). We administered different formulations at a dose of 0.1 mg/g thrice weekly after successful model induction. At 28 days post-induction, we conducted Rotarod tests with starting speeds of 5 rpm or 10 rpm. Additionally, we performed the Morris Water Maze test 21 days post-SAH.

Nissl staining

After dewaxing the tissue section, Nissl staining solution [G1036, Servicebio, China] was applied for 5 min. The section was then rinsed with distilled water until the effluent ran clear. Differentiation of the section was carried out using 0.1% glacial acetic acid. The brain tissue was differentiated until the Nissl bodies appeared deep blue, with the background being light blue or colorless. Finally, excess moisture on the slide was decanted, and the section was mounted with neutral resin.

TUNEL staining

After dehydration, the tissue section was immersed in the TUNEL reaction buffer and incubated at 37 °C for one hour. Subsequently, it was incubated with 1 × DAPI for 30 min. Finally, the section was mounted with an anti-fade mounting medium [12156792910, Roche, Switzerland]. The section was observed under a confocal microscope, where apoptotic neurons appeared red.

FJC staining

The prepared tissue sections were incubated overnight in an anti-NeuN antibody solution at 4 °C. The sections were then rinsed with PBS for 3*5 min, before being transferred to a secondary antibody labeled with Alexa Fluor 594 for incubation at room temperature for 2 h. Following another series of rinses with PBS for 3*5 min, the sections were covered on a slide heater at 50 °C for 30 min until completely dry. They were then incubated with potassium permanganate for 10 min and further incubated with DAPI and FJC staining solution [TR-100-FJ, Biosensis, Australia] for another 10 min. The sections were observed under a confocal microscope, where degenerated neurons appeared green.

Prussian blue staining

After dewaxing the tissue section, the sample was incubated with Prussian blue staining solution for one hour, followed by rinsing with tap water to remove the staining solution [G1029, Servicebio, China]. The cell nuclei were then stained with hematoxylin staining solution.

MDA

The tissue sample was evenly lysed and centrifuged at 10,000 g for 10–15 min. The supernatant was collected and thoroughly mixed with the MDA detection working solution. The mixture was then incubated at 95 °C for 40 min [G4300, Servicebio, China]. Following the incubation, the mixture was immediately placed on ice for 5 min. After the ice bath, the mixture was centrifuged again at 10,000 g for 10 min. A volume of 200 μL of the supernatant was transferred to a transparent 96-well plate, and the absorbance at 532 nm was measured using a microplate reader (Synergy H1, BioTek, USA) to determine the MDA content in the sample.

GSH

The supernatant was collected and mixed thoroughly with the protein removal reagent at a 1:1 ratio. After mixing well, it was centrifuged again at 10,000 g for 10–15 min at 4 °C. The supernatant was then mixed with the GSH detection probe working solution and incubated at room temperature for 5 min [G4305, Servicebio, China]. The absorbance at 412 nm was measured using a microplate reader (Synergy H1, BioTek, USA) to determine the GSH content in the sample.

LPO

The tissue sample was evenly lysed and centrifuged at 3000 g for 10 min. The supernatant was collected and mixed with the LPO working solution and the chromogenic reagent [A106-1, Nanjing Jiancheng Bioengineering Institute, China]. The mixture was incubated at 45 °C for 60 min. After incubation, it was centrifuged again at 4000 g for 10 min. A volume of 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance value of each well was measured at a wavelength of 586 nm using a microplate reader (Synergy H1, BioTek, USA) to determine the LPO content in the sample.

Measurement of iron level

The tissue sample was evenly lysed, and 0.2 mL of Reagent 1 (buffer solution) was added. After mixing thoroughly, it was placed on an ice box to lyse for 10 min. Then, it was centrifuged at 15,000 ×g for 10 min. A volume of 80 μL of the supernatant was taken and mixed with 80 μL of Reagent 2 (chromogenic reagent). The mixture was incubated at 37 °C for 40 min. The absorbance values of the wells were measured at 593 nm using a microplate reader (Synergy H1, BioTek, USA) to determine the iron content in the sample [E-BC-K880-M, Elabscience Biotechnology Co.,Ltd, China].

Synthesis of HA-PEG-UA NPs

This study used solvent evaporation method to prepare HA-PEG-DSPE. Specifically, ursolic acid and HA-PEG-DSPE were dissolved together in 1 ml of ethyl acetate and added dropwise to 3 ml of shaken 2.5% polyvinyl alcohol (PVA) solution. The obtained lotion was ultrasonically treated on ice, poured into 30 ml 0.3% PVA aqueous solution, and stirred overnight. Collect the liquid and centrifuge at 18,000 rpm for 30 min to enrich the product.

Scanning electron microscopy (SEM)

Gold plating was carried out on the samples under vacuum and argon atmosphere with sputtering current of 20 mA for 120 s. (auto fine coater JFC 1600, JEOL Ltd., Japan). SEM imaging was performed with Zeiss SIGMA field emission scanning electron microscope (Zeiss SIGMA, Carl Zeiss AG, UK).

Dynamic light scattering (DLS).

The hydration diameter and zeta potential were measured by Dynamic Light Scatterer (Zetasizer Nano ZSP, Malvern instruments Ltd., UK).

High performance liquid chromatography (HPLC)

The drug loading and encapsulation efficiency of HA-PEG-UA NPs was carried out with an Agilent 1100 (Agilent Technologies, Santa Clara, USA) system. The mobile phase was acetonitrile, ammonium acetate and methanol (67:21:12), the detection wavelength was 210 nm, and the injection volume was 10 μl.

Hemolysis test

10 ml of blood was drawn from the ear vein of a New Zealand rabbit and washed to remove fibrin, leukocytes, and platelets. The washed red blood cells were then suspended in saline to create a 2% cell suspension. Each group received 1 ml of the 2% red blood cell suspension and 1 mg of the drug to be tested. The negative control group was treated with 10μl of PBS, while the positive control group received 10 μl of triton-100. The mixtures were incubated at a constant temperature of 37 °C for one hour, followed by centrifugation at 13000 g for 15 min. Then, 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance value of each well was measured at a wavelength of 545 nm using a microplate reader (Synergy H1, BioTek, USA) to determine the hemolysis rate of the drug.

Fluorescent imaging

Tetraethyl Rhodamine B200 (RB200) is a fluorescent dye used for in vivo imaging. After successful SAH modeling, HA-PEG-UA NPs loaded with RB200 were injected through the tail vein. The mice were euthanized to separate the brain and other organs, and imaged using the IVIS Lumina XRMS Series III imaging system. Quantify the fluorescence intensity in each brain using Living Image 3.0 (Xenogen, USA). After fixing the brain tissue with paraformaldehyde and making frozen sections, observe the distribution of fluorescent particles in the brain.

After euthanizing the mice, the brain was carefully extracted. The wet weight of each brain component was measured. Next, the brain specimen was subjected to drying at a temperature of 110℃ for a duration of 72 h. Once the drying process was complete, the brain specimen was weighed again to obtain its dry weight.

Modified Garcia score

The short-term results were assessed using the Modified Garcia Score (a 3–18 points scoring system) [35]. This score consists of six components: spontaneous activity, spontaneous limb movement, forelimb extension, tactile sensation, body proprioception, and climbing ability. The higher the score, the better the neurological function. A double-blind design was adopted for this scoring to ensure fairness and objectivity in the evaluation process. Data collection and analysis were performed by a third party unaware of the mice’s grouping information.

Neurological function assessment

24 h after modeling, the neurological function assessments were conducted. 0 points: No significant neurological deficits. 1 point: Slight neurological impairment, such as minor curling of the forelimbs or hind limbs when the mouse is subjected to the tail suspension test. 2 points: Moderate neurological impairment, such as the mouse leaning to one side while walking. 3 points: Significant neurological impairment, such as tilting to one side. 4 points: Severe neurological impairment, such as an inability to walk autonomously. 5 points: Extremely severe neurological impairment, such as a complete loss of consciousness. 6 points: Died. The scoring was conducted using a single-blind design, meaning that the evaluator was not informed of the experimental group assignment of the mice. The mice were anonymized through random assignment of unique identifiers.

Rotarod test

Two hours before data collection, the mice were trained to adapt to the rotating rod. Mice were placed on a Treadmill (Media Associates, St Albans, VT). First, the initial Rotarod speed was set to 5 rpm or 10 rpm and the acceleration to 2 revolutions per 5 s. The mice were placed in the roller, and their fall delay was observed. Three experiments were conducted at intervals of no less than 5 min to evaluate the average latency to fall.

Morris water maze

At the beginning of the training, put the platform at the first quadrant, and put the mouse at any point from the four starting points of the pond wall to the pool to the pool. Record the time and swimming path of the mouse to find the platform. Four training will be placed in the water from four different starting points (different quadrants), respectively. After the mice find the platform or the platform cannot be found within 60 s, the experimenters will take it to the platform and rest 15 s on the platform for the next test. Taking the average period of mouse training for 4 times a day as the academic performance of the mouse on the day. On the seventh day, remove the platform and place mice in any position, recording their exploration activity in various quadrants.

Power calculations

This study estimated the sample size required to compare the sample mean with the population mean, where the population mean belonged to the SAH group, and the sample group was the SAH + HA-PEG-UA NPs group. The outcome measure observed was the modified Garcia score. Based on preliminary experimental results, the average modified Garcia score for the SAH group was 10.6 ± 1.8 points, and it was anticipated that the score for the SAH + HA-PEG-UA NPs group would increase to an average of 13.2 ± 1.5 points. Assuming a two-sided α = 0.05 and β = 0.1, PASS 15 software calculated that the required sample size for the treatment group was 8 subjects. Considering mortality and model success rates, the sample size for the treatment group in this experiment was therefore set at 12–13 individuals.

Statistical analysis

All cell and animal experiments were conducted at least three times, with data represented as mean ± standard deviation (SD). The significance between two groups was evaluated using a two-tailed unpaired Student's t-test with SPSS software version 24. Paired-Samples t test was used to evaluated the significance of size stability of nanoparticles (Fig. 4G). A one-way analysis of variance (ANOVA) was used for comparison among multiple groups, followed by a Tukey post hoc test. The Shapiro–Wilk test was employed to assess normality, and the Mann–Whitney U test was used to evaluate data with non-normal distributions. All statistical graphs were created using GraphPad Prism 8.0.

Result and discussion

Ferroptosis is a significant hallmark within the EBI process of SAH

As illustrated in Fig. 2A, SAH models in mice were generated using the intravascular puncture method as previously described [33]. During the surgical procedure, we measured cerebral blood flow, which showed a substantial decrease of over 80% in the middle cerebral artery CBF (Fig. 2B). The mice were euthanized 24 h after SAH induction. It is noteworthy that the observation was made that SAH predominantly occurred at the bifurcation of the left internal carotid artery and the middle cerebral artery, extending into the basal cisterns and the left temporal cortex, with a lesser extent to the right temporal cortex (Fig. 2C).

Fig. 2
figure 2

Ferroptosis of neurons after SAH and Temporal patterns of Nrf-2/SLC7A11 after SAH. A SAH model construction schematic diagram. B ultrasonic Doppler monitoring the changes in cerebral blood flow from the left MCA blood supply area (n = 5). C Representative images of the left blood vessels piercing the sub -absorbial cavity in the region. Scale bar = 100 μm. D Representative images of TUNEL staining in cerebral cortex 24 h after molding. Scale bar = 100 μm. E Representative images of Prussian blue stain in temporal cortex 24 h after molding. The arrows showed the iron deposition in the neuronal cell. Scale bar = 50 μm. F Representative images of FJC staining in temporal cortex 24 h after molding. Scale bar = 100 μm. G Representative TEM images of mitochondria of temo cortex neurons 24 h after molding. The arrows showed the increased membrane density and outer membrane rupture of mitochondria. Scale bar = 500 nm. H Representative western blotting images of SLC7A11 and GPX4 expression in ipsilateral temporal cortex after SAH. I, J, K Relative GSH, LPO and MDA level 24 h after molding (n = 5). **P < 0.01. ***P < 0.001

Full size image

To visualize apoptotic cells, we employed TUNEL staining in the mouse SAH model. This revealed a significant increase in TUNEL-positive cells post SAH induction (Fig. 2D and S1). Similarly, Fluoro-Jade C (FJC) staining, which is typically used to identify degenerated neurons, showed a marked rise in FJC-positive cells in brain tissue following SAH, aligning with the TUNEL staining results (Fig. 1F and S2). Iron deposition, a key hallmark of ferroptosis, was observed through Prussian blue staining, which specifically labels trivalent iron ions. This staining revealed iron deposition in the neuronal cortex after SAH (Fig. 2E and S3). Using transmission electron microscopy (TEM), we examined the mitochondria in neurons from SAH-affected tissue. Compared to normal mitochondria, those in SAH tissue showed distinct ferroptosis characteristics, including a reduced number of cristae, increased membrane density, and ruptured outer membranes (Fig. 2G).

The study further examined the protein expression of SLC7A11 and GPX4, and levels of lipid peroxidation markers in the cerebral cortex post SAH. The results indicated a significant reduction in SLC7A11 and GPX4 expression one day post modeling, followed by a gradual recovery trend after 3 days (Fig. 2H and S4). Consequently, in subsequent experiments, we selected 24 h as our experimental time point. Malondialdehyde (MDA), a key product of membrane lipid peroxidation and an indicator of cell membrane damage, significantly increased after SAH, alongside a noticeable rise in (Lipid Hydroperoxide) LPO. Concurrently, there was a sharp decline in antioxidant glutathione (GSH) levels (Fig. 2I–K).

In summary, these findings suggest that cortical neurons exposed to blood-induced stimuli during subarachnoid hemorrhage undergo ferroptosis in the early stages of brain injury. However, further research is required to understand the patterns of ferroptosis.

UA inhibits neuronal ferroptosis by reducing the degradation of Nrf2

In this study, we focused on exploring the regulatory influence of UA on the neuronal ferroptosis post-SAH. Our initial step involved assessing the potential of UA to counteract neuronal ferroptosis in vitro. We established an in vitro SAH model by co-incubating hemin with the HT22 mouse neuronal cell line [36]. Our preliminary examination involved determining the impact of UA on HT22 cell proliferation using the CCK-8 assay. The IC50 of UA was identified as 10.3 μg/ml. Interestingly, at a working concentration of 2 μg/ml, HT22 cell proliferation was not significantly affected (Figure S5).

Subsequent observations revealed that UA effectively inhibited hemin-induced neuronal apoptosis in HT22 cells (Fig. 3A, S6), reduced MDA and LPO levels, and increased GSH concentrations (Fig. 3C–E). Additionally, UA was found to upregulate Nrf-2 expression (Fig. 3B and S7). To further investigate the role of Nrf2 in the mechanism of UA against ferroptosis, we utilized the Nrf2 transcription inhibitor ML385. The results indicated that the introduction of ML385 reversed the UA-induced elevation of SLC7A11 and GPX4 levels (Fig. 3F, S8). Upon further isolation of nuclear proteins, we discovered that UA facilitated the nuclear translocation of Nrf2, which was inhibited by ML385 (Fig. 3G and S9). Inhibition of protein synthesis using Chx revealed that UA could delay Nrf-2 degradation (Fig. 3H and S10). These results indicated that UA primarily exerted its anti-neuronal ferroptotic effects by preventing the degradation of Nrf2 and enhancing Nrf2 nuclear translocation.

Fig. 3
figure 3

Effect of UA on ferroptosis of neurons in vitro. A Representative images of TUNEL staining in HT22 cells 24 h after incubated with hemin. Scale bar = 100 μm. B The expression level of Keap1 and Nrf-2 in HT22 cells after UA treatment. C–E Relative MDA, LPO and GSH level of HT22 cells 24 h after incubated with hemin. F The expression level of SLC7A11, GPX4 and Nrf-2 in HT22 cells after incubated with Nrf-2 inhibitors ML385. G The expression level of nucleus Nrf-2 in HT22 cells after incubated with Nrf-2 inhibitors ML385. H The expression level of Nrf-2 in HT22 cells after Chx treatment. I and J The molecular simulation diagram of the UA and Nrf-2 combined with the KEAP1DC region, respectively

Full size image

Considering Nrf2’s binding to Keap1 via the ETGE sequence, we hypothesized that UA might mimic the action of the ETGE sequence of Nrf2. To test this hypothesis, we utilized auto-docking software to analyze the binding interactions of Keap1 with both the ETGE sequence and UA (Fig. 3I and J). Our results showed that UA and ETGE both formed hydrogen bonds when binding to Keap1’s DC domain. Quantitatively, each UA molecule released approximately 52.22 kJ of energy upon binding to Keap1, which exceeded the 41.13 kJ released per ETGE molecule, indicating a more stable binding. However, this is only our preliminary inference, more experiments such as bicore or hydrogen–deuterium exchange mass spectrometry need to be done to further prove the binding of UA to Keap1.

Based on these observations, we preliminarily concluded that UA could competitively bind to the Keap1 protein in conjunction with Nrf2, leading to a reduction in Keap1-mediated degradation of Nrf2 and an increase in nuclear translocation levels. Subsequently, Nrf2 promoted the transcription of SLC7A11 and GPX4, ultimately exerting an anti-neuronal ferroptotic effect.

Synthesis and characterization of HA-PEG-UA NPs

UA has shown potential in activating the Keap1/Nrf-2/SLC7A11/GPX4 signaling pathway, marking it as a promising natural inhibitor of ferroptosis. However, UA faces several challenges typical of many natural bioactive compounds, including limited water solubility, low bioavailability, restricted tissue targeting, and poor penetration through the BBB. These limitations significantly hinder its in vivo application. Building on our initial research, we have successfully synthesized self-assembling UA nanoparticles (UA NPs) using UA as the foundational material.

To improve the biological effectiveness of UA NPs, we explored the addition of polyethylene glycol (PEG) modifications to the surface of these nanoparticles, resulting in PEG-modified UA NPs (PEG-UA NPs). Concurrently, our observations of increased CD44 protein expression in brain tissue post-SAH under blood stimulation (Fig. 4H and 4I) led us to develop UA NPs coated with hyaluronic acid (HA)-PEG-DSPE. This design leverages the targeting interaction between HA and CD44. Scanning electron microscopy (SEM) revealed the spherical nature of the UA NPs, PEG-UA NPs, and HA-PEG-UA NPs, showing similar morphologies but differing in particle size (Fig. 4A). Transmission electron microscopy (TEM) further confirmed the presence of substantial PEG rings on the surfaces of PEG-UA NPs and HA-PEG-UA NPs, indicating successful synthesis (Fig. 4B).

Fig. 4
figure 4

Synthesis and Characterization of HA-PEG-UA NPs. A Representative TEM image of HA-PEG-UA NPs. Scale bar: 200 nm. B Representative SEM image of HA-PEG-UA NPs. Scale bar: 50 nm. C Mean sizes of HA-PEG-UA NPs detected by DLS. D Zeta potential of HA-PEG-UA NPs. E FTIR of HA-PEG-UA NPs. F XRD of HA-PEG-UA NPs. G The stability of HA-PEG-UA NPs stored at room temperature, in a dry and dark environment. H, I Representative confocal (H) and western blotting (I) images of CD44 in SAH brain. Scale bar = 20 μm

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Dynamic light scattering (DLS) analysis showed that the particle size of the PEG-UA NPs was about 159 nm, slightly larger than the 143 nm of the UA NPs (Fig. 4C). The HA-PEG-UA NPs were slightly larger still, measuring 173 nm, likely due to the longer chain length of HA-PEG-DSPE compared to PEG-DSPE. The zeta potential of the HA-PEG-UA NPs demonstrated a significant decrease after the encapsulation of HA-PEG-DSPE (Fig. 4D). As shown in Fig. 4E, the FTIR of various nanoparticles were measured. Compared to the single substance of UA and UA NPs, HA-PEG-UA NPs exhibited an overlapping state of absorption peaks for various functional groups. This indicates that it is a mixed component, proving the successful doping of HA-PEG-DSPE. As shown in Fig. 4F, the XRD patterns of each group of nanoparticles were measured. UA displayed sharp characteristic peaks at 2θ positions such as 10.9° and 14.3°, indicating high crystallinity. Compared with free UA, there were no sharp peaks in the spectra of UA-NPs, PEG-UA NPs, and HA-PEG-UA NPs, further suggesting that UA was no longer in a crystalline phase but in an amorphous state. The XRD results confirmed that UA was successfully self-assembled into nanoparticles. The encapsulation of HA-PEG onto the surface of UA NPs was further corroborated using H1 nuclear magnetic resonance (H1 NMR) (Figure S11). To investigate the stability of HA-PEG-UA NPs after lyophilization, we stored them under room temperature in a dark and dry environment and continuously measured their particle size changes. The study found that there was no significant change in the particle size of HA-PEG-UA NPs within 48 days (Fig. 4G and S12).

Cellular uptake and biodistribution of HA-PEG-UA NPs

To assess the cellular uptake capabilities of different UA nanoparticle formulations, we incorporated fluorescent dyes coumarin 6 (C6) and rhodamine B200 (RB200) into these nanoparticles. Considering the innate absence of fluorescence in UA, any detected fluorescence signals could be confidently attributed to the dyes. We ensured uniformity in drug concentration across all experimental groups by standardizing based on fluorescence intensity, thus maintaining consistent dye content.

Our initial experiments centered on the in vitro neuronal uptake of these UA nanoparticles. We introduced C6-loaded nanoparticles into HT22 cell culture media, incubating them for 20 min before assessing uptake using fluorescence microscopy and flow cytometry (Fig. 5A-C). Intriguingly, no notable differences emerged in the cellular uptake between UA NPs, PEG-UA NPs, and HA-PEG-UA NPs, indicating that the substantial negative potential of HA-PEG-UA NPs did not impede their cellular uptake.

Fig. 5
figure 5

Cellular uptake and biodistribution of HA-PEG-UA NPs. A, B, C Confocal images and flow cytometry detection of cell uptake and mean fluorescence intensity of HT22 cells after incubation with different formulations. Scale bar = 20 μm. D Ex vivo fluorescence imaging of HA-PEG-UA NPs loaded with RB200 (red) in the harvested brains by an IVIS system. E Representative images of HA-PEG-UA NPs in the regions of SAH brain. Scale bar = 50 μm

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Expanding our focus to in vivo applications, we first examined the blood compatibility of HA-PEG-UA NPs. Hemolysis tests showed hemolysis rates for the positive and negative controls at 100% and 0%, respectively. Significantly, the hemolysis rates for UA NPs, PEG-HA NPs, and HA-PEG-UA NPs were all below 0.5%, indicating excellent blood compatibility and minimal hemolysis induction (Figure S13). We used HPLC to study the drug release pattern of HA-PEG-UA NPs, as shown in Figure S14, 53% of the UA was released at 12 h, and 71% was released at 24 h. We then tracked the blood concentration of UA over time following intravenous injection of RB200-labeled HA-PEG-UA NPs in mice. By measuring fluorescence intensity (subtracting spontaneous blood fluorescence) as a surrogate for UA concentration, we observed that PEG-UA NPs and HA-PEG-UA NPs had notably prolonged circulation times compared to standard UA NPs, largely due to the effect of PEG (Figure S15).

Further, we investigated the distribution of UA in various organs. After administering RB200-loaded HA-PEG-UA NPs intravenously in SAH mice, we conducted IVIS imaging of retrieved organs after 24 h. The imaging results revealed that free dyes primarily accumulated in the liver, with minimal BBB penetration into brain tissue. Contrastingly, UA NPs were effectively enriched in the SAH injury area, with HA-PEG-UA NPs showing even more pronounced enrichment in the SAH-affected brain tissue (Fig. 5D). It is postulated that UA NPs are capable of penetrating the brain tissue through the blood–brain barrier, which has been compromised following SAH [37]. Given that larger molecular particles are unlikely to be metabolized by brain tissue after SAH, UA NPs can also accumulate in the brain tissue to a certain extent. Furthermore, due to the targeting ability of HA towards CD44, HA-PEG-UA NPs are able to enrich more significantly in the brain tissue subsequent to SAH. Confocal microscopy of brain tissue sections confirmed a high concentration of intact fluorescent particles in the vicinity of neurons in the HA-PEG-UA NPs group, highlighting their targeted delivery capabilities to SAH tissues (Fig. 5E). To further investigate the correlation between the targeting ability of HA-PEG-UA NPs and CD44 expression, we measured the levels of CD44 expression at various time points following SAH and detected the content of HA-PEG-UA NPs in brain tissues. It was observed that the content of HA-PEG-UA NPs in brain tissues positively correlated with the levels of CD44 expression at different time points after drug administration (Figure S16). In summary, these results demonstrate that HA-PEG-UA NPs exhibit enhanced bioavailability and superior targeting efficiency to SAH-affected brain tissue compared to conventional UA NPs.

Effect of HA-PEG-UA NPs on short-term neurological function and cortical neuronal injury.

In our study, after examining the distribution of HA-PEG-UA NPs in SAH-affected mice, we next assessed their therapeutic efficacy in the same model. As shown in Experiment 2 of Fig. 1, Upon successful establishment of the SAH model in mice, we administered various formulations at a dose of 0.1 mg/g immediately post-induction. After a 24-h period, the mice were subjected to neurological deficit scoring, followed by euthanasia for brain tissue collection and SAH severity assessment. The analysis of SAH scores showed no notable differences among the treatment groups (Fig. 6A). However, using the modified Garcia score and neurological system score, we observed significant neurological dysfunction in mice 24 h post SAH induction (Fig. 6B). Treatment with unmodified UA NPs improved neurological function, and a more pronounced enhancement was seen with HA-PEG-UA NPs administration (Fig. 6C).

Fig. 6
figure 6

Effect of HA-PEG-UA NPs on short-term neurological function and cortical neuronal injury. A Quantification of SAH grade. B, C Quantification of neurological function with Modified Garcia score systems (B) and neurological score systems (C) at 24 h after molding. *P < 0.05. **P < 0.01. ***P < 0.001. D Quantification of brain water content 24 h after molding. *P < 0.05. **P < 0.01. ***P < 0.001. E Representative images of TUNEL staining in temporal cortex 24 h after molding (n = 3). *P < 0.05. **P < 0.01. ***P < 0.001. Scale bar = 50 μm. F Representative images of Nissl staining in temporal cortex 24 h after molding (n = 3). *P < 0.05. **P < 0.01. ***P < 0.001. Scale bar = 100 μm

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Brain edema, a critical outcome of ischemia and hypoxia post-SAH, serves as an independent predictor of poor prognosis. Compared to the sham group, we noted a substantial increase in brain tissue water content in the SAH group at the 24 h mark. Notably, HA-PEG-UA NP treatment significantly reduced brain tissue water content compared to treatment with UA NPs alone (Fig. 6D).

We further investigated cell damage and neuronal activity using TUNEL and Nissl staining on brain tissue samples. The results indicated that UA NP treatment decreased the proportion of TUNEL-positive cells and increased the presence of Nissl bodies compared to the SAH group (Fig. 6E, F, S17 and S18), highlighting the neuroprotective effects of UA NPs. Importantly, the enhanced efficacy of HA-PEG-UA NPs over standard UA NPs could be attributed to their increased targeting specificity to SAH-affected tissue, resulting in a higher local concentration of NPs.

Effect of HA-PEG-UA NPs on neuronal ferroptosis in vivo.

In our study, we extracted proteins from the temporal lobe cortex, specifically from areas affected by blood stimulation, for Western blot analysis. Consistent with our cellular level observations, the results revealed that UA NPs increased Nrf-2 expression in brain tissue post-SAH. This increase was accompanied by elevated protein levels of the anti-ferroptosis genes SLC7A11 and GPX4 (Fig. 7A-C). The effects were more pronounced with PEG-UA NPs and HA-PEG-UA NPs than with UA NPs alone, likely due to the enhanced bioavailability and targeted delivery afforded by the PEG modification.

Fig. 7
figure 7

Effect of HA-PEG-UA NPs on ferroptosis of neurons in vivo. A, B, C Representative western blotting images and quantitative analyses (n = 3). *P < 0.05. **P < 0.01. ***P < 0.001. D, E, F, G Relative GSH, LPO MDA and Iron level 24 h after molding (n = 8). *P < 0.05. **P < 0.01. ***P < 0.001

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Our investigation also included an analysis of changes in GSH, LPO, MDA, and trivalent iron ion concentrations. The results, obtained 24 h post-SAH, showed a significant decrease in the antioxidant GSH and notable increases in LPO, MDA, and iron ion levels (Fig. 7D–G). While treatment with UA alone did not result in significant improvements, the administration of UA NPs effectively increased GSH levels, reduced LPO and MDA accumulation, and decreased the concentration of iron ions in the tissues affected by SAH. These findings indicate that HA-PEG-UA NPs are capable of reducing lipid peroxidation, limiting the accumulation of trivalent iron ions, and mitigating neuronal ferroptosis.

ML385 inhibits Nrf2 reversed the anti-ferroptosis effect of HA-PEG-UA NPs on neurons in vivo

To further investigate the mechanism of action of HA-PEG-UA NPs in vivo, we administered the Nrf2 inhibitor ML385 via intracerebroventricular injection in SAH mice and assessed the expression of ferroptosis-related proteins. The results indicated that the introduction of ML385 reversed the elevation of SLC7A11 and GPX4 levels induced by HA-PEG-UA NPs (Figs. 8A–C). Further examination of ferroptosis-related markers revealed that the introduction of ML385 also reversed the increase in GSH, and the reduction in LPO, MDA, and iron accumulation caused by HA-PEG-UA NPs (Figs. 8D–G). These findings suggest that HA-PEG-UA NPs mitigate neuronal ferroptosis by reducing lipid peroxidation through Nrf2 and limiting the accumulation of trivalent iron.

Fig. 8
figure 8

ML385 inhibits NRF2 reversed the anti-ferroptosis effect of HA-PEG-UA NPs on neurons in vivo. A, B, C Representative western blotting images and quantitative analyses (n = 3). *P < 0.05. **P < 0.01. ***P < 0.001. D, E, F, G Relative GSH, LPO MDA and Iron level 24 h after molding (n = 7). *P < 0.05. **P < 0.01. ***P < 0.001

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Effect of HA-PEG-UA NPs on persistent neurological function and hippocampus injury

Given the robust anti-ferroptotic effects of HA-PEG-UA NPs, there was an intention to further investigate its impact on the long-term prognosis of SAH mice, aiming to holistically assess the role of HA-PEG-UA NPs in the treatment of SAH in mice. This would provide a basis for the subsequent development and clinical application of HA-PEG-UA NPs.

In our investigation into the long-term neurological effects of UA in a mouse model of subarachnoid hemorrhage (SAH), we administered different formulations at a dose of 0.1 mg/g thrice in the first week after successful model induction. At 28 days post-induction, we conducted Rotarod tests with starting speeds of 5 rpm or 10 rpm. Relative to the sham group, mice in the SAH group exhibited a significant decrease in the time they remained on the rotating rod. In contrast, treatment with UA NPs, PEG-UA NPs, and HA-PEG-UA NPs notably extended this latency, effectively mitigating the deficits induced by SAH (Fig. 9A and B).

Fig. 9
figure 9

HA-PEG-UA NPs Reduce neuronal damage. A, B Fall latency of mice in the Rotarod test at initial speeds of 5 rpm (A) and 10 rpm(B) (C, D, E, F) Swimming distance, platform crossover times, probe quadrant duration, and escape latency of Morris water maze (n = 7). *P < 0.05. **P < 0.01. ***P < 0.001. G Representative swimming trajectories. H Representative images showing Nissl staining of CA1 CA3 and DG regions. Scale bar = 100 μm

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Additionally, As shown in Experiment 4 of Fig. 1, we performed the Morris Water Maze (MWM) test 21 days post-SAH. Initial results showed no significant differences in swimming distance and escape latency on the first day among all groups, ensuring baseline uniformity in swimming skills (Fig. 9C and F). However, from the second to the sixth day, compared to the sham group, the SAH group demonstrated increased escape latency and swimming distance, along with fewer platform crossings (Fig. 9D, S19, S20), as depicted in their distinctive swimming paths (Fig. 9G). When the underwater platform was removed, the SAH group showed limited exploration in the quadrant where the platform had been, indicating severe learning and memory impairments likely due to hippocampal neuronal damage (Fig. 9 E). Treatment with UA NPs notably improved these parameters from days 3 to 5, exhibiting reduced escape latency, decreased swimming distances, increased platform crossings, and more time spent in the target quadrant. These results highlight the therapeutic potential of UA NPs in alleviating persistent hippocampal damage resulting from SAH. Notably, HA-PEG-UA NPs demonstrated even greater efficacy than UA NPs alone.

Subsequent Nissl staining of brain tissue revealed a significant reduction in intact neurons within the DG, CA1, and CA3 regions in the SAH group compared to the sham group. While UA treatment did not fully restore neuronal counts to normal levels, HA-PEG-UA NP treatment led to a substantial increase in viable neuron counts (Fig. 9H). These findings suggest that HA-PEG-UA NPs can effectively mitigate long-term neurological dysfunction by reducing oxidative stress and minimizing hippocampal neuronal damage post-SAH.

Conclusion

In summary, our findings highlight the pronounced emergence of neuronal ferroptosis during the initial phases of brain injury following subarachnoid hemorrhage. By modifying the surface of UA NPs with HA, we successfully synthesized brain-targeted UA NPs. The resulting HA-PEG-UA NPs exhibit an impressive capacity to selectively target the CD44-enriched region affected by SAH. Most notably, brain-targeted UA NPs hold the potential to enhance SAH prognosis by limiting neuronal ferroptosis via modulation of the Nrf-2/SLC7A11/GPX4 signal.

Availability of data and materials

No datasets were generated or analysed during the current study.

References

  1. Macdonald RL, Schweizer TA. Spontaneous subarachnoid haemorrhage. Lancet. 2017;389(10069):655–66.

    Article  PubMed  Google Scholar 

  2. Mowry EM, Ontaneda D, Evangelou N, Newsome SD. Pragmatic clinical trials for treating relapsing multiple sclerosis. Lancet Neurol. 2019;18(12):1075.

    Article  PubMed  Google Scholar 

  3. Sehba FA, Hou J, Pluta RM, Zhang JH. The importance of early brain injury after subarachnoid hemorrhage. Prog Neurobiol. 2012;97(1):14–37.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol. 2021;18(5):280–96.

    Article  CAS  PubMed  Google Scholar 

  5. Hu X, Zhang H, Zhang Q, Yao X, Ni W, Zhou K. Emerging role of STING signalling in CNS injury: inflammation, autophagy, necroptosis, ferroptosis and pyroptosis. J Neuroinflammation. 2022;19(1):242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shuili Jing, Ye Liu, Zhifei Ye, Abdullkhaleg Ali Ghaleb Al-bashari, Heng Zhou and Yan He.  Ferrostatin-1 loaded Gelatin methacrylate scaffold promotes recovery from spinal cord injury via inhibiting apoptosis and ferroptosis. Nano TransMed.2023.100005.

    Google Scholar 

  7. Zhou H, Li Z, Jing S, Wang B, Ye Z, Xiong W, Liu Y, Liu Y, Xu C, Kumeria T, He Y, Ye Q. Repair spinal cord injury with a versatile anti-oxidant and neural regenerative nanoplatform. J Nanobiotechnology. 2024;22(1):351.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, Fulda S, Gascón S, Hatzios SK, Kagan VE, Noel K, Jiang X, Linkermann A, Murphy ME, Overholtzer M, Oyagi A, Pagnussat GC, Park J, Ran Q, Rosenfeld CS, Salnikow K, Tang D, Torti FM, Torti SV, Toyokuni S, Woerpel KA, Zhang DD. Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease. Cell. 2017;171(2):273–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lee J, Roh JL. SLC7A11 as a gateway of metabolic perturbation and ferroptosis vulnerability in cancer. Antioxidants. 2022;11(12):2444.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ghoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD, Corey E, Paulmurugan R, Stoyanova T. Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer. Cancer Res. 2021;81(6):1583–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Koppula P, Zhuang L, Gan B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell. 2021;12(8):599–620.

    Article  CAS  PubMed  Google Scholar 

  13. Cuadrado A, Rojo AI, Wells G, Hayes JD, Cousin SP, Rumsey WL, Attucks OC, Franklin S, Levonen AL, Kensler TW, Dinkova-Kostova AT. Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat Rev Drug Discov. 2019;18(4):295–317.

    Article  CAS  PubMed  Google Scholar 

  14. Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol. 2019;23: 101107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jiang L, Kon N, Li T, Wang SJ, Su T, Hibshoosh H, Baer R, Gu W. Ferroptosis as a p53-mediated activity during tumour suppression. Nature. 2015;520(7545):57–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wang L, Liu Y, Du T, Yang H, Lei L, Guo M, Ding HF, Zhang J, Wang H, Chen X, Yan C. ATF3 promotes erastin-induced ferroptosis by suppressing system Xc. Cell Death Differ. 2020;27(2):662–75.

    Article  CAS  PubMed  Google Scholar 

  17. Zhang T, Wu P, Budbazar E, Zhu Q, Sun C, Mo J, Peng J, Gospodarev V, Tang J, Shi H, Zhang JH. Mitophagy reduces oxidative stress via Keap1 (Kelch-Like epichlorohydrin-associated protein 1)/Nrf2 (nuclear factor-E2-related factor 2)/PHB2 (prohibitin 2) pathway after subarachnoid hemorrhage in rats. Stroke. 2019;50(4):978–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou YS, Ueno I, Sakamoto A, Tong KI, Kim M, Nishito Y, Iemura S, Natsume T, Ueno T, Kominami E, Motohashi H, Tanaka K, Yamamoto M. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat Cell Biol. 2010;12(3):213–23.

    Article  CAS  PubMed  Google Scholar 

  19. Yamamoto M, Kensler TW, Motohashi H. The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis. Physiol Rev. 2018;98(3):1169–203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Si L, Meng K, Tian Z, Sun J, Li H, Zhang Z, Soloveva V, Li H, Fu G, Xia Q, Xiao S, Zhang L, Zhou D. Triterpenoids manipulate a broad range of virus-host fusion via wrapping the HR2 domain prevalent in viral envelopes. Sci Adv. 2018;4(11):8408.

    Article  Google Scholar 

  21. Dinkova-Kostova AT, Liby KT, Stephenson KK, Holtzclaw WD, Gao X, Suh N, Williams C, Risingsong R, Honda T, Gribble GW, Sporn MB, Talalay P. Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress. Proc Natl Acad Sci USA. 2005;102(12):4584–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ikeda Y, Murakami A, Ohigashi H. Ursolic acid: an anti- and pro-inflammatory triterpenoid. Mol Nutr Food Res. 2008;52(1):26–42.

    Article  CAS  PubMed  Google Scholar 

  23. Gautam R, Jachak SM. Recent developments in anti-inflammatory natural products. Med Res Rev. 2009;29(5):767–820.

    Article  CAS  PubMed  Google Scholar 

  24. Zhang Y, Li X, Ciric B, Curtis MT, Chen WJ, Rostami A, Zhang GX. A dual effect of ursolic acid to the treatment of multiple sclerosis through both immunomodulation and direct remyelination. Proc Natl Acad Sci USA. 2020;117(16):9082–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhang T, Su J, Guo B, Zhu T, Wang K, Li X. Ursolic acid alleviates early brain injury after experimental subarachnoid hemorrhage by suppressing TLR4-mediated inflammatory pathway. Int Immunopharmacol. 2014;23(2):585–91.

    Article  PubMed  Google Scholar 

  26. Wang Y, Li L, Deng S, Liu F, He Z. Ursolic acid ameliorates inflammation in cerebral ischemia and reperfusion injury possibly via high mobility group box 1/toll-like receptor 4/NFκB pathway. Front Neurol. 2018;18(9):253.

    Article  Google Scholar 

  27. Bhattacharjee S, Dashwood RH. Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals. Antioxidants. 2020;9(9):865.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lei P, Li Z, Hua Q, Song P, Gao L, Zhou L, Cai Q. Ursolic acid alleviates neuroinflammation after intracerebral hemorrhage by mediating microglial pyroptosis via the NF-κB/NLRP3/GSDMD pathway. Int J Mol Sci. 2023;24(19):14771.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Naß J, Abdelfatah S, Efferth T. The triterpenoid ursolic acid ameliorates stress in Caenorhabditis elegans by affecting the depression-associated genes skn-1 and prdx2. Phytomedicine. 2021;15(88): 153598.

    Article  Google Scholar 

  30. Kashyap D, Tuli HS, Sharma AK. Ursolic acid (UA): a metabolite with promising therapeutic potential. Life Sci. 2016;146:201–13.

    Article  CAS  PubMed  Google Scholar 

  31. Mlala S, Oyedeji AO, Gondwe M, Oyedeji OO. Ursolic acid and its derivatives as bioactive agents. Molecules. 2019;24(15):2751.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Li Y, Zhao L, Zhao Q, Zhou Y, Zhou L, Song P, et al. Ursolic acid nanoparticles for glioblastoma therapy. Nanomedicine. 2023;50: 102684.

    Article  CAS  PubMed  Google Scholar 

  33. Tian Q, Guo Y, Feng S, Liu C, He P, Wang J, et al. Inhibition of CCR2 attenuates neuroinflammation and neuronal apoptosis after subarachnoid hemorrhage through the PI3K/Akt pathway. J Neuroinflammation. 2022;19(1):312.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sugawara T, Ayer R, Jadhav V, Zhang JH. A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model. J Neurosci Methods. 2008;167(2):327–34.

    Article  PubMed  Google Scholar 

  35. Garcia JH, Wagner S, Liu KF, Hu XJ. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats statistical validation. Stroke. 1995;26(4):627–34.

    Article  CAS  PubMed  Google Scholar 

  36. Liu C, Yao K, Tian Q, Guo Y, Wang G, He P, Wang J, Wang J, Zhang Z, Li M. CXCR4-BTK axis mediate pyroptosis and lipid peroxidation in early brain injury after subarachnoid hemorrhage via NLRP3 inflammasome and NF-κB pathway. Redox Biol. 2023;68: 102960.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Solár P, Zamani A, Lakatosová K, Joukal M. The blood-brain barrier and the neurovascular unit in subarachnoid hemorrhage: molecular events and potential treatments. Fluids Barriers CNS. 2022;19(1):29.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank the personnel in the department of neurosurgery and the central laboratory at the Renmin Hospital of Wuhan University.

Funding

This work was supported by the National Natural Science Foundation of China (No.82001311), the Knowledge Innovation Program of Wuhan-Shuguang Project (No.2022020801020483), and the Health China·BuChang ZhiYuan Public welfare projects for heart and brain health under Grant No. HIGHER2022096.

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Author notes

  1. Yong Li, Xinyi Zhu and Wei Xiong have contributed equally.

Authors and Affiliations

  1. Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Yong Li & Xiaobing Jiang

  2. Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan, China

    Xinyi Zhu, Qingyu Zhao, Youdong Zhou, Yujia Guo, Baohui Liu, Mingchang Li, Qianxue Chen, Yangzhi Qi & Gang Deng

  3. Center of Regenerative Medicine, Department of Stomatology, Renmin Hospital of Wuhan University, Wuhan, China

    Wei Xiong & Qingsong Ye

  4. Sydney Dental School, The University of Sydney, Camperdown, NSW, 2050, Sydney, Australia

    Qingsong Ye

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Contributions

GD, YL, QSY and YZQ designed this study. YL, XZ, WX, QZ, YZ, YG, BL, HZ, CW, ML, QX and GD performed the data and wrote the paper. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xiaobing Jiang, Yangzhi Qi, Qingsong Ye or Gang Deng.

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The reason for using animals is that research requires them to develop new therapies for treating diseases. We conducted tests on animals to observe whether HA-PEG-UA NPs are safe and effective against SAH mice. A total of 226 C57 BL/6 male mice were used, with an average age of 8 weeks and a body weight between 22-26 g. All mice were purchased from China Slake Experimental Animal Co., Ltd. The mice were kept at a constant temperature under a 12 h dark light cycle, and were fed freely with water and standard laboratory feed. Conduct the experiment after one week of adaptation. At the end of study, the animals were sacrificed with cervical dislocation method following anesthesia with 1.5% isoflurane. The Laboratory Animal Ethics Committee of Renmin Hospital of Wuhan University approved all experiments with animals in this study (WDRY20210305A). All methods are reported in accordance with ARRIVE guidelines.

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Li, Y., Zhu, X., Xiong, W. et al. Brain-targeted ursolic acid nanoparticles for anti-ferroptosis therapy in subarachnoid hemorrhage. J Nanobiotechnol 22, 641 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-024-02866-x

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-024-02866-x

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Journal of Nanobiotechnology

ISSN: 1477-3155

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