Neuronal Ferroptosis Attenuation by RKIP Inhibition Following Spontaneous Intracerebral Hemorrhage via NRF2/HO-1 Pathway Activation

Summary

By conducting both organismal and cell culture-based studies, it was demonstrated that inhibiting Raf kinase inhibitor protein (RKIP) can mitigate neuronal ferroptosis following spontaneous intracerebral hemorrhage, thereby reducing brain injury. The observed cytoprotection arises through NRF2/HO-1 signaling axis modulation.

Abstract

Raf kinase inhibitor protein (RKIP), a primary member of the phosphatidylethanolamine-binding protein family, is recognized for its involvement in various physiological processes, including cell differentiation, migration, cell cycle, and apoptosis. This study explored its function in neuronal ferroptosis following spontaneous intracerebral hemorrhage (ICH) and the associated mechanisms. A cellular model of spontaneous ICH was developed using cultured rat pheochromocytoma (PC12) cells stimulated with hemin. Cell viability, RNA expression, reactive oxygen species (ROS) levels, and lipid hydroperoxide (LPO) were assessed. Additionally, protein expression levels were measured. This study confirmed that RKIP inhibition is neuroprotective in vitro following ICH. This effect was associated with increased expression of glutathione peroxidase 4 (GPX4) and decreased expression of acyl-CoA synthetase long-chain family 4 (ACSL4), along with ROS and LPO levels in neurons. RKIP inhibition demonstrated a protective role against neuronal ferroptosis in vitro following ICH, potentially via activation of the nuclear factor E2-related factor 2/heme oxygenase-1 (NRF2/HO-1) pathway. This mechanism may offer insights into therapeutic strategies targeting neuronal ferroptosis in ICH.

Introduction

Spontaneous intracerebral hemorrhage (ICH) is a cerebrovascular condition characterized by bleeding due to intracranial vascular damage, necrosis, and vessel rupture, commonly affecting the basal ganglia and representing a major subtype of hemorrhagic stroke¹. The mortality rate among patients diagnosed with ICH is approximately 50%, and a significant proportion of survivors experience severe loss of independence². Current treatment options for spontaneous ICH are limited, with no existing therapies shown to significantly reduce mortality or markedly enhance neurological outcomes post-ICH.

Ferroptosis, an iron-dependent form of programmed cell death, is defined by lipid peroxidation, accumulation of ferrous ions, and depletion of glutathione, distinguishing it from other forms of programmed cell death in genetic, morphological, and biological terms³. Increasing evidence highlights the role of neuronal ferroptosis in the pathology of ICH. Raf kinase inhibitor protein (RKIP), also known as phosphatidylethanolamine-binding protein 1 (PEBP1), is involved in neural development and forms the 15LOX/PEBP1 complex through its binding with 15-lipoxygenase (15LOX), a key regulator of ferroptosis⁴. However, the specific role and mechanisms of RKIP in ferroptosis linked to the progression of spontaneous ICH remain unexplored.

This study examined the anti-ferroptosis effects of RKIP inhibition on neurons, demonstrating that inhibition of RKIP expression could suppress neuronal ferroptosis following ICH, potentially through activation of the nuclear factor E2-related factor 2/heme oxygenase-1 (NRF2/HO-1) pathway. These findings are significant for the development of novel therapeutic strategies targeting the pathogenesis of ICH.

Protocol

Culture of PC12 cells
The PC12 cell line was propagated in Roswell Park Memorial Institute 1640 (RPMI-1640) growth medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, with routine maintenance under standard culture conditions (37 °C, 5% CO₂ humidified atmosphere). For experimental procedures, adherent cells were plated onto culture vessels and allowed to proliferate until achieving near-confluent monolayers (approximately 90% surface coverage). Cellular dissociation was accomplished through enzymatic treatment using 0.25% trypsin-EDTA solution, followed by three successive subculturing cycles to ensure population stability. Processed cells were subsequently allocated for downstream experimental applications and analytical assessments.

Cell viability assays
Cell viability was assessed to evaluate the cytotoxicity of hemin and Locostatin on PC12 cells, following the instructions provided by the assay kit's manufacturer. PC12 cells were uniformly seeded in 96-well plates and cultured with hemin or Locostatin for 24 h. Following washing with phosphate-buffered saline (PBS), the cells were incubated in RPMI-1640 medium containing 10% tetrazolium salt solution for 90 min at 37 °C. Optical Density (OD) values were then measured at 450 nm using a microplate reader.

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
Total RNA was extracted from PC12 cells using the RNA extraction reagent. First, the sample was homogenized in the extraction reagent, ensuring thorough lysis. Chloroform was added to the mixture, shaken vigorously, and centrifuged to separate the phases (12,000 × g, 15 min, 4 °C). The aqueous phase containing the RNA was collected, and the RNA was precipitated by adding isopropanol (aqueous phase : iPrOH = 1:1) and incubating at room temperature. Centrifugation was done to pellet the RNA (12,000 × g, 10 min, 4 °C), 1 mL of 70% ethanol was added to remove impurities, and finally, the RNA pellet was dissolved in RNase-free water for storage at -80 °C. Complementary DNA (cDNA) templates were generated by reverse transcription with the RT Master Mix. The resulting templates were then diluted at a 1:5 ratio and subjected to quantitative real-time PCR on a real-time PCR instrument. Each sample was amplified in triplicate, and the relative amount of PCR product was averaged. The primers used are listed in Table 1, with β-actin serving as the housekeeping gene. Relative mRNA concentration was determined using the formula E = 2^−ΔΔCt, and the Critical Threshold Cycle (CT) Value was recorded for each reaction.

Intracellular reactive oxygen species (ROS) and lipid hydroperoxide (LPO) assays
ROS and LPO levels were measured using flow cytometry. PC12 cells were treated with hemin (80 µM), Locostatin (5 µM), and ML385 (5 µM) for 24 h and washed 3x with PBS in dishes. The cells were then incubated at 37 °C with 5% CO₂ for 40 min in the presence of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), a ROS probe, and BODIPY 581/591 C11, an LPO probe. Following PBS washes to remove excess probes, fluorescence intensity was measured by flow cytometry. A consistent gating strategy was applied across all samples: first, cells were gated on FSC-A vs. SSC-A to exclude debris, then, single cells were selected by FSC-A vs. FSC-H gating. Unstained cells and cells treated with hemin alone were used as negative and positive controls to establish the fluorescence compensation and gates. Data were analyzed using the linked software.

Protein extraction and western blot
Total protein extraction 
The supernatant of treated PC12 cells was discarded, followed by three washes with ice-cold PBS. Cells were harvested using trypsin and centrifuged at 200 × g for 5 min. After removing the supernatant, the cell pellet was homogenized in cold Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing 10% protease inhibitor and 10% phosphatase inhibitor. After a 30 min incubation on ice, the lysate was centrifuged at 12,000 × g for 15 min at 4 °C. The clear supernatant was mixed with Loading Buffer (4:1) and heated at 95 °C for 5 min to denature proteins. Protein samples were stored at -80 °C for subsequent use.

Western blot analysis 
Proteins were separated by SDS-PAGE using a stacking gel and resolving gel, with samples loaded into wells. Electrophoresis was performed at 80 V for the stacking gel and 120 V for the resolving gel. Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (pre-activated in methanol for 1 min) using a transfer system at a constant current of 300 mA for 1-2 h. The membrane was blocked with 5% skim milk in TBST for 2 h at room temperature to prevent nonspecific binding. After three washes with TBST (10 min each), the membrane was incubated overnight at 4 °C with the following primary antibodies diluted in TBST: Rabbit anti-ACSL4 (1:1,000), Rabbit anti-GPX4 (1:1,000), Rabbit anti-HO-1 (1:2,000), Rabbit anti-NRF2 (1:1,000), Rabbit anti-RKIP (1:1,500), and Mouse anti-β-actin (1:5,000). The membrane was washed for 3 x 10 min with TBST and incubated with HRP-conjugated secondary antibodies for 2 h at room temperature: HRP-labeled Goat Anti-Mouse IgG (1:1,000), HRP-labeled Goat Anti-Rabbit IgG (1:1,000). After additional TBST washes for 3 x 5 min, protein signals were visualized using an ECL Western Blot Kit and quantified using ImageJ software.

Immunofluorescence staining
Cells were seeded onto coverslips pre-placed in culture plates and allowed to adhere. After experimental treatments, the supernatant was discarded, and cells were washed 3x with PBS. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, followed by three PBS washes. Subsequently, cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature and washed 3x with PBS. Nonspecific binding was blocked by incubating with 3% bovine serum albumin (BSA) for 30 min at room temperature. Primary antibodies were applied overnight at 4 °C: Rabbit anti-HO-1 (1:500), Rabbit anti-NRF2 (1:500). After three PBS washes the next day, cells were incubated with fluorescent secondary antibodies in the dark for 1 h at room temperature: Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (1:500). Following the final washes, the samples were mounted with mounting medium containing 4',6-diamidino-2'-phenylindole (DAPI) for nuclear counterstaining. Images were captured using a confocal laser scanning microscope.

Statistical analysis
Measurement data were expressed as mean ± standard deviation (S.D.). Quantitative data representing results from three independent replicate assays were taken for analysis. For comparisons between two groups, a t-test was applied, while a one-way analysis of variance (ANOVA) was used for comparisons among multiple groups, followed by Tukey's post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.

Results

Hemin can induce plasma membrane damage, thus it is frequently used for the construction of in vitro cell models of ICH. This study investigated the effects of hemin treatment at varying concentrations and durations on PC12 cell viability, while simultaneously analyzing RKIP protein expression dynamics under corresponding experimental conditions through western blot analysis (Figure 1A). Cell viability was significantly reduced at hemin concentrations of 60 µM or higher (

Discussion

This study investigated the effects and underlying mechanisms of RKIP knockdown on ferroptosis induced by ICH in an in vitro setting. Stimulation of cells with hemin triggers oxidative stress, accompanied by excessive generation of ROS and LPO, thereby promoting ferroptosis. Inhibition of endogenous RKIP expression accelerates the liberated NRF2 to translocate into the nucleus and bind to the Antioxidant Response Element (ARE), initiating the transcription of downstream antioxidant gen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488-labeled Goat Anti-Rabbit IgG	Beyotime Biotech	A0423
Anti-ACSL4 antibody	Cell signaling	38493T
Anti-GPX4 antibody	Cell signaling	59735S
Anti-HO-1 antibody	MedChemExpress	HY-P80499
Anti-NRF2 antibody	Cell signaling	12721T
Anti-RKIP antibody	Abcam	ab76582
Anti-β-actin antibody	Abcam	ab179467
BeyoECL Plus	Beyotime Biotech	P0018M
BSA	NCM Biotech	WB6504-100g
Cell Counting Kit-8	Beyotime Biotech	C0037
CELLSAVING	NCM Biotech	C40050
ChamQ SYBR Color qPCR Master Mix	Vazyme	Q411-02
DMSO	Beyotime Biotech	ST038
Fetal Bovine Serum	Gibco	A5670801
GraphPad Prism
Hemin	MedChemExpress	HY-19424
HiScript Q RT SuperMix for qPCR (+gDNA wiper)	Vazyme	R123-01
HRP-labeled Goat Anti-Mouse IgG	Thermo	C31430100
HRP-labeled Goat Anti-Rabbit IgG	Thermo	C31460100
Lipid Peroxide (LPO) Content Assay Kit	Solarbio	BC5245
Locostatin	MedChemExpress	HY-W013411A
ML385	MedChemExpress	HY-100523
Mounting Medium?antifading (with DAPI)	Solarbio	S2110
NON-FatPowderedMilk	Solarbio	D8340
PBS (powder?pH7.2-7.4)	Solarbio	P1010
PC-12Adh	Nanjing BioChannel Biotechnology	BC-C-RA-005
Penicillin-Streptomycin Liquid	Solarbio	P1400
PRMI-1640	Gibco	A1049101
Protease and phosphatase inhibitor cocktail for general use, 50x	Beyotime Biotech	P1045
Protein Marker	Thermo	26617
PVDF	Millipore	RIKB43638
Reactive Oxygen Species Assay Kit	Solarbio	CA1410
RIPA buffer (high)	Solarbio	R0010
SDS-PAGE Gel Quick Preparation Kit	Epizyme	PG110
SDS-PAGE Sample Loading Buffer, 5x	Beyotime Biotech	P0015
TritonX-100	Solarbio	IT9100
TRIzol	Thermo	15596018CN
Trypsin-EDTA solution?0.25% (without phenol red)	Solarbio	T1300