Application of inhibitor of Bach1 in preparation of medicine for treating chemotherapy brain injury induced by chemotherapy medicine

Abstract

The invention discloses an application of a Bach1 inhibitor in preparation of a therapeutic drug for chemotherapy brain injury induced by a chemotherapy drug. The Bach1 inhibitor is prepared from 5-aminolevulinic acid or si-Bach1. In vivo and in vitro studies prove that administration of 5-ALA and overexpression of ALAS2 can prevent harmful effects of doxorubicin, including neuronal synaptic reduction, apoptosis, neurogenesis impairment, oxidative stress and cognitive impairment. The treatment with the 5-ALA or Bach1 inhibitor can enhance Nrf2 activation, reduce oxidative stress response, mitochondrial dysfunction and doxorubicin-induced neuron death. Research results of the invention show that a new strategy for resisting oxidative stress is represented by using 5-ALA to target a Bach1 / Nrf2 pathway, and a powerful neuroprotection effect is provided for doxorubicin-induced neurotoxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of a Bach1 inhibitor in the preparation of a therapeutic drug for chemotherapy-induced brain damage. Background Art

[0002] Doxorubicin (DOX) is a potent anthracycline antineoplastic agent that is widely used in the treatment of malignancies such as breast and esophageal cancer. However, its therapeutic use is greatly limited due to its potential to induce cytotoxicity, particularly affecting the heart and brain. Although the cardiotoxic effects of doxorubicin are well recognized, chemotherapy-induced cognitive impairment, commonly known as "chemo brain," has not received sufficient attention, perhaps due to its subtle symptoms. Although DOX has a limited ability to cross the blood-brain barrier (BBB), long-term administration can still lead to neurotoxicity and may induce neurodegenerative diseases. Notably, up to 70% of cancer survivors receiving chemotherapy, including those treated with DOX, report experiencing chemo-stroke.

[0003] Currently, the main clinical treatments for chemotherapy brain include drug therapy and cognitive behavioral therapy, such as modafinil, methylphenidate, donepezil, astaxanthin, resveratrol, catechins and Ginkgo biloba.

[0004] Numerous studies have shown that DOX can elevate peripheral oxidative stress markers and inflammatory cytokines, which can cross the BBB via receptor-mediated endocytosis and induce central nervous system toxicity. In addition, DOX-induced chemo-brain is associated with DNA damage and disruption of synaptic plasticity. Despite these findings, the comprehensive mechanisms of DOX-induced chemo-brain remain to be fully elucidated. Therefore, the combination of transcriptomics and metabolomics provides a powerful approach to elucidate the interaction between gene expression and cellular metabolism and enhance our understanding of biological processes. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of a Bach1 inhibitor in the preparation of a therapeutic drug for chemotherapy-induced brain damage. The technical solution implemented by the present invention is:

[0006] In one aspect, the present invention provides the use of a Bach1 inhibitor in the preparation of a therapeutic drug for chemotherapy-induced brain damage.

[0007] The inhibitor of Bach1 includes 5-aminolevulinic acid (5-ALA) or si-Bach1.

[0008] Among them, 5-aminolevulinic acid alleviates doxorubicin-induced neurotoxicity.

[0009] Among them, 5-aminolevulinic acid alleviates cognitive impairment caused by doxorubicin-induced chemo-brain.

[0010] Furthermore, 5-aminolevulinic acid alleviated cognitive impairment in doxorubicin-induced chemo-brain via the Bach1 / Nrf2 signaling pathway.

[0011] Among them, si-Bach1 can inhibit Bach1 and thus activate Nrf2.

[0012] The inhibitor of Bach1 is hexaenoyl pyrophosphate (HPPE).

[0013] The chemotherapy drugs include doxorubicin and methotrexate.

[0014] Furthermore, the chemotherapy drug is doxorubicin DOX.

[0015] In another aspect, the present invention also provides a pharmaceutical composition comprising a Bach1 inhibitor as an active ingredient.

[0016] Furthermore, the pharmaceutical composition also contains an excipient.

[0017] The preliminary comprehensive analysis of the present invention showed that DOX downregulated the expression of aminolevulinic acid synthase 2 (ALAS2) gene, resulting in decreased levels of 5-aminolevulinic acid (5-ALA) in mouse hippocampal tissue. As a precursor for heme synthesis, 5-ALA catalyzed by the ALAS enzyme has multifaceted benefits, such as anti-inflammatory and antioxidant capabilities, and has demonstrated potential in anti-tumor and neuroprotective applications. The therapeutic potential of 5-ALA may be related to its ability to inhibit BTB and CNC homolog 1 (Bach1), a protein that is critical in the management of oxidative stress, mitochondrial function, heme homeostasis, immune response, and cancer metastasis progression. Bach1 is believed to be in the regulation of genes that respond to oxidative stress due to its competitive interaction with Nrf2. The Bach1 / Nrf2 pathway in the brain is hypothesized to provide protection against neurodegenerative diseases.

[0018] This study first determined which type of nerve cells were mainly affected by the downregulation of ALAS2 expression caused by DOX, and then clarified that supplementing 5-ALA or enhancing ALAS2 expression could improve doxorubicin-induced neural damage. Finally, this study explored that the protective effect of 5-ALA depends on the Bach1 / Nrf2 pathway. The findings of the present invention pave the way for the development of potential therapeutic strategies for DOX-induced chemo-brain.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention provides the use of Bach1 inhibitors in the preparation of therapeutic drugs for chemo-induced chemo-brain. In vivo and in vitro studies have confirmed that 5-ALA administration and ALAS2 overexpression can prevent the harmful effects of doxorubicin, including neuronal synaptic reduction, cell apoptosis, impaired neurogenesis, oxidative stress and cognitive impairment. 5-ALA or Bach1 inhibitor treatment can enhance Nrf2 activation and reduce oxidative stress response, mitochondrial dysfunction and doxorubicin-induced neuronal death.

[0021] (2) The results of the present invention indicate that targeting the Bach1 / Nrf2 pathway with 5-ALA represents a new strategy for anti-oxidative stress and provides a strong neuroprotective effect against DOX-induced neurotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 , comprehensive metabolomics and transcriptomics analysis showed that ALAS2 and 5-ALA were deficient in the hippocampus tissue of mice after DOX treatment. A is a volcano plot depicting differentially expressed genes. B is a cluster analysis heat map showing significant changes in genes. C is the GO pathway analysis of differentially expressed genes. D is the PPI network enrichment GO pathway analysis. E is the GSEA result of differentially expressed genes. F is the network PPI analysis of differentially expressed genes. G is a volcano plot depicting the differential expression of metabolites. H is the KEGG pathway enrichment analysis of differentially expressed metabolites. Data are Mean±SD. p values ​​are from unpaired Student's t-test.

[0024] Figure 2 , ALAS2 deficiency was only present in neurons, but not in DOX-treated neurons or astrocytes and microglia in the mouse hippocampus. A is a representative Western blot and statistical analysis of ALAS2 protein levels in mouse hippocampus. B is the relative expression of ALAS2 mRNA in mouse hippocampus. C is the colocalization of ALAS2 and neuronal marker MAP2. D is the colocalization of microglial marker Iba-1 and E is the colocalization of astrocyte marker GFAP in mouse hippocampus. F is the corresponding statistical analysis; scale bar: 10 μm. GH is the colocalization of ALAS2 and MAP2 in primary neurons, and the corresponding statistical analysis is performed; scale bar: 10 μm. IJ is a representative Western blot and statistical analysis of ALAS2 protein levels in primary neurons. Data are Mean ± SD. p values ​​are from unpaired Student's t test. **p < 0.01 compared with the control group.

[0025] Figure 3 Supplementation of 5-ALA or ALAS2 overexpression can improve DOX-induced neuronal damage and cognitive impairment. A is an image of the swimming tracks of the CTRL, DOX, DOX+5-ALA, and DOX+ALAS2 groups in the MWM test. B is the escape latency during training. CD are representative Western blots and statistical analysis of PSD95 and SYN protein levels in mouse hippocampal tissue. E is a representative Golgi staining image of mouse hippocampal neuron morphology and dendritic spine density. Scale bar: upper 50μm, lower 5μm. FG is a statistical analysis of dendritic branches and spine numbers. H is a representative image of the apoptosis rate of primary neuronal cells detected by flow cytometry. I is a representative example: immunofluorescence image of primary neuronal cells. Scale bar: 20μm. J is a statistical graph of the Sholl analysis results showing the number of intersections per radius around the neuronal body. All data are Mean±SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p<0.01 compared with the control group. Compared with the DOX group, p<0.01.

[0026] Figure 4 , 5-ALA supplementation or ALAS2 overexpression attenuates DOX-induced chemo-brain by promoting the degradation of Bach1. A is a schematic diagram of the hypothesized mechanism of 5-ALA regulation of Bach1. BC is a statistical analysis of the relative content of PpXI and heme. D is a representative immunofluorescence colocalization image of Bach1 and DAPI. Scale bar: 10 μm. EF is an immunoblot image and statistical analysis of Bach1 degradation after CHX inhibition of protein synthesis. G is co-immunoprecipitation of flag-tagged E3 and myc-tagged Bach1. H is a representative immunoblot analysis of Bach1 protein for ubiquitin-mediated proteasomal degradation in the presence and absence of the proteasome inhibitor MG132. All data are Mean ± SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p < 0.01 compared with the control group. p < 0.01 compared with the DOX group.

[0027] Figure 5, 5-ALA or HPPE attenuated oxidative stress by inhibiting Bach1 and further activating Nrf2. AC is a representative image of TUNEL staining and DHE fluorescence of primary neurons and a statistical graph of the percentage of positively stained cells. Scale bar: 20μm. D is a representative image of cell apoptosis rate detected by flow cytometry. E is a flow cytometry detection of intracellular ROS generation. FG is an immunoblot image and statistical analysis of Nrf2 degradation in the presence and absence of Bach1 after CHX inhibition of protein synthesis. H is a representative Western blot of Nrf2 in the cytoplasm and nucleus. I is the relative expression of Nrf2 target genes HO-1, Nqo1, GCLc, and GCLm mRNA. J is an image of the movement trajectory of the NOR test and the exploration time of new and old objects. K is a representative image of DHE fluorescence and TUNEL staining in the mouse hippocampus and a statistical graph of the percentage of positively stained cells. Scale bar: 50μm. All data are Mean±SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **Compared with the control group, p<0.01. Compared with the DOX group, p<0.01.

[0028] Figure 6 , 5-ALA or HPPE alleviated mitochondrial dysfunction in primary neurons treated with DOX. A is the co-immunofluorescence staining of MitoTracker (green) and MitoSOX (red) to detect mitochondrial ROS. Scale bar: 5 μm. B is a representative fluorescence image of JC-1 to detect mitochondrial membrane potential. Scale bar: 10 μm. C is the relative fluorescence intensity of MitoSOX. DE is the relative fluorescence intensity of JC-1 aggregates and JC-1 monomers. F is the statistical analysis of the relative content percentage of ATP. G is the OCR measurement of primary neurons. H is the statistical analysis of baseline respiratory capacity, ATP-coupled respiratory capacity, and maximum respiratory capacity. I is a schematic diagram of the simplified 13C metabolic tracing analysis of uniformly labeled [U-13C6]-glucose. Solid circles, 13C; open circles, 12C. JK is the statistical analysis of the content of 13C-labeled TCA cycle metabolites, including citrate, α-ketoglutarate, fumarate, and malate. L is a transmission electron microscopy image of mitochondria. Scale bar: 1 μm. M is the statistical analysis of the relative content percentage of normal mitochondria. Data are Mean ± SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p<0.01 compared with the control group. p<0.01 compared with the DOX group.

[0029] Figure 7, 5-ALA supplementation can alleviate DOX-induced damage in zebrafish. A is a transmission electron microscopy image of zebrafish mitochondrial morphology. Scale bar: 1μm. B is a statistical analysis of the relative percentage of abnormal mitochondrial content. C is a representative image of apoptotic cells detected by AO staining of zebrafish brain tissue. Scale bar: 50μm. D is a representative fluorescence image of ROS accumulation detected by DCFH-DA staining. Scale bar: 50μm. E is a statistical analysis of the relative percentage of apoptotic cells. F is a quantitative analysis of the relative fluorescence intensity of DCFH-DA. G is a representative track map of zebrafish. All data are Mean±SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p<0.01 compared with the control group. Compared with the DOX group, p<0.01.

[0030] Figure 8 , 5-ALA in the potential pathogenesis of DOX-induced chemical brain.

[0031] Fig. 9 , the quality of the metabolomics model is good. A is a schematic diagram of the experimental process of DOX-treated mouse hippocampal tissue and the comprehensive metabolomics and transcriptomics analysis process. B is an orthogonal partial least squares discriminant analysis (OPLS-DA) diagram of the metabolite data set. C is a permutation test indicating that the quality of the OPLS-DA calculation model is good (R2=(0.0,0.8925),Q2=(0,-0.61)). Data are Mean±SD (n=8), and p values ​​are from unpaired Student's t test.

[0032] Fig.10 , 5-ALA or ALAS2 overexpression can improve DOX-induced neuronal damage and cognitive impairment in the mouse hippocampus. A is a representative trajectory map of mice in OFT. B is the residence time in the central zone. C is a representative image of TUNEL and MAP2 staining in the mouse hippocampus. Scale bar: 30μm. D is a statistical graph of the percentage of tunel-positive cells. E is a representative immunofluorescence image of BrdU and DAPI in the mouse hippocampus. Scale bar: 30μm. F is a statistical graph of the percentage of BrdU-positive cells. G is a representative Western blot and statistical analysis of PSD95 and SYN protein levels in primary neuronal cells. H is a representative image of TUNEL and MAP2 staining in primary neuronal cells. Scale bar: 20μm. Data are all Mean±SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p<0.01 compared with the control group. p<0.01 compared with the DOX group.

[0033] Fig.11, 5-ALA or ALAS2 overexpression can alleviate DOX-induced oxidative stress. AC is the quantification of SOD, MDA levels and relative fluorescence intensity of DHE in mouse hippocampal tissue. D is a representative image of DHE and DAPI. Scale bar: 50μm. EF is the determination of SOD and MDA content in primary neuronal cells. G is the detection of intracellular ROS generation by flow cytometry. All data are Mean±SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p<0.01 compared with the control group. Compared with the DOX group, p<0.01.

[0034] Fig.12 , 5-ALA or HPPE attenuates oxidative stress by activating Nrf2. A is a representative Western blot of Nrf2 and HO-1. B is an image of the swimming tracks of the CON, DOX, and DOX+HPPE groups in the MWM test. C is the escape latency during training. D is a statistical chart of the time spent in the target quadrant. E is the number of crossings beyond the original platform position. F is the relative mRNA expression of Nrf2 target genes HO-1, Nqo1, GCLc, and GCLm in the mouse hippocampus. GJ is a representative fluorescent image and statistical chart of DHE staining of the hippocampus of WT or Nrf2 knockout mice. Scale bar: 30μm. All data are Mean±SD. p values ​​are from one-way ANOVA and post hoc multiple comparisons. **p<0.01 compared with the control group. p<0.01 compared with the DOX group. p<0.01 compared with the DOX+5-ALA group. Compared with the DOX+HPPE group, p<0.01. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] Doxorubicin, a widely used chemotherapeutic drug, is known to cause chemotherapy-induced cognitive impairment, or “chemo-brain,” but the underlying mechanisms are not fully understood. Our combined transcriptomic and metabolomic analysis of doxorubicin-treated mice revealed downregulation of the ALAS2 gene and reduced expression of 5-aminolevulinic acid (5-ALA) metabolites in hippocampal tissue, suggesting perturbations in heme synthesis. Validation confirmed that ALAS2 deficiency was specific to hippocampal neurons, with no changes in astrocytes or microglia. In vivo and in vitro studies confirmed that 5-ALA administration and ALAS2 overexpression protected against the deleterious effects of doxorubicin, including neuronal synaptic loss, apoptosis, impaired neurogenesis, oxidative stress, and cognitive impairment. Doxorubicin disrupts the Bach1 ubiquitin-proteasome degradation pathway by promoting nuclear retention and inhibiting heme synthesis, an effect that was mitigated by 5-ALA or ALAS2 overexpression. In addition, treatment with 5-ALA or Bach1 inhibitors enhanced Nrf2 activation and alleviated oxidative stress, mitochondrial dysfunction, and doxorubicin-induced neuronal death. The zebrafish model further highlighted the protective effects of 5-ALA against doxorubicin-induced neurotoxicity and cognitive impairment. In summary, the Bach1 / Nrf2 pathway plays a key role in doxorubicin-related chemo-brain, and 5-ALA can exert neuroprotective effects by regulating this pathway.

[0037] Embodiment 1,

[0038] 1.1. Animals and drug treatment 8-week-old male C57BL / 6 mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. Nrf2 knockout (Nrf2- / -) mice were prepared and maintained. These mice were adapted to a regulated environment of 22±1°C, 55±5% humidity, and a 12-h light-dark cycle with unlimited access to food and water. All experimental protocols were approved by the Animal Ethics Committee of Jining First People's Hospital and followed the guidelines established by the China Laboratory Animal Care and Use Committee.

[0039] The animal study was conducted using a three-stage design. In the first stage, mice were randomly assigned to a control group (CTRL) and a DOX group to investigate transcriptomic and metabolomic analyses after DOX exposure. Preliminary analysis revealed that the perturbation of the ALAS2 gene and 5-ALA in the mouse hippocampus attracted our attention. In the second stage, to determine whether supplementation with 5-ALA or enhancement of ALAS2 expression could improve cognitive deficits in DOX-exposed mice, mice were randomly divided into four groups: CTRL, DOX, DOX+5-ALA, and DOX+ALAS2 (n=8). In the final stage, to explore the important role of the Bach1-Nrf2 pathway in DOX-induced chemo-brain, mice were divided into the following groups: CTRL, DOX, DOX+5-ALA, DOX+HPPE (hexaenoyl pyrophosphate, a Bach1 inhibitor), and DOX+5-ALA+Nrf2.

[0040] DOX+HPPE+Nrf2 gene knockout group (n=8).

[0041] The DOX group was intraperitoneally injected with DOX every other day at a dose of 2.5 mg / kg for a total of 7 times;

[0042] The DOX+5-ALA group was injected intraperitoneally with 5-ALA (40 mg / kg / day) every day for 14 days, and the DOX treatment was the same as the DOX group;

[0043] The DOX+HEEP group was given intragastric administration at a dose of 10 mg / kg, twice a day, with an interval of 12 hours, for a total of 14 days, and the DOX treatment was the same as the DOX group;

[0044] In the CTRL group, control mice were treated in the same manner with an equal dose of solvent;

[0045] The DOX+5-ALA+Nrf2 knockout group underwent the same operation as the DOX+5-ALA group, except that the mice were replaced with Nrf2 knockout mice. The DOX+HPPE+Nrf2 knockout group underwent the same operation as the DOX+HPPE group, except that the mice were replaced with Nrf2 knockout mice.

[0046] Mice in the DOX group were injected intraperitoneally with DOX (2.5 mg / kg) every two days for a total of 7 injections. For stereotaxic injections, mice were anesthetized with isoflurane and fixed in a stereotaxic apparatus. Subsequently, 1 μL of pAV-FH control vector or pAV-ALAS2 (WZ Biosciences, China) was slowly injected into both sides of the hippocampus (Bregma, 2.3 mm; lateral, 1.8 mm; longitudinal, 2.0 mm for anesthetized mice). The injection was performed using a 10 μL Hamilton syringe with a 30-gauge bevel needle at an injection rate of 0.25 μL / min. After the injection, the needle remained in place for 5 minutes and then gently withdrawn to prevent viral reflux. 5-ALA (40 mg / kg / day) was injected intraperitoneally every day for two consecutive weeks. HPPE (MCE, China) was formulated as a suspension in sodium phosphate buffer, mixed with 1% methylcellulose, PEG400, and 40% dissolving alcohol HS15, and administered orally at a dose of 10 mg / kg twice a day with an interval of 12 hours. In contrast, the control group was administered with an equal volume of the drug-carrying solution without any active substance.

[0047] 1.2. Maintenance of zebrafish and embryo collection Zebrafish (Danio rerio, wild-type AB, 3-4 months old) were purchased from Yaoshunyu Biotechnology Co., Ltd. (Nanjing, China) and males and females were housed separately. Zebrafish were housed in a constant temperature environment of 28 ± 1 °C with a 14 h light / 10 h dark photoperiod per day. Zebrafish were fed freshly hatched brine shrimp every day.

[0048] Embryo collection began the night before the experiment. Zebrafish were placed in an incubator at a 2:1 ratio of male to female. The incubator was then placed in a dark temperature-controlled incubator at 28.5°C. The next morning, the isolation plate was removed to allow natural spawning. One hour after fertilization, embryos were collected in a clean culture dish and gently mixed to minimize differences between groups. After a short incubation, embryos at a similar developmental stage (approximately 4 hours after fertilization) were selected and divided into three groups: CTRL group, 20 μM DOX treatment group, 48 hours, DOX+5-ALA treatment group, 5 mg / L 5-ALA pretreatment for 2 hours, and then 20 μM DOX was added (n = 15 per group). DOX (20 μM) was dissolved in embryo culture water and cultured under a 14-hour light and 10-hour dark environment, and the culture medium was replaced every 24 hours. The control group was cultured in embryo culture water under a 14-hour light and 10-hour dark environment, and the culture medium was replaced every 24 hours.

[0049] 1.3. Cell culture and treatment

[0050] Primary neurons were isolated from newborn C57BL / 6J mice for in vitro culture. Mouse brain blocks were removed using sterile techniques and then minced with scissors. After digestion with 0.125% trypsin (Gibco, Shanghai, China) at 37°C for 30 minutes, trypsin activity was inhibited with DMEM medium containing 10% fetal bovine serum (FBS). The suspension was then filtered through a 400 mesh sieve and centrifuged at 1000 rpm for 5 minutes. Cells were cultured on poly-d-lysine coated plates. Neurobasal medium was used to culture primary neurons. B27 (Gibco, Shanghai, China), streptomycin / penicillin and glutamate were added to the culture medium to provide necessary nutrients.

[0051] The experimental design aimed to evaluate the effect of DOX on ALAS2 by dividing primary neurons into CTRL and DOX groups. The mechanistic study was performed in a two-stage approach. In the first stage, primary neurons were randomly divided into four groups: CTRL, DOX, DOX+5-ALA, and DOX+ALAS2. In the second stage, neurons were divided into CTRL, DOX, DOX+5-ALA, and DOX+HPPE or DOX+siBach1 groups, with five replicates in each group. The DOX group was exposed to 2 mg / mL DOX for 24 h, while the DOX+5-ALA group was pretreated with 200 μM 5-ALA for 2 h before the 24-h DOX exposure. Recombinant adenoviruses for ALAS2 overexpression or Bach1 knockdown were constructed by OriGene, Shanghai, China. The DOX+HPPE group was pretreated with 5 μM HPPE for 2 h before the 24-h DOX exposure. The control group was treated with an equal amount of vehicle solution without any active compound.

[0052] Si-Bach1, Bach1 (Mouse)-3 unique 27mer siRNA duplexes, recombinant adenovirus constructed by OriGene, Shanghai, China, CAT#: SR419715. Cells were transfected according to the manufacturer's instructions.

[0053] 1.4. Transcriptome analysis According to the instructions, RNA was extracted from mouse hippocampal tissue using TRIzol reagent, and its quantity and purity were analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Sequencing libraries were constructed using high-quality RNA samples. These libraries were sequenced on the Illumina NovaSeq 6000 platform to generate 150bp paired-end reads. Subsequently, clean reads were mapped to the mouse reference genome (GRCm38.p6) using HISAT2. Gene expression levels were quantified using FPKM (fragments per kilobase of transcript per million sequenced). The DESeq R software package was used to analyze differentially expressed genes, with P values ​​<0.05 and |log2FC|≥1 as criteria for screening differential genes.

[0054] 1.5. Metabolomics analysis Consistent with previous methods, metabolomics analysis was performed on mouse samples from the CTRL and DOX groups. Each sample (20 mg) was mixed with 400 μL of methanol and acetonitrile (4:1) containing l-2-chlorophenylalanine (4 μg / mL), ground, sonicated, and centrifuged (13000 rpm, 30 min). The supernatant was dried and resuspended for LC-MS / MS analysis. LC-MS / MS was performed on a Dionex Ultimate 3000RS UHPLC system using a Q Exactive+ mass spectrometer, an ACQUITYUPLC HSS T3 column, 45 °C, and a flow rate of 0.35 mL / min. The ESI source alternated between positive and negative ionization modes, and the settings optimized the mass range (100-1200 m / z) and resolution (70,000 for full MS and 17,500 for MS / MS). The data were peak identified and normalized using Progenesis QIV2.3, and then R analysis was performed using orthogonal partial least squares discriminant analysis (OPLS-DA) and Student's t test to identify significant metabolite differences (VIP>1.0, P<0.05).

[0055] 1.6. Immunofluorescence Assay Immunofluorescence staining was performed on frozen hippocampal sections or paraffin-embedded cells. The samples were washed three times with PBS and incubated with primary antibodies in a humidified chamber at 4 °C overnight. The following primary antibodies were used: anti-ALAS2 (Invitrogen; 1:100 dilution), anti-BACH1 (Proteintech; 1:100), anti-GFAP (1:500, Servicebio, Wuhan, China), anti-IBA-1 (1:500, Servicebio, Wuhan, China), anti-MAP2 (1:500, Abcam, Shanghai, China), and anti-Brdu (1:100 dilution). After primary incubation, the sections were treated with secondary antibodies conjugated to Alexa-488 (green, Invitrogen, Shanghai, China), Alexa-594 (red, Invitrogen, Shanghai, China), and Alexa-647 (red, Invitrogen, Shanghai, China) at room temperature in the dark for 2 h. The sections were then mounted with medium containing 50% glycerol. Examination involved five random fields per section and was performed under a fluorescence microscope (Olympus, Tokyo, Japan) by an observer blinded to the experimental groups. Images were captured and analyzed using ImageJ software with standardized settings for all samples.

[0056] 1.7. Immunoprecipitation and Western blotting Co-immunoprecipitation (Co-IP) assays were performed using the Co-IP kit from Absin Bioscience, China, following the manufacturer’s protocol. Cells were transfected with Flag-E3 and / or Myc-Bach1 plasmids. Subsequently, cell lysates were incubated with anti-Flag antibody overnight at 4°C. Samples were immunoprecipitated with anti-Flag antibody and also stored at 4°C overnight. The precipitated proteins were then eluted from the beads and separated by SDS-PAGE. For Co-IP assays without tags, cell lysates were incubated with anti-Bach1 antibody overnight at 4°C. After incubation, proteins were eluted from the beads and analyzed by SDS-PAGE. For protein stability experiments, cells were collected at the 0 h mark before the addition of cycloheximide (CHX). Time points of 2, 4, 6, and 8 h after CHX treatment were then collected using freshly prepared RIPA buffer.

[0057] Western blotting was performed by lysing hippocampal tissue or cultured cells in RIPA buffer and extracting total protein. Protein concentration was determined using a BCA assay kit. Samples (50 μg) were electrophoresed on a 12% SDS-PAGE gel, transferred to a PVDF membrane, and blocked in 10% skim milk for 1 hour at room temperature. Subsequently, the membrane was incubated with the following primary antibodies at 4°C overnight: anti-ALAS2 (1:1000, Invitrogen), anti-PSD95 (1:1000, Santa Cruz), anti-synaptophysin (1:1000, Cell Signaling Tech), anti-BACH1 (1:1000, Proteintech), anti-NRF2 (1:1000, Proteintech), anti-LAMIN B (1:1000, Affinity), anti-ubiquitin (1:1000, Abcam), anti-β-Actin (1:2000, Proteintech), anti-NQO1 (1:1000, Proteintech), anti-HO-1 (1:1000, Cell Signaling Tech), Proteintech). The blot was then washed three times with Tris-buffered salt-tween-20 (TBST) and incubated with secondary antibodies. Finally, the signals were detected using an ECL kit (Thermo Fisher, Shanghai, China). Band intensities were analyzed using Image J software and normalized to GAPDH or β-actin.

[0058] 1.8. Nuclear and cytoplasmic extracts To determine the localization of proteins, cells were separated into cytoplasmic and nuclear fractions using a kit from Solarbio (Beijing, China), following the manufacturer's protocol. Briefly, cells were suspended in a hypotonic buffer and lysed with the kit's proprietary detergent. The lysate was then centrifuged at 800 × g for 10 min at 4 °C. The resulting supernatant was further centrifuged at 16,000 × g for 5 min to obtain the cytoplasmic protein fraction. The original pellet was resuspended in nuclear extraction buffer and incubated on ice for 40 min with intermittent vortexing. After salt extraction, the nuclear pellet was centrifuged at 16,000 × g for 10 min. The supernatant of this step was collected as the nuclear extract. All extracts were stored at -80 °C for further use.

[0059] 1.9. Fluorescence quantitative PCR (RT-qPCR) To determine the mRNA levels of nrf2-regulated genes HO-1, Nqo1, GCLc, and GCLm, we extracted total RNA from the samples using Trizol reagent (Invitrogen, Shanghai). Then cDNA was synthesized using a library construction kit (Tiangen, Beijing). The primer sequences are listed in Table 1. We used a BioRad CFX96TM RT-qPCR was performed using SYBR PCR Master Mix (Tiangen, Beijing, China) on the system. The protocol included denaturation at 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. β-actin was used for data normalization.

[0060] Table 1 Primer sequences used for qPCR

[0061]

[0062] 1.10. Behavioral assessment The learning and memory abilities of mice were tested by behavioral tests, including Morris water maze test (MWM), open field test, and novel object recognition. These methods were performed according to previous studies. Behavioral test analysis was performed using VisuTrack 3.0 software (Shanghai Xinruan, China).

[0063] Zebrafish larvae 72 h post fertilization were randomly selected from each group and placed in a 24-well plate (1 per well). The plate was then placed in a zebrafish behavior analysis system (Noldus, The Netherlands). After 5 min of acclimation, the behavior of the larvae was tracked. Data were collected at regular intervals. After the behavior was recorded, statistical analysis was performed to evaluate the average speed, distance traveled, and motor activity.

[0064] 1.11. Golgi-Cox staining Mouse brain was subjected to Golgi-Cox staining using a kit (Servicebio, Wuhan, China). After anesthesia induced by sodium pentobarbital, the brain was quickly removed, cleaned, and soaked in solutions A and B in a dark and ventilated room for 14 days. Then, they were transferred to solution C and soaked for another 7 days. The sections (100 μm) were cut with a microtome, placed on a slide, and stained. After alcohol treatment, they were cleared and covered with xylene. All sections were then observed using an optical microscope (Olympus Microscope System BX51, Olympus Corporation, Tokyo, Japan), scanned using a digital slice scanner, and subsequently analyzed using Fiji software.

[0065] 1.12. Flow cytometry to detect cell apoptosis and reactive oxygen species (ROS) To confirm cell apoptosis, we collected cells and stained them using the Annexin V-FITC / PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA). The cells were resuspended in 200 μL of binding buffer at a concentration of 1×10 -6Cells / mL. Subsequently, 5 mL of Annexin-FITC and 5 mL of PI were mixed with the cell suspension, incubated in the dark for 15 min, and then apoptosis was analyzed by flow cytometry (DxFLEX, Beckman Coulter, USA). To measure the intracellular ROS level, the cells were treated for 12 h and then stained with 10 μM DCFH-DA for 30 min at room temperature using a ROS detection kit. Finally, the cells were collected and analyzed using the same flow cytometer.

[0066] 1.13. MAP2-SHALL analysis MAP2-SHALL analysis was used to assess neurite complexity. After fixation, primary neurons were stained with mouse anti-MAP2 antibody (1:1000 dilution, Millipore). Images were captured using a double-blind confocal microscope (Zeiss LSM710, 20x objective, 488nm laser). Four neurite growth parameters were analyzed using Image J software (Neuron J and ShollAnalysis plugin). Typically, 100 neurons per group were imaged, and the experiment was repeated three times independently.

[0067] TUNEL assay

[0068] TUNEL apoptosis detection kit was purchased from Beyotime (Beijing, China). After glutamate treatment, cells were collected by trypsinization, washed with PBS, and centrifuged. Then staining was performed according to the manufacturer's instructions. The labeled nuclei were visualized and imaged using a fluorescence microscope (Olympus, Japan).

[0069] 1.15. Determination of intracellular and mitochondrial ROS

[0070] Dihydroethidium (DHE, Beyotime) was used to measure ROS levels. Cells were treated with DHE at 37°C for 30 min, rinsed three times, and ROS levels were observed under an inverted fluorescence microscope. Mitochondrial ROS generation was assessed using MitoTracker Green and MitoSOX red dyes. Hippocampal neurons were incubated with 4 μM MitoTracker Green for 20 min and rinsed twice with PBS. Fluorescence images were captured using an inverted microscope (Olympus, Japan), and the average fluorescence density per field of view was calculated using ImageJ.

[0071] 1.16. Mitochondrial membrane potential measurement (ΔΨm)

[0072] ΔΨm was measured by flow cytometry using the JC-1 detection kit (Beyotime, Suzhou, China) according to the manufacturer's guidelines. After drug treatment, cells were harvested and incubated with 0.5 mL of JC-1 staining solution at 37°C for 20 min. The cells were then centrifuged at 600 × g for 3 min at 4°C and washed twice with JC-1 buffer. Fluorescence images were captured by an inverted microscope (Olympus, Japan). The average fluorescence density of each field of view was calculated using ImageJ software.

[0073] ATP measurement

[0074] Adenosine triphosphate (ATP) content was determined using a colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's guidelines.

[0075] 1.18.5-ALA and heme concentration determination

[0076] 5-ALA concentration was determined by liquid chromatography-mass spectrometry (LC-MS / MS). The LC-MS / MS device consisted of a Waters ACQUITY-UPLC system and a Waters Quattro Premier XE triple quadrupole mass spectrometer. The flow rate was set at 0.3 mL / min. Mass spectrometry analysis used pure standard solutions with positive electrospray ionization (ESI + ) mode. The optimized analysis parameters were: source temperature 120°C, capillary voltage 3.0 kV, dissolved gas (N2) temperature 300°C, flow rate 650 L / h, cone gas (N2) flow rate 650 L / h, cone voltage 20 V, collision gas (Ar) flow rate 0.22 mL / min.

[0077] Chromatographic separation: A C18 chromatographic column (ODS Ultrasphere 5 μm, 150×4.6 mm id) was used, and the mobile phase was 95% 50 mM phosphoric acid solution (containing 5 mM octyl sulfate) and 5% acetonitrile.

[0078] Heme levels were measured using a heme assay kit (K672-100, BioVision) according to the manufacturer's instructions. 5 The concentration of cells / mL was incubated overnight in a 6-well plate. The cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours. After treatment, the cells were collected, rinsed with PBS, and lysed in a solution of 1% NaOH and 5% Triton. The lysate was adjusted to pH 7.5 for heme quantification.

[0079] 1.19. PpIX production in cells

[0080] Cells were lysed in 100 μL lysis buffer (GenDEPOT, Barker, TX, USA). Lysates were then analyzed by quantitative fluorescence. Fluorescence measurements were performed using a Tecan M200 spectrometer (Tecan, Salzburg, Austria), with excitation and emission wavelengths set to 485 nm and 635 nm, respectively. PpIX levels were then normalized to protein content and determined using a BCA protein assay kit (Pierce Pharmaceuticals, Rockford, IL, USA) according to the manufacturer's protocol.

[0081] 1.20. Oxygen consumption rate (OCR)

[0082] Oxygen consumption rate (OCR) was measured using the Seahorse XF24 extracellular flux analyzer. Neurons (10,000 per well) were seeded in XF24 plates and cultured for 24 hours to form a uniform monolayer. They were then treated with 50 μM corticosterone (CORT) for 12 hours before OCR testing.

[0083] The culture medium was exchanged for culture medium and the plate was equilibrated for 1 h at 37°C in a CO2-free atmosphere before measurement. After three initial readings, 1 μM oligomycin, 0.5 μM FCCP, and 0.5 μM antimycin A / rotenone were added sequentially at 10-min intervals and OCR was recorded. OCR values ​​were normalized to protein concentration and determined by plotting the oxygen (O2) tension of the medium in the chamber as a function of time (pmoL / min).

[0084] 1.21. 13 C metabolic tracer analysis 13 C metabolic tracer analysis was performed according to previously established methods. Cells (2 × 10 cells per well) were plated in six-well plates. -5 Cells) were supplemented with 10 mM 13 C6 glucose (4mM 12 C-glutamine), 4mM 13 C5 Glutamine (10 mM 12 C glucose) or 10 mM 12 C glucose and 4mM 12 C-glutamate in DMEM for 16 h. The cells were then harvested on dry ice using 80% methanol in water. After freeze-thaw cycles at -80°C, the cell supernatant was centrifuged at 20,000 × g for 10 min and then dried using a speed vacuum for 3 h in preparation for LC-MS / MS analysis.

[0085] 1.22. Transmission Electron Microscopy Cells were fixed with 2.5% glutaraldehyde, rinsed three times with PBS for 5 minutes each time, and then fixed with 1% osmium tetroxide for 1 hour. After further PBS washing, cells were dehydrated through a graded propanol series and then embedded in epoxy resin after saturation with 2% uranyl acetate. Mitochondrial morphology was observed using a Hitachi HT7700 transmission electron microscope (Tokyo, Japan).

[0086] 1.23. Detection of ROS and apoptosis in zebrafish For ROS detection, larvae were treated with 10 mM DCFHDA (Aladdin Holding Group Co., Ltd., China) working solution at room temperature in the dark for 1 h. After washing three times with PBS, the stained larvae were anesthetized with 0.03% MS-222 (Aladdin Holding Group Co., Ltd., China) and observed under a stereo fluorescence microscope. The fluorescence intensity was quantified using Image Plus software.

[0087] Apoptosis detection was performed by staining with 5 mg / mL acridine orange (AO) (Aladdin Holding Group Co., Ltd., China) for 30 min. After staining, the larvae were extensively washed with PBS. Apoptotic cells showing green fluorescence were observed under a fluorescence microscope.

[0088] Statistical analysis

[0089] Statistical analysis was performed using SPSS software (IBM Corp., Armonk, NY, USA, Version 29.0), and the results were expressed as mean ± standard deviation (SD). The analysis included unpaired Student's t-test for pairwise comparisons and one-way or two-way analysis of variance (ANOVA) for evaluating differences in multiple groups. After ANOVA, post hoc Tukey's test was used to determine specific inter-group differences. A p value of less than 0.05 was used as the criterion for statistical significance.

[0090] Example 2, Results

[0091] 2.1. Integrated transcriptomic and metabolomic analysis revealed that the hippocampus of DOX-treated mice lacked ALAS2 and 5-ALA. Transcriptomic analysis revealed that after exposure to doxorubicin (DOX), gene expression in the hippocampus of mice was significantly altered, with 105 genes upregulated and 114 genes downregulated. Notably, ALAS2 was identified as one of the most significantly downregulated genes, while other genes critical for hemoglobin synthesis, including Hba-a1, Hba-a2, Hbb-bt, and Hbb-bs, were also downregulated ( Figure 1 A). The heat map clearly depicts the substantial differences in gene expression between the control and DOX-treated groups ( Figure 1B). Gene ontology (GO) enrichment analysis highlighted that the differentially regulated genes were mainly associated with cellular components, such as the hemoglobin complex and the haptoglobin-hemoglobin complex ( Figure 1 C). To integrate gene-level information, we constructed a protein-protein interaction (PPI) network, revealing that the genes in the final PPI network were mainly enriched in hemoglobin complex, oxygen carrier activity, oxygen binding, and haptoglobin-hemoglobin complex ( Figure 1 D, E). To further elucidate the pathways most enriched in gene expression patterns during DOX exposure, we performed gene set enrichment analysis (GSEA) using the read count data matrix. Comparison between the control and DOX groups showed that the most regulated gene sets were mainly enriched in pathways related to abnormal hemoglobin ( Figure 1 F).

[0092] We then performed metabolomic analysis of hippocampal tissues from mice exposed to DOX. Strikingly, 5-ALA, a key precursor for heme synthesis, was found to be significantly downregulated ( Figure 1 G). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis highlighted the involvement of these metabolites in pathways such as glycine, serine, and threonine metabolism, as well as arginine and proline metabolism ( Figure 1 H). Transcriptomic and metabolomic analyses revealed that differentially abundant genes and metabolites were associated with abnormal heme synthesis, with a particular emphasis on downregulation of ALAS2 and 5-ALA expression. Data for these significantly differentially expressed genes and metabolites are provided in Supplementary Tables 1 and 2. The timeline of DOX treatment and metabolomic OPLS-DA plots are shown in Fig. 9 .

[0093] ALAS2 deficiency was only observed in neurons, but not in astrocytes and microglia

[0094] Western blot and LC-MS / MS confirmed the downregulation of ALAS2 and 5-ALA in DOX-treated mice ( Figure 2 A and B). Further immunofluorescence revealed that ALAS2 deficiency was specific to hippocampal neurons and absent in astrocytes and microglia ( Figure 2 CF). In vitro studies confirmed that DOX reduced the expression of ALAS2 in primary neurons as shown by western blotting and immunofluorescence ( Figure 2 GJ).

[0095] 2.3.5-Neuroprotective effects of ALA administration and ALAS2 overexpression against DOX-induced neurotoxicity

[0096] Representative MWM navigation test results are as follows: Figure 3 A. DOX-treated mice showed significantly prolonged escape latency compared with control mice, indicating impaired spatial memory. However, this cognitive deficit was significantly reversed by 5-ALA administration or ALAS2 overexpression, suggesting that it has a protective effect against DOX-induced chemo-brain ( Figure 3 B). Western blot analysis showed that the expression levels of synaptophysin (SYN) and postsynaptic density protein 95 (PSD95) in the DOX-treated group were significantly decreased compared with those in the control group. Overexpression of 5-ALA or ALAS2 could restore the expression of SYN and PSD95 in the DOX-treated group. Figure 3 C and D). Golgi-Cox staining confirmed that DOX significantly reduced the number of dendritic branches and spines, and both 5-ALA and ALAS2 overexpression ameliorated this degeneration ( Figure 3 EG). The neuroprotective effects of 5-ALA and ALAS2 were further verified by in vitro experiments. Cell apoptosis was quantitatively evaluated by flow cytometry ( Figure 3 H), and the branching morphology of neurons was assessed using Sholl analysis ( Figure 3 IJ). The results showed that DOX induced strong apoptosis and reduced crossing in primary neuronal cells, and overexpression of 5-ALA or ALAS2 alleviated this effect. Fig.10 and 11 Additional evidence was provided that 5-ALA supplementation and ALAS2 overexpression could prevent DOX-induced inhibition of neurogenesis, apoptosis, and oxidative stress.

[0097] 2.4.5-ALA supplementation or ALAS2 overexpression alleviates DOX-induced chemotherapy-induced brain damage by promoting Bach1 ubiquitin-dependent degradation

[0098] Figure 4 A describes a potential mechanism linking heme synthesis to inhibition of Bach1. Our results showed that DOX significantly reduced PpIX and heme levels, whereas supplementation with 5-ALA or overexpression of ALAS2 could effectively restore these levels ( Figure 4 B, C). We found that DOX enhanced the nuclear localization of Bach1 ( Figure 4 D) and inhibited its degradation in primary neuronal cells ( Figure 4 E, F). In contrast, supplementation with 5-ALA or overexpression of ALAS2 reversed these effects. In addition, Co-IP results showed that Bach1 interacts with E3 ubiquitin ligase ( Figure 4 G). Further data showed that DOX inhibited the ubiquitin-dependent degradation of Bach1, whereas 5-ALA supplementation or ALAS2 overexpression increased the ubiquitin-dependent degradation of Bach1 ( Figure 4 H).

[0099] 2.5.5-ALA or Bach1 inhibition attenuates oxidative stress via Nrf2 activation

[0100] Our study found that si-Bach1 significantly reduced the number of TUNEL-positive and DHE-positive cells in DOX-induced primary neurons ( Figure 5 AC). Flow cytometry analysis further confirmed the antioxidant effect of si-BACH1 ( Figure 5 D, E). Bach1 is known to compete with Nrf2 in yeast cells and participate in the transcriptional regulation of oxidative stress genes. We also studied the effect of Bach1 on Nrf2 metabolism in primary neurons and found that Bach1 overexpression significantly reduced Nrf2 levels ( Figure 5 F, G). Subsequently, we detected the expression of Nrf2 and its downstream genes in DOX-induced neuronal cells and found that Nrf2 protein and its downstream genes HO-1, Nqo1, GCLc and GCLm, the latter of which was reversed by 5-ALA or si-Bach1 ( Figure 5 HI and Fig.12 AB). These results indicate that 5-ALA or si-Bach1 can activate Bach1 and inhibit Nrf2.

[0101] Additional in vivo studies showed that inhibition of Bach1 using HPPE significantly increased the exploration time of DOX-treated mice in a novel object recognition test ( Figure 5 J). Immunofluorescence analysis showed that HPPE significantly reduced DOX-induced apoptosis and ROS production in mouse hippocampal cells ( Figure 4 K). In addition, 5-ALA and HPPE were beneficial in increasing hippocampal Nrf2 levels and improving cognitive performance in the water maze test ( Fig.12 CH). The antioxidant effects of 5-ALA and HPPE were abolished in Nrf2 knockout mice, as shown in Figure Fig.12 IM shown.

[0102] 2.6.5-ALA and HPPE protect against mitochondrial dysfunction in doxorubicin neurotoxicity

[0103] It has been reported that the loss of antioxidant defense is accompanied by mitochondrial dysfunction. We further elucidated the effects of 5-ALA supplementation and HPPE on mitochondrial health in primary neurons. As expected, DOX induced a significant increase in mitochondrial ROS production and a substantial decrease in mitochondrial membrane potential and ATP content compared with the control group, which was abolished by 5-ALA or HPPE treatment ( Figure 6AF). In addition, mitochondrial respiratory activity was assessed, and the results showed that DOX significantly reduced basal respiration, ATP production, and maximal respiratory capacity. These parameters were restored after 5-ALA or HPPE treatment ( Figure 6 G, H). To evaluate the effect of 5-ALA or HPPE on glucose metabolism, homogeneously labeled 13 C6 glucose-treated primary neurons. In DOX-treated cells, glucose-derived citrate, α-ketoglutarate, fumarate, and malate 13 The C-labeled isotope fraction was significantly reduced, and 5-ALA and HPPE treatment significantly reversed this effect ( Figure 6 K). TEM was used to further examine the mitochondrial morphology of neurons. DOX treatment resulted in increased mitochondrial damage, as evidenced by loss of double membrane structure, disordered mitochondrial cristae, and blurred edges. Notably, 5-ALA and HPPE treatment increased the proportion of normal mitochondria in DOX-treated neurons ( Figure 6 LM).

[0104] 2.7. Zebrafish model highlights the protective effect of 5-ALA against DOX-induced neurotoxicity and cognitive impairment

[0105] To provide further evidence to support the hypothesis that 5-ALA treatment alleviates DOX-induced chemo-brain, we performed additional validation in a zebrafish model. TEM analysis revealed that DOX-exposed zebrafish exhibited significant mitochondrial dysfunction, as evidenced by mitochondrial swelling, disordered mitochondrial cristae, and mitochondrial fragmentation. Importantly, 5-ALA intervention was found to effectively restore mitochondrial integrity and significantly reduce the incidence of abnormal mitochondria ( Figure 7 A, B). In addition, AO staining showed that DOX exposure led to an increase in apoptotic cells, and 5-ALA treatment could attenuate this response. Thus, neuronal apoptosis was reduced ( Figure 7 C, E). DCFH-DA staining was used to evaluate oxidative stress in the zebrafish larvae brain, showing that the fluorescence intensity of the DOX-treated group increased significantly, while that of the 5-ALA-treated group decreased significantly ( Figure 7 D, F). In addition, behavioral analysis showed that DOX-exposed zebrafish had reduced exploratory activity in the central area, and this behavior returned to normal after 5-ALA treatment ( Figure 7 G).

[0106] Collectively, these results suggest that 5-ALA alleviates DOX-induced neurotoxicity and associated cognitive impairment, with potential mechanisms such as Figure 8 shown.

[0107] ALAS2 is a member of the ALAS family and plays a key role as the rate-limiting enzyme in heme synthesis. This process begins with the production of 5-ALA, which is mainly expressed in erythroid precursors, especially in bone marrow and brain tissues. Our novel findings show that ALAS2 deficiency is only observed in hippocampal neurons of DOX-treated mice, but not in astrocytes or microglia. Interestingly, previous studies have shown that ALAS1 expression is specifically downregulated in cardiomyocytes after DOX treatment. These findings emphasize the role of ALAS proteins in apoptosis in response to DOX-mediated toxicity. Previous studies have demonstrated that ALAS2 deficiency is associated with symptoms such as cognitive and psychiatric disorders. Notably, the relative expression of ALAS mRNA in the brain of AD patients was significantly reduced by nearly 90% compared with that of controls. Daily oral administration of 5-ALA to transgenic AD (3xTg-AD) model mice maintained mitochondrial enzyme activity in the brain, thereby contributing to the preservation of neural activity.

[0108] Recent studies have shown that 5-ALA can downregulate Bach1 in multiple cell types, such as renal tubules and macrophages. This study further demonstrated that 5-ALA also has this downregulatory effect on neurons. The degradation of Bach1 is a two-step process, including nuclear export and ubiquitin-proteasomal degradation after heme binding. Our results suggest that DOX disrupts the Bach1 ubiquitin-proteasomal degradation pathway by enhancing its nuclear retention and inhibiting heme synthesis, leading to neuronal oxidative stress and apoptosis. Further experiments showed that supplementation with 5-ALA or overexpression of ALAS2 could counteract these effects.

[0109] Bach1 acts as a competitive inhibitor of Nrf2 and can degrade and upregulate Nrf2 expression. The Bach1 / Nrf2 signaling pathway is essential for cellular antioxidant defense, and Bach1-targeted degradation is viewed as a potential strategy for cognitive enhancement. Preclinical studies have shown that Nrf2 activators have significant neuroprotective effects due to their antioxidant properties and ability to restore mitochondrial function. However, the high electrophilicity of these activators makes them less suitable for the treatment of neurodegenerative diseases. Therefore, inhibiting Bach1 to activate Nrf2 presents a more effective and feasible therapeutic strategy. Our study highlights the importance of 5-ALA in regulating the Bach1 / Nrf2 pathway. Furthermore, the neuroprotective effects of 5-ALA supplementation or Bach1 inhibition were negated in Nrf2 knockout mice, emphasizing the necessity of Nrf2 activation for the neuroprotective effects of 5-ALA against DOX-induced neurotoxicity.

[0110] Recent studies have shown that 5-ALA can reduce oxidative stress and mitochondrial dysfunction and has neuroprotective effects. Our study suggests that 5-ALA may also reverse DOX-induced cognitive decline through the Bach1 / Nrf2 pathway. Notably, previous studies have shown that 5-ALA can inhibit iron overload and lipid peroxidation in cardiomyocytes and mice, thereby preventing DOX-induced cardiomyopathy. As an endogenous substance, 5-ALA has a well-established safety profile and has been approved by the US Food and Drug Administration (FDA) for use in photodynamic therapy (PDT). Although PDT using 5-ALA has ample evidence in cancer treatment, recent basic research has highlighted the direct antitumor effects of 5-ALA. Studies have shown that exogenous supplementation with 5-ALA significantly inhibits the proliferation of ovarian and breast cancer cells by enhancing heme synthesis and inhibition of oxidative metabolism, while slowing the migration of both cell types by disrupting Bach1 stability. Therefore, for cancer patients receiving DOX therapy, adjuvant 5-ALA treatment may provide multiple benefits, including neuroprotection, cardioprotection, and potential enhanced antitumor activity. Further clinical trials are essential to verify these potential benefits.

[0111] Example 3, a pharmaceutical composition

[0112] A method for treating chemo-induced chemo brain comprising an inhibitor of Bach1 as an active ingredient.

[0113] The pharmaceutical composition further comprises an excipient.

Claims

1. Application of Bach1 inhibitors in the preparation of therapeutic drugs for chemotherapy-induced brain damage.

2. The use according to claim 1, characterized in that: The inhibitor of Bach1 includes 5-aminolevulinic acid or si-Bach1.

3. The use according to claim 2, characterized in that: 5-Aminolevulinic acid alleviates doxorubicin-induced neurotoxicity.

4. The use according to claim 3, characterized in that: 5-Aminolevulinic acid alleviates cognitive impairment in doxorubicin-induced chemo-brain via the Bach1 / Nrf2 signaling pathway.

5. The use according to claim 3, characterized in that: si-Bach1 inhibits Bach1 and thus activates Nrf2.

6. The use according to claim 2, characterized in that: The inhibitor of Bach1 is hexaenoyl pyrophosphate (HPPE).

7. The use according to any one of claims 1 to 6, characterized in that: The chemotherapy drugs include doxorubicin and methotrexate.

8. The use according to any one of claims 1 to 6, characterized in that: The chemotherapy drug is doxorubicin.

9. A pharmaceutical composition, characterized in that Contains an inhibitor of Bach1 as an active ingredient.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further comprises an excipient.

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