Biochanin A inhibits excitotoxicity-triggered ferroptosis by targeting GPX4 in hippocampal neurons

doi:10.21203/rs.3.rs-4071868/v1

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Biochanin A inhibits excitotoxicity-triggered ferroptosis by targeting GPX4 in hippocampal neurons

https://doi.org/10.21203/rs.3.rs-4071868/v1

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Excitatory neurotransmitter-induced neuronal ferroptosis has been implicated in multiple neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Although there are several reports pertaining to the pharmacological activities of biochanin A, the effects of this isoflavone on excitotoxicity-triggered neuronal ferroptosis remain unclear. In this study, we demonstrate that biochanin A inhibits ferroptosis of mouse hippocampal neurons induced by glutamate or the glutamate analog, kainic acid. Biochanin A significantly inhibited accumulation of intracellular iron and lipid peroxidation in glutamate- or kainic acid-treated mouse hippocampal neurons. Furthermore, biochanin A regulated the level of glutathione peroxidase 4, a master regulator of ferroptosis, by modulating its autophagy-dependent degradation. We observed that biochanin A reduced the glutamate-induced accumulation of intracellular iron by regulating expression of iron metabolism-related proteins including ferroportin-1, divalent metal transferase 1, and transferrin receptor 1. Taken together, these results indicate that biochanin A effectively inhibits hippocampal neuronal death triggered by glutamate or kainic acid. Our study is the first to report that biochanin A has therapeutic potential for the treatment of diseases associated with hippocampal neuronal death, particularly ferroptosis induced by excitatory neurotransmitter.

Neurodegenerative disease

Ferroptosis

Biochanin A

Excitotoxicity

Glutathione peroxidase 4

Ferroportin-1

Glutamate is the major excitatory neurotransmitter of the central nervous system. The concentration of glutamate is approximately 10 mM in the vesicles of neurons, and 1 mM in the synaptic cleft (Dzubay and Jahr, 1999). Elevated glutamate levels in the hippocampal tissue are related to neurologic diseases such as Alzheimer’s, Parkinson’s, epilepsy, multiple sclerosis, and stroke (Ashraf et al., 2020; Zhao et al., 2023; Green et al., 2021; Muhlert et al., 2014; Wang et al., 2021). Glutamate toxicity is mediated by the two channel proteins N-methyl-D-aspartate receptor (NMDAR) and cystine/glutamate antiporter (xCT) (Schubert and Piasecki, 2001). NMDAR- and xCT-mediated glutamate toxicity leads to ferroptosis, an iron-dependent cell death (Dixon et al., 2012). Glutathione peroxidase 4 (GPX4) plays a critical role in resistance to ferroptotic cell death in many nerve cells by converting glutathione to oxidized glutathione (Dixon et al., 2012). In a recent study, autophagy-mediated GPX4 degradation was demonstrated to be the main cause of ferroptosis induced by glutamate in HT-22 mouse hippocampal cells (Wu et al., 2019). In fact, blockade of autophagic GPX4 degradation by the autophagy inhibitor, chloroquine, attenuated ferroptosis (Wu et al., 2019). Glutamate also increased accumulation of intracellular iron and lipid peroxidation by suppressing ferroportin 1 expression and accelerating GPX4 degradation, respectively (Wu et al., 2019; Xu et al., 2018). Furthermore, several ferroptosis inhibitors such as ferrostatin-1, liproxstatin-1, and deferoxamine almost entirely inhibited glutamate-induced cell death in HT-22 mouse hippocampal cells (Jiang et al., 2020). Ferrostatin-1 attenuated glutamate-induced excitotoxic cell death in mouse hippocampal slice cultures (Li et al., 2017). Similarly, the glutamate analog, kainic acid, induces the excitotoxic death of neurons in the CA1 and CA3 regions of the hippocampus, and is therefore employed as an in vivo model of glutamate-induced ferroptosis (Andrade-Talavera and Rodríguez-Moreno, 2023; Xie et al., 2022). Neurons in the CA1 and CA3 areas reportedly function to drive memory and learning, and neuronal death in these areas is associated with diverse neurological diseases including Alzheimer’s, stroke, and epilepsy (Choi et al., 2023; Medvedeva et al., 2017; Ioannou et al., 2019). Comparable with glutamate-induced HT22 neuronal death, ferroptosis was also observed in neurons of the hippocampus in mice administered with kainic acid (Xie et al., 2022; Choi et al., 2023; Wang et al., 2022a). Indeed, biomarkers of ferroptosis including increased levels of iron and lipid peroxide and decreased level of GPX4 are readily observed in the hippocampus of these mice (Xie et al., 2022). Additionally, ferroptosis inhibitors such as ferrostatin-1 and quercetin have been reported to inhibit the death of hippocampal neurons, especially in the CA1 and CA3 regions (Xie et al., 2022; Ye et al., 2020). It is therefore of paramount importance to identify bioactive molecules that have neuroprotective effects by inhibiting ferroptosis.

Biochanin A is one of the most abundant isoflavones in natural plants such as Trifolium pratense L. and soybeans (Sarfraz et al., 2020). Biochanin A possesses antioxidant, anti-inflammatory, anti-cancer, and anti-menopausal biological activities (Sarfraz et al., 2020). In addition, biochanin A also effectively inhibits several types of cell death, including apoptosis, necrosis, autophagy, and ferroptosis (Sarfraz et al., 2020; Dong et al., 2022; He et al., 2023). Accordingly, this molecule protects multiple organs in diverse disease models such as sepsis, osteoarthritis, cancer, and ischemia/reperfusion injury (Sarfraz et al., 2020; He et al., 2023; Liu et al., 2016; Guo et al., 2019). Biochanin A is also protective against iron dextran-induced knee osteoarthritis by regulating iron accumulation and GPX4 expression (He et al., 2023). Further, biochanin A mitigated symptoms of memory decline in a cuprizone-induced multiple sclerosis mouse model by inhibiting histological changes in the hippocampus (Aldhahri et al., 2022). Biochanin A also attenuated cognitive impairment in ovariectomized APP/PS1 mice by suppressing hippocampal mitochondrial damage (Hou et al., 2022).

Despite the diverse biological activities of biochanin A, the effects of this compound in excitotoxicity-triggered ferroptotic neuronal death remains unclear. In the present study, we explored the activity of biochanin A in a model of ferroptotic cell death induced by glutamate in HT-22 mouse hippocampal cells, and in a model of kainic acid-induced hippocampal neuronal death in mice. Our findings suggest that biochanin A suppresses glutamate- or kainic acid-induced ferroptotic cell death in hippocampal neurons, and suggest a role for autophagy-dependent inhibition of GPX4 degradation in this process.

2.1. Materials

Chloroquine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), glutamic acid sodium salt, ML-385, and a primary antibody against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies specific for ferroptosis suppressor protein 1 (FSP1), divalent metal transferase 1 (DMT1), LC3-I/II, and p62 were purchased from SantaCruz biotechnology (Dallas, TX, USA). Kainic acid, monoclonal antibodies specific for GPX4 and transferrin receptor 1 (Tfr1) were purchased from Abcam (Cambridge, UK). Polyclonal antibodies specific for ferroportin 1 (FPN1) and PARP1 were purchased from NOVUS biologicals (Centennial, CO, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively.

2.2. Cell culture

HT22 mouse hippocampal cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic cocktail (100 U/mL penicillin and 100 µg/mL streptomycin) at 37℃ with 5% CO₂.

2.3. Cytotoxicity assays

To assess cell viability and cytotoxicity, we employed an MTT assay and lactate dehydrogenase (LDH) release assay. For the MTT assay, HT22 cells were plated at 4 × 10⁴ cells/well in 24-well plates. Following incubation of the cells with the indicated reagents for the indicated time, MTT solution (0.5 mg/mL in phosphate buffered saline) was added to the culture medium and cells were incubated for an additional 1 h. After removal of the medium, formazan crystals formed in living cells were dissolved in acidified isopropanol. Absorbance was then measured at 570 nm using a Multiskan™ GO microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA). For the LDH release assay, culture medium from cells treated with the indicated reagents was collected. The amount of LDH released into the medium was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA, Cat# G1780). Absorbance was measured at 490 nm using a Multiskan™ GO microplate spectrophotometer (Thermo Scientific).

2.4. Measurement of lipid peroxidation

To measure lipid peroxidation, we used the fluorescent probe BODIPY-C11 (581/591) (Invitrogen, Waltham, MA, USA) and a lipid peroxidation assay kit (colorimetric/fluorometric) (Invitrogen, Waltham, MA, USA). HT22 cells treated with the indicated reagent for 8 h were treated with 200 nM BODIPY-C11 for 30 min at 37℃. After incubation, cells were washed with PBS and fluorescence was detected using a Ti2 fluorescence microscope (Nikon, Tokyo, Japan). Fluorescence intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA).

2.5. Detection of intracellular malondialdehyde (MDA)

To measure intracellular MDA, HT22 cells or mouse hippocampal tissues were lysed in MDA lysis buffer with butylated hydroxytoluene. Following centrifugation, supernatants were collected and reacted with thiobarbituric acid at 95℃ for 60 min. A Multiscan GO microplate UV-spectrophotometer (Thermo Scientific) was subsequently used to measure the absorbance at 510 nm.

2.6. Measurement of intracellular iron levels

Intracellular iron levels were quantified using a fluorescent probe and ferene-based calorimetric method. The fluorescent probe calcein-AM green acetoxymethyl ester (BD Biosciences, Franklin Lakes, NJ, USA) was used to determine the intracellular iron level in HT22 cells as described previously (Won et al., 2023). Briefly, HT22 cells treated with the indicated reagents for 8 h were incubated with 100 nM calcein-AM at 37℃ for 30 min. After incubation, the cells were washed twice with PBS and trypsinized. Cellular fluorescence intensity was subsequently analyzed using a BD FACSCaliburTM flow cytometer (BD Biosciences) or a Ti2 fluorescence microscope (Nikon). For the ferene-based method, intracellular iron levels in mouse hippocampal tissues were determined as described previously (Abbasi et al., 2021). Briefly, hippocampal tissues were lysed in radioimmunoprecipitation assay buffer and sonicated 2 times for 10 s at a frequency of 20 kHz. After centrifugation for 10 min at 21000 x g, the supernatant was collected and reacted with 100 µL of ammonium acetate buffer (pH 4.5, 2.5 M) and 120 µL working solution (5 mM ferene and 10 mM ascorbic acid in ammonium acetate buffer). Following centrifugation of reactants for 5 min at 15000 x g, absorbance of the supernatants was measured at 595 nm in a Multiskan GO microplate UV-spectrophotometer (Thermo Scientific). Iron concentrations were calculated using an iron standard curve and normalized to the amount of protein.

2.7. Measurement of total glutathione

An OxiSelect Total Glutathione (GSSG/GSH) Assay Kit was used to determine intracellular total glutathione according to the manufacturer’s instructions (Cell biolabs, CA, USA). Briefly, HT22 cells were washed with PBS and trypsinized. Detached cells were resuspended with ice-cold 5% meta-phosphoric acid and sonicated 2 times for 10 s at a frequency of 20 kHz. After centrifugation at 16000 x g for 5 min, the supernatant was collected and reacted with glutathione reductase and NADPH. A Multiscan GO microplate UV-spectrophotometer (Thermo Scientific) was used to measure the absorbance at 405 nm.

2.8. Western blot analysis

Mouse hippocampal tissues and HT22 cells were lysed in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea). Extracted proteins were quantified using a Bradford assay. An equal amount of protein (10 − 20 µg) was separated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilion-P polyvinylidene difluoride membranes (Merck, Darmstadt, Germany). Membranes were blocked at room temperature for 1 h with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and washed three times for 30 min with TBS-T. Membranes were subsequently incubated overnight at 4 ℃ with the indicated primary antibodies diluted in TBS-T. Membranes were washed three times for 30 min with TBS-T and incubated with a horseradish peroxidase-conjugated secondary IgG antibody at room temperature for 1 h. Membranes were washed three times for 30 min and chemiluminescence was visualized using WesternBright ECL (Advansta Inc., MenloPark, CA, USA, CAT#K-12045-D50).

2.9. Real-time polymerase chain reaction (RT-PCR) analysis

Total RNA from HT22 cells treated with biochanin A for the indicated time was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and complementary DNA (cDNA) was synthesized from 2 µg of total RNA by reverse transcription using a TOPscript RT DryMIX kit (Enzynomics, Daejeon, Korea). An aliquot of cDNA was added to a 10 µL reaction mix containing 10 pM primers, before combining with 2 x Real-time PCR smart mix (Solgent, Daejeon, Korea). The amplification conditions using a LightCycler 96 instrument (Roche Diagnostics, Basel, Switzerland) were as follows: one cycle of initial denaturation for 15 min at 95°C, 50 cycles of 10s at 95°C, 20 s at 55°C, and 30 s at 72°C. The following primers were used: GPX4, 5’-CCATGCACGAATTCTCAGCC-3’ and 5’-GGTGACGATGCACACGAAAC-3’. GAPDH, 5’-CATGGCCTTCCGTGTTCCTA-3’ and 5’-CCTGCTTCACCACCTTCTTGAT-5’.

2.10. Animal experiments

All animal studies were approved by the Institutional Animal Care and Use Committee of Konkuk University (approval number: KU23233). Male C57BL/6 mice (7-week-old, 20–25 g) were obtained from Orient Bio (Seongnam, Korea). After 1 week of acclimatization, hippocampal neuronal death was induced by injecting 2 µg kainic acid (5 µL injection volume) intracerebroventricularly (i.c.v.) in both ventricles. In subsequent experiments, mice were randomly divided into the following groups: control (saline), 2 µg kainic acid, and 2 µg kainic acid plus 40 mg/kg biochanin A. Biochanin A (40 mg/kg/day) was administered via intraperitoneal (i.p.) injection for 2 weeks every day prior to kainic acid administration. Hippocampal tissues were obtained by sacrifice 3 days after kainic acid administration. To administer kainic acid by i.c.v. injection, mice were anesthetized with isoflurane (induction: 3–4%, maintain: 1–2%) and injected with 2 µg kainic acid into each ventricle.

2.11. Cresyl violet staining (Nissl staining) of hippocampal tissues

Cresyl violet staining (Nissl staining) was performed on hippocampal tissues harvested from mice euthanized 24 h after kainic acid (2 µg) injection and fixed in 4% paraformaldehyde for 24 h. For the preparation of paraffin blocks, tissues were dehydrated using alcohol and xylene, and blocks were prepared by infiltrating paraffin using an autofluidizer (Sakura 5, Finetek, Japan). The paraffin blocks were cut into 3.5 µm thick sections. Sections were subsequently deparaffinized by immersing in xylene solution, followed by hydration using gradually reduced ethanol concentrations from 100–70%, and rinsing with tap water. The Nissl bodies (subcellular structures found in nerve cell bodies and dendrites) were stained in 0.1% cresyl violet acetate solution before dehydration using increasing concentrations of ethanol solution from 70–100%. The sections were immersed in xylene and observed under an optical microscope (Nikon). Neuronal death was scored as the loss of the pyramidal layer in the hippocampal CA1 and CA3 regions on a scale from 0–6 as follows: 0, no damage; 1, less than 10%; 2, 11–25%; 3, 26–50%; 4, 51–75%; 5, 76–90%; 6, more than 91% of observable cells lost.

2.12. Statistical analysis

Data were expressed as means ± standard error (SE). Statistical significance was determined using a Student’s t-test to compare two variables or a one-way ANOVA followed by Tukey's post-hoc test to compare three or more variables. Analyses were conducted using Statistical Package for Social Sciences (SPSS, Version 25; IBM Corp., New York, USA).

3.1. Biochanin A inhibits glutamate-induced ferroptotic cell death in HT22 cells.

To examine the effects of biochanin A on glutamate-induced ferroptotic cell death, we first defined the optimal concentrations of both glutamate and biochanin A in our model system. When cells were exposed to varying concentrations of glutamate for 8 h, cellular toxicity became evident at 3 mM glutamate, and at 10 mM cell viability LDH release measurements indicated death of approximately two-thirds of all cells (Supplemental Fig. 1A and 1B). We next performed a glutamate treatment time course to define the optimal time point to observe cell death. When cells were treated with 10 mM glutamate, cells remained viable for the first 4 h, but a decrease in cell viability and increased LDH release were observed at 8 h, which continued to increase up until 24 h post-treatment (Supplemental Fig. 1C and 1D). Based on these results, cells were treated with 10 mM glutamate for 8 h in subsequent experiments. We also investigated potential toxicity of biochanin A across a range of concentrations in our system in the absence of glutamate. Following an 8 h incubation, cell viability was not affected up to 100 µΜ biochanin A (Supplemental Fig. 1E and 1F). Thus, 100 µM biochanin A was the predominant concentration used in subsequent experiments.

To determine whether biochanin A inhibited glutamate-induced ferroptotic cell death in HT22 cells, cells were treated with an increasing concentration of biochanin A for 8 h in the presence of 10 mM glutamate. Biochanin A significantly suppressed glutamate-induced ferroptotic cell death in a dose-dependent manner. Biochanin A at 100 µM most potently inhibited cell death, as determined by trypan blue exclusion and LDH release assay (Fig. 1A and 1B). Biochanin A displayed similar anti-ferroptotic effects to the known ferroptosis inhibitors ferrostatin-1, liproxstatin-1, and deferoxamine in HT22 cells treated with glutamate (Fig. 1C and 1D).

3.2. Biochanin A attenuates glutamate-induced iron accumulation and lipid peroxidation in HT22 cells.

Intracellular iron accumulation is the main cause of ferroptotic cell death (Dixon et al., 2012). Calcein-AM, a fluorescent probe, was used to detect intracellular iron levels by fluorescence microscopy and fluorescence-activated cell sorting (FACS) analyses. Microscopy revealed that biochanin A significantly attenuated the decrease in fluorescence intensity induced by glutamate (Fig. 2A and 2B). This phenotype was recapitulated in the FACS analysis, indicating that biochanin A inhibits glutamate-induced iron accumulation in HT22 cells (Fig. 2C and 2D). The iron chelator deferoxamine (DFO) was used as a positive control in these assays and displayed similar effects to those of biochanin A in HT22 cells treated with glutamate (Fig. 2A-2D).

Accumulated iron induces lipid peroxidation through the Fenton reaction (Spangler et al., 2016). To quantify lipid peroxidation, we imaged the fluorescent probe BODIPY-C11 by fluorescence microscopy. Consistent with our observations of iron accumulation, biochanin A decreased the fluorescence intensity of oxidized BODIPY-C11 (green) in glutamate-treated HT22 cells (Fig. 3A and 3B). Similarly, biochanin A also inhibited glutamate-induced accumulation of the lipid peroxidation product MDA (Fig. 3C). The ferroptosis inhibitor ferrostatin-1 showed a similar inhibitory effect on lipid peroxidation to that of biochanin A in glutamate-treated HT22 cells (Fig. 3A-3C).

3.3. Biochanin A rescues the levels of GPX4 protein and total glutathione in glutamate-treated HT22 cells.

Decreased levels of GPX4 protein are commonly observed in glutamate-induced ferroptotic cell death in HT22 cells (Wu et al., 2019). Western blot analysis revealed that biochanin A recovered the decrease in GPX4 protein levels induced by glutamate in HT22 cells (Fig. 4A and 4B). Under control conditions, GPX4 inhibits lipid peroxidation by oxidizing GSH to GSSG (Wu et al., 2019). Consequently, biochanin A rescued the levels of GSSG induced by glutamate in HT22 cells (Fig. 4C). The positive control compound ferrostatin-1 displayed similar effects to those of biochanin A in HT22 cells treated with glutamate (Fig. 4A-4C). Instead, the glutathione-independent ferroptosis suppressor protein, FSP1, was not changed by treatment with biochanin A (Supplementary Fig. 2A and 2B).

3.4. Biochanin A inhibits autophagy-mediated GPX4 degradation in HT22 cells.

To clarify the mechanisms by which biochanin A regulates GPX4 protein levels, we analyzed the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway. A recent study demonstrated that GPX4 protein levels are regulated by Nrf2 signaling, and that decreased levels of GPX4 sensitize cells to ferroptosis inducers such as glutamate (Ma et al., 2022). To confirm whether Nrf2 signaling is associated with the protective effects of biochanin A in glutamate-induced ferroptotic death of HT22 cells, cells were treated with ML385 to inhibit Nrf2 transcriptional activity. Although ML385 affected cell viability in the glutamate-treated group, there were no differences in the biochanin A co-treatment group (Supplementary Fig. 3A and 3B). Despite observations that GPX4 mRNA and protein expression was not regulated by biochanin A (Supplemental Fig. 4A-4C), the glutamate-triggered decrease in GPX4 was rescued by biochanin A (Fig. 4A and 4B). We therefore considered whether biochanin A could modulate the autophagy-mediated degradation of GPX4. As expected, glutamate reduced GPX4 levels in HT22 cells, and biochanin A attenuated this phenotype, and, also inhibited the glutamate-induced change in p62 and LC3-II protein levels (Fig. 5A and 5B). To confirm whether biochanin A suppresses GPX4 protein degradation by inhibiting autophagy, we used chloroquine to block autophagosome fusion with lysosomes. Chloroquine restored the levels of p62 and GPX4, similarly to biochanin A in glutamate-treated HT22 cells (Fig. 5C and 5D). Consistent with these observations, cellular toxicity was also restored by chloroquine in glutamate-treated HT22 cells, indicating that biochanin A-mediated regulation of GPX4 is a key event regulating glutamate-triggered ferroptotic cell death in HT22 hippocampal cells (Fig. 5E and 5F).

3.5. Biochanin A regulates expression of proteins associated with iron metabolism in HT22 cells.

To elucidate the biochanin A-mediated mechanisms governing inhibition of iron accumulation in our model, we examined the levels of iron metabolism-related proteins in glutamate-treated HT22 cells. Biochanin A inhibited the change in levels of FPN1 (an iron exporter), DMT1 (an iron importer), and TfR1 (a transferrin receptor) induced by treatment with glutamate (Fig. 6A-6D). Consistent with these results, ferrostatin-1 also showed similar effects of biochanin A in glutamate-treated HT22 cells, indicating that biochanin A is a potent inhibitor of glutamate-triggered ferroptosis (Fig. 6A-6D).

3.6. Biochanin A inhibits kainic acid-induced ferroptosis in mouse hippocampal neurons.

To confirm the inhibition of excitotoxicity-triggered ferroptosis by biochanin A, we established an in vivo model of hippocampal neuronal death by intracerebroventricular injection of C57BL/6 male mice with the glutamate analog kainic acid (Xie et al., 2022). Based on previous studies (Tan et al., 2013; Wang et al., 2015; Hanke and Rami, 2023; Thayyullathil et al., 2021), we administered biochanin A at 40 mg/kg/day intraperitoneally for 2 weeks, and sacrificed mice 3 days following kainic acid administration (Fig. 7A). Histological analysis of the hippocampus with cresyl violet revealed a decrease in the number of viable neurons in the CA1 and CA3 regions in mice injected with kainic acid (Fig. 7B). The mean cell death scores in the CA1 and CA3 of the hippocampus injected with kainic acid were 4.3 ± 1.6 (n = 3) and 5 ± 0.6 (n = 3), respectively (Fig. 7C). Pathology was significantly reduced by pretreatment of mice with 40 mg/kg biochanin A, with histological scores of 1 ± 0.6 (n = 3) and 0.3 ± 0.3 (n = 3) in CA1 and CA3 regions of the hippocampus, respectively (Fig. 7C). Consistent with the cresyl violet analysis, kainic acid-induced PARP-1 cleavage, a marker for cell death (Wang et al., 2022b), was also decreased in the group pretreated with biochanin A (Fig. 7D and 7E). To clarify whether ferroptosis was the mode of neuronal death induced by kainic acid in the hippocampus, we examined the accumulation of iron and lipid peroxides, which are typical markers of ferroptosis. The kainic acid-induced increase of hippocampal labile iron and MDA was significantly reduced by biochanin A pretreatment (Fig. 7F and 7G). Pretreatment with biochanin A also restored GPX4 protein levels, which were reduced by kainic acid challenge, comparable with our in vitro observations in HT22 cells (Fig. 7H and 7I). These results indicate that biochanin A suppresses neuronal cell death by inhibiting ferroptosis in the hippocampus of mice treated with kainic acid.

This is the first report demonstrating that biochanin A is a potent inhibitor of ferroptosis induced by kainic acid and glutamate in the murine hippocampus and in HT22 cells. We show that in glutamate-challenged HT22 cells, biochanin A significantly reduced the accumulation of lipid peroxidation, and that turnover of the master cellular peroxidation regulator GPX4 is regulated through an autophagy-dependent mechanism. Biochanin A also reduced the accumulation of intracellular iron through transcriptional regulation of iron metabolism-related proteins, including FPN1, DMT1, and TfR1. Furthermore, biochanin A dramatically inhibited hippocampal neuronal death by regulating cellular iron/MDA levels and GPX4 expression in kainic acid-injected mice. These observations indicate that biochanin A inhibits glutamate- or kainic acid-triggered ferroptosis in hippocampal neurons, demonstrating its therapeutic potential for the treatment of excitotoxicity-associated modes of neuronal death such as ferroptosis.

An increase in cellular antioxidant capacity is one of the strategies to resist ferroptosis. Iron accumulation induces lipid peroxidation through the Fenton reaction, which alters the physical properties of the cell membrane, leading to cell death (Dixon et al., 2012). Indeed, the mechanism of action of the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 are to directly inhibit peroxidation of phospholipids without iron accumulation (Dixon et al., 2012). Several studies have described the antioxidant activity of biochanin A, and this activity is suggested to contribute to suppression of ferroptosis by inhibiting lipid peroxidation. Biochanin A was shown to inhibit tert-butyl hydroperoxide (t-BHP)-induced cell death in HepG2 cells by activation of Nrf2/HO1/NQO1 axis (Liang et al., 2019). Biochanin A also prevented arsenic-induced liver damage in mice by increasing the expression of multiple antioxidant enzymes such as superoxide dismutase and catalase (Jalaludeen et al., 2016). Further, Biochanin A inhibited amyloid-beta- or glutamate-induced apoptosis in PC12 cells by preventing mitochondrial dysfunction (Tan and Kim, 2016; Tan et al., 2013). Biochanin A also attenuated lipopolysaccharide-induced dopaminergic neuronal damage in mice by inhibiting MDA accumulation and activation of antioxidant enzymes such as superoxide dismutase, NAD(P)H quinone oxidoreductase 1, and GPX4 (Wang et al., 2015). Considering these studies, the anti-ferroptotic effects of biochanin A may be derived from its antioxidant activity in glutamate- or kainic acid-treated hippocampal neuronal cells.

Although biochanin A did not alter the expression of GPX4, a key protein regulating ferroptosis, biochanin A maintained cellular level of GPX4 by suppressing its degradation by autophagy. Indeed, previous studies have demonstrated that glutamate-induced cell death in HT22 cells is associated with autophagy-mediated GPX4 degradation (Dixon et al., 2012; Wu et al., 2019; Xu et al., 2018). The autophagy inhibitor 3-methyladenin suppressed erastin-induced ferroptosis in HT22 cells by preventing degradation of GPX4 (Hanke and Rami, 2023). Erastin- or glutamate-mediated activation of acid sphingomyelinase, a key enzyme regulating sphingolipid metabolism, also induced ferroptosis by promoting autophagic degradation of GPX4 in HT-1080 cells (Thayyullathil et al., 2021). Erastin- or glutamate-induced ferroptosis is also associated with chaperone-mediated GPX4 degradation (Wu et al., 2019). Thus, regulation of autophagy is a major strategy to resist glutamate-induced ferroptosis in HT22 cells. Biochanin A was demonstrated to inhibit autophagy by regulating the AMP-activated protein kinase/unc-51-like kinase 1 pathway in glioblastoma (Dong et al., 2022). Biochanin A also inhibited rotenone-induced dopaminergic neuronal death in mice by regulating expression of the autophagy component beclin-1 (El-Sherbeeny et al., 2020). Considering these studies, recovery of GPX4 levels by biochanin A is likely mediated through an autophagy-dependent mechanism in mouse hippocampal neurons and HT22 cells.

Accumulation of intracellular iron induces cell death through Fenton reaction- mediated oxidative stress and lipid peroxidation, highlighting the importance of intracellular iron homeostasis (Dixon et al., 2012). Iron metabolism is regulated by several proteins including FPN1 (an iron exporter), DMT1 (an iron importer), and TfR1 (a transferrin receptor). Several studies have described inhibition of iron accumulation by regulating expression of these proteins. The glucagon-like peptide-1 receptor agonist liraglutide attenuated accumulation of intracellular iron by decreasing TfR1 expression and increasing FPN1 expression in db/db mice (Song et al., 2022). The peroxisome proliferator-activated receptor delta agonist GW501516 suppressed 6-hydroxydopamine-induced iron accumulation by regulating expression of DMT1 protein in SH-SY5Y cells (Lee et al., 2022). Biochanin A was also shown to suppress intracellular iron levels by regulating the expression of TfR1 and FPN1 in iron overloaded mice (Ma et al., 2022). Consistent with these previous reports, we have demonstrated that biochanin A inhibits accumulation of intracellular iron by regulating the expression of iron metabolism-related proteins, highlighting a role for this pharmacological activity in the inhibition of ferroptosis in glutamate- or kainic acid-treated hippocampal neuronal cells.

In agreement with in vitro observations in HT22 hippocampal cells, biochanin A effectively attenuated kainic acid-induced ferroptosis in mouse hippocampal neurons by inhibiting the accumulation of iron and lipid peroxide. Ferroptosis is reportedly involved in the death of neuronal cells in the hippocampus, leading to neurodegenerative diseases including epilepsy, seizure, and Alzheimer’s disease (Reichert et al., 2020). In fact, hippocampal neuronal ferroptosis was observed in Alzheimer’s disease model APPswe/PS1De9 mice (Bao et al., 2021). Similarly, neurotoxic A1 astrocytes induced ferroptosis in mouse hippocampal tissue, which was suppressed by ferrostatin-1 treatment (Liang et al., 2023). Ferroptosis of hippocampal neuronal cells triggered by sepsis-associated encephalopathy was inhibited by irisin-associated upregulation of GPX4 expression (Wang et al., 2022c). Collectively, biochanin A-mediated modulation of GPX4 may contribute to its inhibition of ferroptosis in kainic acid-challenged mouse hippocampal neurons, placing biochanin A as a candidate therapy for ferroptosis-related neurodegenerative diseases.

This study demonstrates that biochanin A inhibits kainic acid- and glutamate-induced ferroptosis by regulating cellular levels of GPX4, iron, and lipid peroxides both in vivo and in vitro. These findings support the use of biochanin A in the treatment or prevention of diseases associated with hippocampal neuronal death. We have also identified the potential for biochanin A to be used as a potent ferroptosis inhibitor.

N-methyl-D-aspartate receptor

NMDAR

Glutathione peroxidase 4

GPX4

Cystine/glutamate antiporter

xCT

Ferroptosis suppressor protein 1

FSP1

Divalent metal transferase 1

DMT1

Transferrin receptor 1

Tfr1

Ferroportin 1

FPN1

Malondialdehyde

MDA

Lactate dehydrogenase

LDH

Nuclear factor erythroid-2-related factor 2

Nrf2

Fluorescence-activated cell sorting

FACS

Deferoxamine

DFO

Intracerebroventricularly

i.c.v.

Intraperitoneal

i.p.

Cornu Ammonis

Declaration of Competing Interest

The authors declared no competing interests.

Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2021R1A2C1007526).

Author Contribution

Jun Pil Won designed the study, performed the experiments, analyzed the data and wrote the manuscript. Hyuk Gyoon Lee and Han Jun Yoon assisted in animal test. Han Geuk Seo contributed to the experimental design, revised the manuscript, funding support and supervised the whole project. All authors read and approved the final manuscript.

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Biochanin A inhibits excitotoxicity-triggered ferroptosis by targeting GPX4 in hippocampal neurons

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Cell culture

2.3. Cytotoxicity assays

2.4. Measurement of lipid peroxidation

2.5. Detection of intracellular malondialdehyde (MDA)

2.6. Measurement of intracellular iron levels

2.7. Measurement of total glutathione

2.8. Western blot analysis

2.9. Real-time polymerase chain reaction (RT-PCR) analysis

2.10. Animal experiments

2.11. Cresyl violet staining (Nissl staining) of hippocampal tissues

2.12. Statistical analysis

3. Results

3.1. Biochanin A inhibits glutamate-induced ferroptotic cell death in HT22 cells.

3.2. Biochanin A attenuates glutamate-induced iron accumulation and lipid peroxidation in HT22 cells.

3.3. Biochanin A rescues the levels of GPX4 protein and total glutathione in glutamate-treated HT22 cells.

3.4. Biochanin A inhibits autophagy-mediated GPX4 degradation in HT22 cells.

3.5. Biochanin A regulates expression of proteins associated with iron metabolism in HT22 cells.

3.6. Biochanin A inhibits kainic acid-induced ferroptosis in mouse hippocampal neurons.

4. Discussion

5. Conclusion

Abbreviations

Declarations

Declaration of Competing Interest

Funding

Author Contribution

References

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