Chrysin ameliorates methotrexate-induced hippocampal neurogenesis impairment by suppressing of oxidative stress and upregulating antioxidant enzyme activity in rodents

TL;DR

Chrysin helps protect the brain from methotrexate's harmful effects by reducing oxidative stress and boosting antioxidant activity in rats.

Contribution

This study demonstrates chrysin's neuroprotective effects against methotrexate-induced neurogenesis impairment in rodents.

Findings

Methotrexate reduced antioxidant enzyme activity and caused oxidative stress in rats.

Chrysin reversed methotrexate's negative effects on neurogenesis and antioxidant enzyme activity.

Chrysin improved the expression of proteins related to neurogenesis and antioxidant pathways.

Abstract

Methotrexate (MTX) is used in treating several malignancies. However, MTX neurotoxicity remains a significant clinical side effect, leading to cell division malformation, and neurogenesis impairment. Chrysin, a flavonoid compound found in natural products, demonstrates various biological characteristics, including neuroprotective and antioxidant properties. The purpose of this study was to investigate the ameliorative effect of chrysin on oxidative damage and neurogenesis impairment caused by MTX. Male Sprague-Dawley rats were randomly divided into four groups, including the vehicle, MTX (75 mg/kg), chrysin (10 mg/kg), and chrysin+MTX groups. Chrysin was orally administered for 15 days. MTX was administered intravenously on days 8 and 15. The hippocampal neural stem cells were evaluated using sex determining region Y-box 2 (sox2) and nestin immunofluorescence staining. Antioxidant enzyme expression and the levels of oxidative stress marker were assessed. Additionally, the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), brain-derived neurotrophic factor (BDNF), cAMP-response element binding (CREB), and phosphorylated CREB (pCREB) were evaluated using Western blotting. Results showed that MTX significantly decreased the activity of antioxidant enzymes and produced oxidative stress. MTX also impaired neurogenesis, evidenced by decreased sox2 and nestin-positive cells and decreased expression of Nrf2, BDNF, CREB, and pCREB in the hippocampus and prefrontal cortex. However, chrysin significantly reversed the effects of MTX on these parameters. In conclusion, chrysin exhibits neuroprotective effects against MTX-induced neurogenesis impairment by upregulating antioxidant enzyme activity, reducing oxidative stress, and improving protein expression related to neurogenesis.

Figures

Experimental timeline.The overall drug administration schedule. MTX: methotrexate. LCV: leucovorin.

Effect of chrysin and MTX on biochemical assessment parameters (a-d).Data are shown as mean ± SEM (n = 6/group). #p < 0.05, ##p < 0.01, ###p < 0.005 versus vehicle, *p < 0.05, p < 0.01, *p < 0.005 versus MTX.

The protein expression of Nrf2 (68 kDa, a-b) and BDNF (14 kDa, c-d).Data are shown as mean ± SEM (n = 6/group). #p < 0.05, ##p < 0.01, ###p < 0.005 versus vehicle, *p < 0.05, p < 0.01, *p < 0.005 versus MTX.

Effect of chrysin and MTX on CREB, pCREB (43 kDa, a-b) protein expression, total CREB density (c-d) and pCREB/total CREB ratio (e-f) in the hippocampus and prefrontal cortex.The results are reported as mean ± SEM (n = 6/group). #p < 0.05, ##p < 0.01, ###p < 0.005 versus vehicle, *p < 0.05, p < 0.01, *p < 0.005 versus MTX.

Effects of chrysin and MTX on sox2 immunofluorescence staining in the hippocampus.Sox2 positive cells (red, white arrow) co-labeled with DAPI nuclear staining marker (blue) (A-D). The results showed significantly decreased sox2 cells in the MTX group versus the vehicle group (E). Chrysin ameliorated the effect of MTX by increasing sox2 cells in the chrysin+MTX group. The results are reported as mean ± SEM (n = 6/group). #p < 0.05 versus vehicle, p < 0.01 versus MTX.

Effects of chrysin and MTX on nestin immunofluorescence staining in the hippocampus.Nestin-positive cells were labeled green as shown at the white arrow head (A-D). The section was counterstained with propidium iodide (red). The results showed chrysin significantly improved the nestin expression versus the MTX group (E). Data are shown as mean ± SEM (n = 6/group). ####p < 0.0001 versus vehicle, *p < 0.001 versus MTX.

Full text

Introduction

Chemotherapy-induced memory impairment, also called “chemo-brain” or “chemofog”, describes changes in cognitive function reflecting systemic chemotherapy’s central nervous system toxicity. Methotrexate (MTX), a widely used chemotherapy drug [1], penetrates cells and blocks the enzyme dihydrofolate reductase, which plays a critical role in the synthesis of DNA and RNA, leading to the inhibition of cell proliferation, a mechanism that has also been implicated in the development of MTX-induced neurotoxicity [2]. In Thailand, a retrospective study conducted between 1990 and 2021 reported that the incidence of neurotoxicity following MTX administration in cancer patients was 5.22% [3]. These findings provide important reference data, indicating that MTX-induced neurotoxicity is not uncommon and represents a clinically significant concern that warrants careful attention. In addition, previous studies have demonstrated that MTX treatment can cause cognitive impairments and reduce the number of immature neurons, cell survival, and proliferation in hippocampal dentate gyrus through the induction of oxidative stress [4–6].

Adult neural stem cells (NSCs) are undifferentiated cells responsible for generating new neurons, a process called neurogenesis [7]. Several factors control the generation of NSCs, such as brain-derived neurotrophic factor (BDNF), which is essential for neuronal survival, synaptic plasticity, and neuronal function [8]; sex-determining region Y-box 2 (sox2), a transcription factor involved in maintaining the properties of neural progenitor stem cells [9] and nestin, a cytoskeleton protein that regulates and remodels the cell cytoskeleton during development [10].

Nowadays, natural products are introduced to reduce complications that occur after chemotherapy. Chrysin (5,7-dihydroxyflavone) is a flavonoid naturally found in propolis, honey, and various plant species like Oroxylum indicum, Passiflora incarnata, and Pelargonium crispum [11,12]. Many studies have demonstrated biological qualities of chrysin, including anti-inflammatory, neuroprotective, anti-apoptotic, and cognitive-improving properties [13]. Additionally, chrysin has strong antioxidant properties [14], enhancing antioxidant enzyme activities [15] and decreasing lipid peroxidation [16,17]. Chrysin also attenuated memory decline by enhancing the level of BDNF in the hippocampus and prefrontal cortex and ameliorated learning and memory impairment by increasing cell proliferation, immature neurons, and cell survival in the sub granular zone (SGZ) of the hippocampus [18].

Therefore, the objective of this study was to investigate the neuroprotective effects of chrysin on hippocampal neurogenesis, with particular emphasis on neural stem cells. In addition, this study aimed to examine the effects of chrysin on key mechanisms related to oxidative stress and antioxidant enzyme activity in MTX-treated rats.

Materials and methods

Chemicals

Chrysin (5, 7-Dihydroxyflavone, 97% purity, lot number: WXBD6380V) was purchased from Sigma-Aldrich, Inc., USA. MTX (Emthexate PF, Batch number: NN1175A) contains a sterile isotonic solution of the sodium salt of MTX without preservatives. Leucovorin (LCV; Rescuvolin, Batch number: 20C25KI) contains calcium folinate, the formyl derivative of tetrahydro folic acid in the form of calcium salt. LCV was administered as a folinate rescue therapy in a treatment with MTX. MTX and LCV were sourced from Pharmachemie B.V., Netherlands.

Animals and experimental protocols

Male Spraque-Dawley rats, five weeks old and weighing 180−200 g, were procured from Nomura Siam International Company Limited, Bangkok, Thailand. The animals were housed under controlled condition with a 12-hour light/dark cycle at a temperature of 23 ± 2 °C at the Northeast Laboratory Animal Center, Khon Kaen, Thailand. The animals were randomly assigned to four groups: the vehicle group (received 0.9% saline solution, propylene glycol 1 ml/kg, and LCV), the MTX group (administered MTX 75 mg/kg BW via intravenous injection on days 8 and 15, as described in previous studies [19,20]), the chrysin group (received chrysin 10 mg/kg BW dissolved in propylene glycol (Ajax Finechem Pty Ltd., Australia) via oral gavage for 15 days) [21], and the chrysin+MTX group. For rats treated with MTX, LCV was administered by intraperitoneal injection at 6 mg/kg BW after 18 hours and at 3 mg/kg BW at 26, 42, and 50 hours for reducing the side effects of MTX (Fig 1). Following drug administration, the animals were euthanized by cervical dislocation and decapitation. The brains were extracted and divided into two hemispheres. One hemisphere was fixed in a 30% sucrose solution for 5 hours, then embedded in an optimal cutting temperature (OCT) compound (Thermo Fisher Scientific Inc., Germany), snap-frozen in liquid nitrogen, and stored at −80 °C for the immunofluorescence analysis. The hippocampus and prefrontal cortex from the other hemisphere were isolated for biochemical evaluation and Western blot analysis.

Experimental timeline.The overall drug administration schedule. MTX: methotrexate. LCV: leucovorin.

Ethic approval

All experimental procedures adhered to the ARRIVE and IACUC guidelines for the Care and Use of Laboratory Animals. This study was approved by the Khon Kaen University Ethics Committee in Animal Research (Approval number: IACUC-KKU-119/64).

Biochemical assessments

The tissue of the hippocampus and prefrontal cortex was homogenized in deionized water using a pellet pestle on ice. The samples were centrifuged at 13,000 rounds per minute at 4°C for 10 minutes. The supernatant was collected, and protein concentration was measured using a nanodrop spectrophotometer (Thermo Fisher Scientific, USA). The samples were loaded in triplicate into a 96-well plate. Optical density for all biochemical analyses was assessed using a microplate reader (Tecan, Austria GmbH, Austria) in conjunction with Magellan data analysis software. Each assessment was replicated 3 times.

Superoxide dismutase (SOD) activity.

The measurement of the SOD activity was performed as previously described [22]. The samples were added with 10.7 mM ethylenediaminetetraacetic acid solution, 216 mM potassium phosphate buffer (pH 7.8), 1.1 mM cytochrome C solution, 0.108 mM xanthine solution, and xanthine oxidase enzyme (XOD) solution. After that, the absorbance was determined at 540 nm at 0 and 5 minutes. The SOD enzyme solution (Sigma Aldrich, Inc., USA) was used as a standard solution. The SOD level was calculated from standard curve and expressed as unit per milligram protein (unit/mg protein).

Glutathione peroxidase (GPx) activity.

The protocol of GPx activity assessment was applied from a previous report [22]. The samples were mixed with a cocktail containing sodium phosphate buffer, sodium azide solution, glutathione solution, β-Nicotinamide adenine dinucleotide phosphate, and glutathione reductase (GR) solution (100 units/ml). Then, 5,5’-Dithiobis-(2-nitrobenzoic acid) solution was added. The mixture was incubated for 10 minutes in the dark room. After that, a 30% H_2_O_2_ solution was added, and then the color reaction was determined at 405 nm. The GPx enzyme solution (Sigma Aldrich, Inc., USA) (20 unit/ml) was used as a standard solution. The GPx level was calculated from the standard curve and expressed as unit/mg protein.

Catalase (CAT) activity.

The protocol for the assessment of the CAT enzyme activity was followed and applied from the previous report [22]. The samples were mixed with a sulfuric acid solution, a 30% H_2_O_2_ solution, and 50 mM potassium phosphate buffer. The mixture was incubated in a dark room for 10 minutes, followed by adding a potassium permanganate solution. The absorbance was determined at 540 nm. The CAT enzyme solution (100 units/ml) was used as a standard. The CAT level was represented as units per mg protein.

Malondialdehyde (MDA) levels.

The MDA level measurement protocol was applied from a previous study [20]. The sample was mixed with a cocktail solution (8.1% sodium dodecyl sulfate, 0.8% thiobarbituric acid, and 20% acetic acid solution) and then heated at 95 °C for 1 hour. The mixture was mixed with n-Butanol and pyridine. After centrifugation (4,000 round per minute, 25°C, 10 minutes), the absorbance was determined at 540 nanometers (nm). 1,1,3,3-Tetraethoxypropane (TEP) was used as a standard. The MDA level was expressed as nanomoles per mg.

Western blotting

The hippocampus and prefrontal cortex tissues were homogenized in lysis buffer (pH 7.6, 1 ml/100 mg) for 10 minutes. The samples were centrifuged at 13,000 rounds per minute for 10 minutes at 4°C. The supernatant was collected, and the protein concentration was evaluated using a nanodrop spectrophotometer and then incubated with 5% bovine serum albumin diluted in Tris-buffer saline 0.1% tween-20 blocking solution for 60 minutes. The protein samples (30 µg) were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was probed at 4 °C overnight with rabbit polyclonal nuclear factor erythroid 2-related factor 2 (Nrf2) antibody or rabbit recombinant monoclonal BDNF antibody (1:1000, Abcam, USA) or mouse monoclonal cAMP-response element binding (CREB) or phosphorylated CREB (pCREB) antibody (1:1000, Santa Cruz Biotechnology, USA) or GAPDH antibody (1:20,000, Abcam, USA). On the following day, the membrane was treated with anti-rabbit or anti-mouse secondary antibody (1:2000, DAKO, Denmark) for 60 minutes. The membrane was treated in ECL solution (GE healthcare Amersham, UK) for visualizing the protein bands. Finally, the protein bands analysis was performed using ImageQuant^TM^400 (GE Healthcare Life Science, Piscataway, NJ, USA) and the image of protein bands was quantified as the relative optical density using Image J software following previously protocol [19]. The relative optical density percentages of Nrf2, BDNF, CREB, and pCREB were compared to GAPDH (internal control).

Immunofluorescence assays

The frozen brain was sectioned at a thickness of 40 µm using a cryostat (Cryostat Series HM 550 Microm International; Walldorf, Germany) to obtain the entire dentate gyrus of the hippocampus. The sections were collected every 8^th^ section (a total of 9 sections per brain) for sox2 and nestin immunofluorescence staining. For sox2 staining, the sections were blocked with 10% normal goat serum (Abcam, USA) for 1 hour and then incubated overnight at 4 °C with sox2 primary antibody (1:400, Abcam, USA) diluted in the blocking solution. The following day, the sections were treated with Alexa Fluor 568 goat anti-rabbit secondary antibody (1:500, Invitrogen, USA) for 1 hour. Counterstain was performed using DAPI staining solution for 30 seconds. For nestin staining, the sections were blocked with 1% bovine serum albumin (Sigma-Aldrich Inc., USA.) for 1 hour and then incubated overnight at 4 °C with nestin primary antibody (1:400, Merck Milipore, Germany). Alexa Fluor 488 rabbit anti-mouse secondary antibody (1:500, Invitrogen, USA) was treated to sections for 1.5 hours. Counterstain was performed using propidium iodide for 30 seconds. The positive cells were counted in the subgranular zone of the hippocampus under 40X magnification of an immunofluorescence microscope (Nikon Eclipse 80i, USA). The sum of the counted cells from 9 sections is multiplied by 8.

Statistical analysis

All data were evaluated using GraphPad Prism (Version 8.0; GraphPad Software Inc., San Diego, CA, USA). The results were reported as mean ± standard error of the mean (SEM), n = 6. The analysis of cell counting in the immunofluorescence study, the determination of biochemical assessments, and the percentage of relative optical density in western blotting were performed using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc test. Statistical significance was set at p-value <0.05.

Results

Chrysin prevented oxidative stress and up-regulated antioxidant enzyme activity against MTX

SOD levels.

SOD is a key enzyme in the antioxidant defense mechanism, playing a crucial role in maintaining equilibrium between ROS and antioxidants. We found that MTX significantly decreased SOD activity in the hippocampus and prefrontal cortex [hippocampus: F(3, 21) = 9.794, p < 0.001; prefrontal cortex: F(3, 26) = 5.825, p < 0.05, Fig 2a, S1 Table]. Additionally, the SOD activity in the chrysin+MTX group was significantly higher than in the MTX group (Bonferroni’s post hoc test, p < 0.01). This suggests that chrysin up-regulated SOD activity in the hippocampus and prefrontal cortex against MTX.

*Effect of chrysin and MTX on biochemical assessment parameters (a-d).Data are shown as mean ± SEM (n = 6/group). #p < 0.05, ##p < 0.01, ###p < 0.005 versus vehicle, *p < 0.05, **p < 0.01, **p < 0.005 versus MTX.

GPx levels.

GPx is the defensive antioxidant enzyme responsible for converting H_2_O_2_ to water. In the hippocampus and prefrontal cortex tissue, GPx levels in the MTX group were significantly lower versus the vehicle group [hippocampus: F(3, 23) = 10.60, p < 0.01; prefrontal cortex: F(3, 22) = 5.505, p < 0.05, Fig 2b, S1 Table]. However, co-administration with chrysin induced a significant elevation of GPx activities versus the MTX group (Bonferroni’s post hoc test, p < 0.001). This data further confirms the antioxidant properties of chrysin against MTX.

CAT levels.

The function of the CAT enzyme is to convert H_2_O_2_ into water and oxygen. MTX significantly reduced the activity of CAT compared to the vehicle [hippocampus: F(3, 20) = 7.652, p < 0.01; prefrontal cortex: F(3, 24) = 8.653, p < 0.01, Fig 2c, S1 Table]. Interestingly, the results demonstrated a significant increase in CAT concentration in animals co-treated with chrysin versus the MTX group (Bonferroni’s post hoc test, p < 0.001), highlighting the antioxidative potential of chrysin against MTX-induced oxidative stress.

MDA levels.

In this study, MTX significantly raised MDA concentration compared to the vehicle group [hippocampus: F(3, 20) = 17.32, p < 0.001; prefrontal cortex: F(3, 22) = 6.678, p < 0.01, Fig 2d, S1 Table]. On the other hand, the MDA levels in the hippocampus and prefrontal cortex tissues of the chrysin and co-treated groups were significantly reduced versus the MTX group (Bonferroni’s post hoc test, p < 0.05). These results indicate that MTX induces oxidative stress, while chrysin ameliorates oxidative stress, resulting in reduced MDA levels.

Chrysin enhanced Nrf2, BDNF, CREB and pCREB proteins expression

In the hippocampus, MTX significantly reduced the expression of Nrf2 [F(3, 22) = 6.732, p < 0.001, Fig 3a], BDNF [F(3, 24) = 2.130, p < 0.001, Fig 3c], CREB [F(3, 20) = 0.4667, p < 0.01, Fig 4a and c], pCREB [F(3, 20) = 0.6133, p < 0.05, Fig 4a], and pCREB/total CREB ratio (p < 0.05, Fig 4e) versus the vehicle group. Similarly, MTX significantly decreased the expression of Nrf2 [F(3, 24) = 2.333, p < 0.001, Fig 3b], BDNF [F(3, 20) = 0.6441, p < 0.01, Fig 3d], CREB [F(3, 25) = 0.4247, p < 0.01, Figs 4b and d], pCREB [F(3, 20) = 2.803, p < 0.001, Fig 4b], and pCREB/total CREB ratio (p < 0.05, Fig 4f) in the prefrontal cortex. However, co-administration with chrysin effectively improved the protein expression of Nrf2 (p < 0.05 and p < 0.01), CREB (p < 0.01), BDNF (p < 0.05 and p < 0.01), pCREB (p < 0.05), and pCREB/total CREB ratio (p < 0.01 and p < 0.05) compared to MTX alone (S2 Table).

*The protein expression of Nrf2 (68 kDa, a-b) and BDNF (14 kDa, c-d).Data are shown as mean ± SEM (n = 6/group). #p < 0.05, ##p < 0.01, ###p < 0.005 versus vehicle, *p < 0.05, **p < 0.01, **p < 0.005 versus MTX.

*Effect of chrysin and MTX on CREB, pCREB (43 kDa, a-b) protein expression, total CREB density (c-d) and pCREB/total CREB ratio (e-f) in the hippocampus and prefrontal cortex.The results are reported as mean ± SEM (n = 6/group). #p < 0.05, ##p < 0.01, ###p < 0.005 versus vehicle, *p < 0.05, **p < 0.01, **p < 0.005 versus MTX.

Chrysin improved neurogenesis impairment against MTX

An immunofluorescence assay was conducted to evaluate the hippocampal neural stem cells. The results demonstrated that MTX significantly decreased the number of sox2 and nestin positive cells in comparison with the vehicle group [sox2: F(3, 20) = 6.238, p < 0.05, Figs 5B and E; nestin: F(3, 20) = 15.9, p < 0.0001, Figs 6E and B]. Interestingly, chrysin attenuated the effect of MTX by increasing the sox2 and nestin-positive cells in the co-treated group (Bonferroni’s post hoc test, p < 0.01, Figs 5D-E and 6D-E). However, chrysin alone showed no significant differences in the number of sox2 and nestin positive cells when compared to the vehicle group (sox2: p > 0.05, Figs 5C and E; nestin: p > 0.05, Figs 6C and E) (S3 Table).

*Effects of chrysin and MTX on sox2 immunofluorescence staining in the hippocampus.Sox2 positive cells (red, white arrow) co-labeled with DAPI nuclear staining marker (blue) (A-D). The results showed significantly decreased sox2 cells in the MTX group versus the vehicle group (E). Chrysin ameliorated the effect of MTX by increasing sox2 cells in the chrysin+MTX group. The results are reported as mean ± SEM (n = 6/group). #p < 0.05 versus vehicle, *p < 0.01 versus MTX.

*Effects of chrysin and MTX on nestin immunofluorescence staining in the hippocampus.Nestin-positive cells were labeled green as shown at the white arrow head (A-D). The section was counterstained with propidium iodide (red). The results showed chrysin significantly improved the nestin expression versus the MTX group (E). Data are shown as mean ± SEM (n = 6/group). ####p < 0.0001 versus vehicle, **p < 0.001 versus MTX.

Discussion

MTX is a folic acid antagonist that is widely used as a chemotherapy agent in several types of cancer [1]. MTX produces neurotoxicity by actively entering cells through the reduced folate carrier (RFC) [23]. Prior studies have demonstrated that MTX chemotherapy at a dosage of 75 mg/kg can lead to cognitive impairment and decrease cell survival and cell proliferation in the hippocampus [5,6], which results in memory deficits [24]. The current study is designed to ascertain the protective impact of chrysin against MTX-induce oxidative damage and neurogenesis impairment. We found that MTX downregulated antioxidant enzyme activity and induced oxidative stress. Furthermore, MTX led to neurogenesis impairment, evidenced by a decrease in sox2 and nestin cells in the SGZ and reduced Nrf2, BDNF, CREB, and pCREB proteins expression in the hippocampus and prefrontal cortex. In this study, immunofluorescence staining was performed to investigate the effects of MTX and chrysin on the expression of sox2 and nestin in the SGZ of the hippocampus. Sox2 transcription factors is expressed in multipotent NSCs and plays a crucial role in somatic cell reprogramming, reversing the epigenetic configuration of differentiated cells back to a pluripotent embryonic state [25]. Nestin, a class VI intermediate filament protein, is expressed in NSCs and type I progenitor cells and regulates the remodeling of the cell cytoskeleton during development [10]. Prior evidence suggests that MTX dramatically reduces the expression of sox2 and nestin-positive cells in the SGZ of the hippocampal dentate gyrus [26,27], a finding that is consistent with those of our study. Conversely, chrysin co-treatment significantly increased the number of sox2 and nestin-positive cells. This study is the first to demonstrate that chrysin improves the expression of sox2 and nestin against MTX-induced NSC damage in the SGZ. Our previous studies have also found that chrysin improves neurogenesis by increasing cell proliferation, immature neurons, and cell survival in the SGZ of the hippocampus in MTX-treated rats. Moreover, behavioral testing revealed that chrysin mitigates impairments in both spatial and recognition memory against MTX [21].

Oxidative stress is an imbalance between antioxidant defenses and the generation of ROS, resulting in interference with redox signaling and lead to molecular damage [28]. Abnormal conditions in the neurological system, such as brain injury, aging, and neurodegenerative disorders, have been linked to the buildup of ROS [29–31]. One of the targets of ROS is polyunsaturated fatty acids (PUFA). The oxidative damage of PUFA leads to the generation of reactive aldehyde products such as MDA, making MDA a reliable marker of oxidative stress [32].

The antioxidant defensive system plays a role in maintaining equilibrium between ROS and antioxidants. Enzymatic defenses such as SOD, CAT, and GPx, constitute the major antioxidant which protect cells from ROS-induced damage by removing free radicals and converting oxidative products into water [33]. Numerous investigations have discovered that MTX increases oxidative stress and inhibits the activity of antioxidant enzymes in the hippocampus and prefrontal cortex [19,20,22]. These findings are consistent with this study. MTX significantly downregulated SOD, CAT, and GPx activities and increased MDA levels, demonstrating the detrimental impact of MTX on antioxidant function.

Chrysin is a natural flavonoid compound with strong antioxidant properties. Previous research has found that chrysin enhances the activities of antioxidant enzymes, including SOD, GPx, and CAT [34,35], and decreases MDA levels, a common indicator of lipid peroxidation [16,17]. Additionally, chrysin can prevent cell damage by eliminating free radicals that contribute to lipid peroxidation [36]. In this study, chrysin exhibited a positive impact on the co-treated group’s response to MTX by enhancing the activity of SOD, CAT, and GPx and lowering MDA levels. This indicates that chrysin improved antioxidant enzyme activity and reduced oxidative stress in the hippocampus and prefrontal cortex. The possible mechanism of antioxidant activity may be due to chrysin’s molecular structure. The hydroxyl groups at positions 5 and 7 give chrysin a stable structure. After the hydroxyl groups degrade, they attach to free radicals to form stable free radical molecules, thereby mitigating oxidative stress [37]. Additionally, chrysin can alter the nitric oxide (NO) pathway, which is involved in the generation of ROS [38].

Nrf2 is involved in drug detoxification and the oxidative response, protecting cells against toxic and oxidative assaults. Under typical conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm. Under conditions of oxidative stress, ROS causes modifications to Keap1, resulting in the translocation of Nrf2 into the nucleus. Nrf2 then binds to the antioxidant response elements (ARE) to initiate the transcription of antioxidant genes, reducing oxidative stress [33]. The downregulation of Nrf2 in the hippocampus and prefrontal cortex that we observed in the MTX-treated group is consistent with previous reports [19,20,22]. However, chrysin mitigated this effect, thus confirming its antioxidant effect against MTX-induced oxidative stress [34]. The underlying mechanism of chrysin on Nrf2 activation is still unclear. Nevertheless, it has been reported that additional flavonoids, such as epigallocatechin-3-gallate (EGCG), may activate Nrf2 by phosphorylation through the activation of protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), or mitogen-activated protein kinase (MAPK) pathways [37].

BDNF is a member of the neurotrophic family of growth factors. BDNF has several functions in the CNS, including maintaining neuronal survival, enhancing synaptic transmission, synaptic plasticity, and overall neuronal function [37,38]. Evidence suggests that BDNF is also involved in neurogenesis by supporting the differentiation, maturation, and survival of neurons, as well as modulating dendritic growth and lengthening [8,39,40]. BDNF binds with the tropomyosin receptor kinase B (TrkB) receptor, activating several transduction cascades. These include the insulin receptor substrate 1 (IRS-1)/PI3K/protein kinase B (AKT) pathway, which plays a role in activating pro-survival genes, the phospholipase C (PLC)/diacylglycerol (DAG)/inositol 1,4,5-triphosphate (IP3) pathway, which regulates intracellular calcium concentrations and neurite growth, and the RAS/mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERKS) pathway, which is essential for neurogenesis and promoting survival [41]. BDNF is regulated by several transcription factors. cAMP-response element binding (CREB) is one of the transcription factors that play roles in neuronal survival, migration, synaptogenesis modulation, and long-term potentiation activation [42,43]. According to Esvald et al., the CREB family is the major regulator of BDNF gene expression [44]. After neuronal membrane depolarization, voltage-gated calcium channels open, causing an influx of calcium ions into the cell [45]. These calcium ions bind to the calcium response element (CRE) within the BDNF gene, triggering the phosphorylation of CREB and activating BDNF transcription [46]. A previous study demonstrated that pCREB and BDNF are critical factors that enhance cognitive performance and hippocampal neurogenesis during brain development [47], which is consistent with the immunofluorescence results of this study.

In our study, the MTX group reported a decrease in BDNF, CREB, and pCREB levels in the hippocampus and prefrontal cortex, in line with previous findings [25,48]. By contrast, chrysin significantly enhanced BDNF, CREB, and pCREB proteins expression against MTX. This indicates the beneficial effects of chrysin on the expression of proteins related to neurogenesis and neuronal survival. A possible mechanism is that chrysin can regulate membrane estrogen receptor alpha (Erα) and beta (Erβ), which can activate the MAPK/ERK1/2 signaling cascade involved in phosphorylation and subsequent CREB activation, thus promoting an increase in BDNF levels [49,50].

In conclusion, chrysin treatment alleviated neurogenesis impairment by increasing sox2 and nestin expression in the hippocampal dentate gyrus. Furthermore, chrysin enhanced antioxidant enzyme activity, inhibited lipid peroxidation, and improved Nrf2, BDNF, CREB, and pCREB proteins expression. These findings support the neuroprotective and antioxidant properties of chrysin against MTX chemotherapy. Therefore, chrysin could be a potential adjuvant for reducing neurotoxic effects in cancer patients undergoing MTX chemotherapy.

Limitations

The oxidative stress markers were measured in total hippocampal and prefrontal cortex homogenates, which do not allow for assessment specifically within neural stem cells. Future studies targeting oxidative stress at the level of neural stem cells are warranted to provide a more precise understanding of MTX-induced neurotoxicity and the neuroprotective effects of chrysin.

Supporting information

S1 DataRaw data for all figures.(XLSX)

S1 Raw imagesThe original uncropped Western blot image of Nrf2 (68 kDa), BDNF (14 kDa), CREB (43 kDa), pCREB (43 kDa) and GAPDH (internal control; 36 kDa) proteins expression in the hippocampus and prefrontal cortex tissues.(PDF)

S1 TableEffect of chrysin and MTX on antioxidant enzymes activity and oxidative status in the hippocampus and prefrontal cortex.(PDF)

S2 TableEffect of chrysin and MTX on the protein expression of Nrf2, BDNF, CREB, and pCREB/CREB ratio in the hippocampus and prefrontal cortex.(PDF)

S3 TableQuantification data of sox2 and nestin positive cells.(PDF)