Application of GPX4 as target spot in preparation of medicine for treating neurodegenerative diseases

Abstract

The invention discloses application of GPX4 as a target spot in preparation of a medicine for treating neurodegenerative diseases, and discloses a key regulation and control effect of GPX4 protein in neurodegenerative diseases caused by toxoplasma gondii infection for the first time, and particularly, GPX4 plays an important role in regulating and controlling a ferroptosis pathway in the occurrence process of Parkinson's disease. Compared with the prior art, the invention not only provides a new research perspective from the pathological mechanism, but also verifies the effectiveness of GPX4 as a potential therapeutic target through in-vivo and in-vitro experiments, has a remarkable application prospect, can provide a new strategy for early diagnosis and targeted therapy of Parkinson's disease, and has an important clinical transformation value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of GPX4 as a target in the preparation of drugs for treating neurodegenerative diseases. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Neurodegenerative diseases (such as Parkinson's disease, PD) are common central nervous system diseases in the elderly population, characterized by the progressive loss of dopaminergic neurons and the formation of Lewy bodies. However, the pathogenesis of Parkinson's disease is not yet fully understood, which limits the development of effective treatments.

[0004] In recent years, ferroptosis, as a unique form of cell death, has been gradually recognized to play a key role in a variety of neurodegenerative diseases. Ferroptosis is characterized by increased intracellular iron levels and accumulation of lipid peroxides, and glutathione peroxidase 4 (GPX4), as an important member of the antioxidant system, plays a core role in inhibiting ferroptosis.

[0005] On the other hand, Toxoplasma gondii is a globally distributed parasite that is known to invade the central nervous system and may be associated with the occurrence of a variety of neurological diseases. However, the specific mechanism of how Toxoplasma gondii infection induces neurodegenerative diseases is still limited, especially its relationship with ferroptosis has not been studied in depth.

[0006] The prior art has not yet revealed in depth the role of GPX4 in neurodegenerative diseases caused by specific pathogen infection, especially Toxoplasma gondii infection. Summary of the invention

[0007] Although Toxoplasma gondii is a common parasite that can invade the central nervous system, its relationship with neurodegenerative diseases is still unclear, especially its mechanism of action in Parkinson's disease has not been fully studied. Therefore, existing technologies cannot effectively explain and utilize the association between Toxoplasma gondii infection and neurodegenerative diseases, especially Parkinson's disease, resulting in challenges in the early prevention and targeted treatment of related diseases.

[0008] In order to address the deficiencies of the prior art, the present invention provides the use of GPX4 as a target in the preparation of drugs for the treatment of neurodegenerative diseases, revealing that Toxoplasma gondii infection induces neuronal ferroptosis by downregulating GPX4 expression, thereby leading to the occurrence of Parkinson's disease. By establishing the key role of GPX4 in this pathological process, the present invention provides a new theoretical basis and target for the early diagnosis and treatment of Parkinson's disease, filling the gap in the prior art, and also provides support for future drug research on GPX4-related targets, which has important clinical application prospects. The present invention provides the following technical solutions:

[0009] On one hand, the present invention provides the use of GPX4 as a target in the preparation of a drug for treating neurodegenerative diseases, wherein the drug targets the GPX4 protein and promotes the transcription or expression of the GPX4 protein gene.

[0010] The neurodegenerative disease is a neurodegenerative disease induced by Toxoplasma gondii infection.

[0011] Furthermore, the neurodegenerative disease is Parkinson's disease.

[0012] The present invention also provides the use of GPX4 protein as an early diagnostic marker for Parkinson's disease. Studies in the embodiments of the present invention show that based on the downregulation of GPX4 protein in peripheral blood, it can be used as a diagnostic marker for early Parkinson's disease, indicating potential neuronal ferroptosis and pathological progression.

[0013] The present invention also provides a detection kit for early Parkinson's disease, comprising a GPX4-specific antibody.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The innovation of this patent is that it reveals for the first time the key regulatory role of GPX4 protein in neurodegenerative diseases caused by Toxoplasma gondii infection, especially in the occurrence of Parkinson's disease, GPX4 plays an important role by regulating the ferroptosis pathway. Compared with the prior art, the present invention not only provides a new research perspective from the pathological mechanism, but also verifies the effectiveness of GPX4 as a potential therapeutic target through in vivo and in vitro experiments. It has significant application prospects and can provide new strategies for the early diagnosis and targeted treatment of Parkinson's disease, which has important clinical transformation value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 This is an immunohistochemical staining image of TH in the substantia nigra of the midbrain of mice infected with Toxoplasma gondii in Example 1.

[0018] Figure 2 This is the correlation analysis between the detection of cerebrospinal fluid GPX4 levels and Parkinson's disease in Example 2.

[0019] Figure 3 These are the results of the rotarod test for behavioral detection of mice infected with Toxoplasma gondii in Example 1.

[0020] Figure 4 These are the results of the open field test on the behavioral detection of mice infected with Toxoplasma gondii in Example 1.

[0021] Figure 5 This is the transcriptomic and proteomic analysis of brain tissue of mice infected with Toxoplasma gondii in Example 1. DETAILED DESCRIPTION

[0022] 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.

[0023] The present invention provides a method for inducing Parkinson's disease behavioral phenotype in mice by using Toxoplasma gondii soluble tachyzoite antigen (STA), and reveals the role of GPX4 as a new target in the process through a series of experimental techniques.

[0024] Example 1: Use of GPX4 as a target in the preparation of drugs for treating neurodegenerative diseases

[0025] 1. Construction of the Toxoplasma gondii soluble tachyzoite antigen infection model

[0026] 1.1 Preparation of Toxoplasma gondii antigen

[0027] Toxoplasma gondii culture: Toxoplasma gondii tachyzoites were inoculated into human lung cancer cell line H1299 and cultured in an incubator at 37°C and 5% CO2. When the cell infection rate reached 70%-80%, the tachyzoites were collected.

[0028] Antigen extraction: Use phosphate buffered saline (PBS) to ultrasonically disrupt the tachyzoites (10 seconds each time, 5 seconds interval, a total of 30 times), then centrifuge at 4°C and 12,000g for 10 minutes, and collect the supernatant as soluble antigen. Use the BCA protein quantification kit to determine the protein concentration, and then divide the antigen solution into aliquots and store it at -80°C for later use.

[0029] 1.2 Establishment of mouse model

[0030] Experimental animals: 8-week-old healthy male C57BL / 6 mice weighing about 20-25 grams were selected. After one week of adaptive feeding, they were randomly divided into experimental and control groups, with 10-12 mice in each group.

[0031] Stereotaxic injection: Anesthetize mice (using sodium pentobarbital 40 mg / kg intraperitoneal injection), and fix them on a stereotaxic instrument after they are fully anesthetized. The injection coordinates are determined according to the Paxton brain atlas. The injection point of the substantia nigra in the midbrain is: 3.0 mm behind the anterior fontanelle, 1.5 mm lateral to the midline, and 4.5 mm below the brain surface; the injection point of the striatum is: 1.0 mm behind the anterior fontanelle, 2.0 mm lateral to the midline, and 3.5 mm below the brain surface. 3 μL of 0.5 mg / mL Toxoplasma STA antigen was injected into the substantia nigra and striatum of the midbrain respectively by a microinjection pump, and the injection speed was controlled at 0.5 μL / min. After the injection, the needle was retained for 5 minutes to prevent antigen spillage, and then the needle was slowly removed and the wound was sutured. The control group was injected with an equal amount of normal saline.

[0032] Postoperative care: After surgery, mice were revived in a warm environment and housed in individual cages, with sterile water and high-calorie feed provided to promote recovery. The general condition and weight changes of the mice were observed daily.

[0033] 1.3 Behavioral testing

[0034] ①Rotarod Test: The rotarod test was performed on the 7th, 14th, and 30th days after injection. The mice were placed on the rotating rod, and the initial speed was set at 5 rpm and gradually accelerated to 40 rpm. The time it took for the mice to fall off the rotating rod (delay time) was recorded. Each mouse was tested three times in a row with an interval of 10 minutes, and the average value was taken for analysis. Figure 3 As shown, after the experimental group mice were infected with gondii antigen, the rotarod behavior test was performed. Compared with the Ctrl group, the rotarod residence time of the gondii group mice was significantly reduced, indicating that the gondii antigen-infected mice showed Parkinson's-like behavior.

[0035] ②Open Field Test: The open field test was conducted in a quiet environment. The mice were placed in a 50cm x 50cm transparent box and allowed to move freely for 10 minutes. The total movement distance, number of spontaneous activities, and time spent in the central area of ​​the mice were recorded by a video tracking system to evaluate the mice's autonomous activity ability and anxiety behavior, such as Figure 4 As shown, the motor ability of mice infected with M. gondii was significantly reduced in the open field test.

[0036] 2. Histopathological Analysis

[0037] 2.1 Tissue sampling

[0038] Sampling time: After behavioral testing, 3-5 mice were randomly selected for brain tissue sampling on the 14th day and the 30th day. First, the mice were anesthetized with sodium pentobarbital (50 mg / kg), and then perfused with PBS and 4% paraformaldehyde in turn by cardiac perfusion to ensure that the brain tissue was well fixed.

[0039] Brain tissue collection: The mouse brain tissue was removed and washed with ice-cold PBS to locate and separate the substantia nigra and striatum regions of the midbrain. The tissue was divided into two parts, one for paraffin embedding and sectioning, and the other immediately frozen in liquid nitrogen for subsequent molecular biology experiments.

[0040] 2.2 Pathological marker detection

[0041] Immunohistochemistry (IHC): Paraffin-embedded brain tissue sections (5 μm thick) were dewaxed and hydrated. Endogenous peroxidase activity was blocked with 3% H2O2, and then antigen retrieval was performed using sodium citrate buffer (pH 6.0). After blocking the sections with 5% bovine serum albumin (BSA), the primary antibodies for α-synuclein and tyrosine hydroxylase (TH) were incubated overnight (4°C). The next day, after washing, the sections were incubated with biotin-labeled secondary antibodies, DAB color development, and hematoxylin counterstaining. The staining results were observed by optical microscopy to evaluate the expression changes of pathological markers. Figure 1 The results of immunohistochemical staining of TH in the substantia nigra of the mouse midbrain, the gold standard for PD pathological detection, showed that the number of TH-positive cells in the gondii group was significantly lower than that in the Ctrl group, suggesting that Toxoplasma gondii antigen can induce the death of dopaminergic neurons in the substantia nigra of the midbrain.

[0042] Western Blot: The frozen brain tissue was homogenized and lysed to extract total protein. Proteins were separated by SDS-PAGE and then transferred to PVDF membrane. After the membrane was blocked, the primary antibody of GPX4 or TH was incubated, followed by the secondary antibody labeled with horseradish peroxidase. Protein expression was detected by ECL chemiluminescence, and the relative intensity of protein bands was quantitatively analyzed using image analysis software.

[0043] 3. Omics analysis and discovery of GPX4 targets

[0044] 3.1 Omics detection

[0045] RNA extraction and transcriptome analysis: Total RNA was extracted from frozen midbrain substantia nigra and striatum tissues, and the purity and integrity of RNA were detected using NanoDrop and Agilent 2100. RNA-seq analysis was performed using the Illumina sequencing platform to generate high-throughput sequencing data. Differential expression analysis (DESeq2 or EdgeR) was used to screen out genes that were specifically downregulated or upregulated in the experimental group.

[0046] Protein extraction and proteomic analysis: Tissue samples were lysed with RIPA buffer, proteins were extracted, and quantified using the BCA method. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS / MS). MaxQuant software was used to quantitatively analyze mass spectrometry data and screen for differentially expressed proteins.

[0047] Transcriptomics and proteomics results Figure 5 As shown, in the substantia nigra region of the midbrain of gondii-infected mice, the mRNA and protein levels of GPX4 molecules were significantly downregulated compared with those in the Ctrl group.

[0048] 3.2 Identification of GPX4 targets

[0049] Data analysis: RNA-seq and proteomics data analysis revealed that GPX4 expression was significantly downregulated in the brain tissue of the experimental group of mice. Combined with KEGG and GO enrichment analysis, the role of GPX4 in ferroptosis and neurodegenerative diseases was further studied.

[0050] Example 2, Identification of GPX4 Targets

[0051] 1. Related materials and operation methods

[0052] 1: Sample collection

[0053] Peripheral blood or cerebrospinal fluid samples are obtained from patients, and brain tissue samples can also be obtained by stereotactic surgery in laboratory mouse models.

[0054] 2: RNA level detection

[0055] Total RNA from the samples was extracted using an RNA extraction kit, and the expression level of the GPX4 gene was detected by qPCR. The significant downregulation of GPX4 mRNA can be used as one of the markers of early neuronal ferroptosis.

[0056] 3: Protein level detection

[0057] GPX4 protein levels in peripheral blood or cerebrospinal fluid were detected by Western blot or ELISA using GPX4-specific antibodies. If GPX4 protein is significantly downregulated in the patient, it indicates the risk of ferroptosis, suggesting early pathological changes in Parkinson's disease.

[0058] 4: Immunofluorescence / Immunohistochemistry analysis

[0059] In the brain tissue of Parkinson's disease model mice or patients, GPX4 protein was quantitatively detected using immunofluorescence or immunohistochemistry to observe changes in its expression level in the substantia nigra or striatum of the midbrain.

[0060] 2. Experimental results:

[0061] 1: Diagnostic application of peripheral blood GPX4 level detection

[0062] C57BL / 6 mice were used in the experiment to establish a Parkinson's disease model by injecting Toxoplasma gondii tachyzoite antigen. The control group was injected with normal saline. Peripheral blood was collected from the experimental group and the control group at different time points after exposure to Toxoplasma gondii antigen, and the GPX4 protein level in the peripheral blood was detected using an ELISA kit.

[0063] Results: The peripheral blood concentration of GPX4 protein was significantly reduced in the experimental group at 1 week, 2 weeks, and 4 weeks after inoculation of Toxoplasma gondii antigen, while the GPX4 level in the control group remained stable. Based on the downregulation of GPX4 protein in peripheral blood, it can be used as a diagnostic marker for early Parkinson's disease, indicating potential neuronal ferroptosis and pathological progression.

[0064] 2: Analysis of the association between cerebrospinal fluid GPX4 levels and Parkinson's disease

[0065] The cerebrospinal fluid of the model mice was collected and the expression level of GPX4 protein was detected by ELISA. Similar to the results of peripheral blood, Figure 2 As shown, GPX4 expression was significantly downregulated in the cerebrospinal fluid of mice exposed to Toxoplasma gondii antigen, further demonstrating that GPX4 can serve as an early marker for Parkinson's disease.

[0066] It can be seen that the decreasing trend of GPX4 in cerebrospinal fluid can help clinicians predict the early development of neurodegenerative diseases and provide a theoretical basis for the early intervention of Parkinson's disease.

[0067] The present invention found that Toxoplasma gondii infection can induce neuronal ferroptosis by downregulating the expression of GPX4, thereby leading to the occurrence of Parkinson's disease. This discovery reveals a new role of GPX4 in Toxoplasma gondii infection-induced neurodegenerative diseases and provides a potential new target for the treatment or prevention of Parkinson's disease and other related diseases.

[0068] Example 3: Application of GPX4 protein as a diagnostic marker for early Parkinson's disease

[0069] 1: Sample collection

[0070] Obtain peripheral blood or cerebrospinal fluid samples from patients or model animals (such as mice). Peripheral blood collection can be performed using vacuum blood collection tubes, while cerebrospinal fluid collection requires professional operation.

[0071] 2: RNA level detection

[0072] Total RNA was extracted from the collected samples using an RNA extraction kit.

[0073] The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0074] The expression level of GPX4 gene was detected by qPCR kit. GPX4 specific primers were set and GPX4 mRNA level was analyzed by quantitative PCR.

[0075] 3: Protein level detection

[0076] ELISA method: Use ELISA kit to detect GPX4 protein level in peripheral blood or cerebrospinal fluid. Add the sample to the ELISA plate pre-coated with GPX4 antibody, and quantify the GPX4 concentration using the standard curve.

[0077] Western blot: GPX4 protein expression in peripheral blood mononuclear cells or brain tissue was analyzed by Western blot. The samples were subjected to SDS-PAGE electrophoresis and then detected using GPX4-specific antibodies.

[0078] 4: Immunofluorescence / Immunohistochemistry analysis

[0079] The brain tissue of the model animal is fixed and sliced, and the expression of GPX4 is detected using immunofluorescence or immunohistochemistry. GPX4-specific antibodies are added to tissue sections, and fluorescent secondary antibodies are used for labeling to observe the expression of GPX4 in neurons.

[0080] 5: Data Analysis

[0081] The detected GPX4 expression levels were compared with the normal control group. The significant downregulation of GPX4 can serve as an early marker of neuronal ferroptosis, indicating the risk of Parkinson's disease.

[0082] Example 4, Detection Kit for Early Parkinson's Disease

[0083] Includes GPX4-specific antibodies.

[0084] GPX4 protein levels in peripheral blood or cerebrospinal fluid were detected by Western blot or ELISA using GPX4-specific antibodies. If GPX4 protein is significantly downregulated in the patient, it indicates the risk of ferroptosis, suggesting early pathological changes in Parkinson's disease.

[0085] Example 5, a drug for treating neurodegenerative diseases

[0086] The medicine comprises a GPX4 promoter targeting GPX4, and the GPX4 promoter is a medicine or reagent capable of increasing the expression amount of GPX4 protein.

[0087] The drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of GPX4 as a target in the preparation of drugs for the treatment of neurodegenerative diseases.

2. The use according to claim 1, characterized in that: The drug targets the GPX4 protein and promotes the transcription or expression of the GPX4 protein gene.

3. The use according to claim 1, characterized in that: The neurodegenerative disease is a neurodegenerative disease induced by Toxoplasma gondii infection.

4. The use according to claim 1, characterized in that: The neurodegenerative disease is Parkinson's disease.

5. The use according to claim 1, characterized in that: The medicament comprises a GPX4 accelerator.

6. The use according to claim 5, characterized in that: The GPX4 promoter is a drug or reagent that increases the expression level of GPX4 protein.

7. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

8. Application of GPX4 protein as an early diagnostic marker for Parkinson's disease.

9. The use according to claim 7, characterized in that: Based on the downregulation of GPX4 protein in peripheral blood, it can be used as a diagnostic marker for early Parkinson's disease, indicating potential neuronal ferroptosis and pathological progression.

10. A detection kit for early Parkinson's disease, characterized in that: Includes GPX4-specific antibodies.

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