Application of FBXW7 modulators in the preparation of drugs for the prevention or improvement of Parkinson's disease

Abstract

This invention discloses the application of FBXW7 modulators in the preparation of drugs for the prevention or improvement of Parkinson's disease. It relates to the field of biomedical technology. Specific applications, pharmaceutical compositions, and screening methods are provided. This invention, by upregulating FBXW7 expression, can inhibit the increase of p-STAT3 levels and improve impaired mitochondrial function. This indicates that FBXW7 can serve as a novel target for Parkinson's disease intervention, and formulations or compositions based on upregulated FBXW7 expression can be used to prevent, delay, or improve Parkinson's disease, providing a new technological basis for the screening of related candidate drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of FBXW7 modulators in the preparation of drugs for the prevention or improvement of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain, decreased dopamine levels in the striatum, and progressively worsening motor and nonmotor symptoms. Current treatments primarily focus on symptom relief, lacking effective intervention strategies to slow disease progression. Therefore, identifying novel molecular targets and developing corresponding prevention and treatment methods are crucial issues that urgently need to be addressed in this field.

[0003] A growing body of research indicates that mitochondrial dysfunction plays a crucial role in the development and progression of Parkinson's disease. Mitochondrial fission / fusion imbalance, decreased membrane potential, reactive oxygen species accumulation, and impaired energy metabolism can exacerbate neuronal damage and promote disease progression. Simultaneously, abnormal activation of the STAT3 signaling pathway is also considered associated with neuroinflammation, cellular stress, and mitochondrial homeostasis imbalance. However, current research on upstream regulatory factors in Parkinson's disease remains insufficient, particularly regarding the key molecular targets that simultaneously link abnormal STAT3 activation and mitochondrial damage, which remain unclear.

[0004] FBXW7 (F-box and WD repeat domain-containing 7) is an important substrate recognition subunit of the SCF-type E3 ubiquitin ligase complex, participating in the regulation of various cellular processes, including cell proliferation, differentiation, metabolism, stress response, and protein degradation. Previous studies have suggested that FBXW7 plays an important role in tumors, metabolic diseases, and some neurological disorders. However, there are currently no clear reports on the role of FBXW7 in Parkinson's disease, particularly whether it participates in the pathological process of Parkinson's disease by regulating p-STAT3 activation, abnormal mitochondrial dynamics, and apoptosis. Furthermore, there are no systematic application protocols for using FBXW7 as a target for the prevention, treatment, auxiliary diagnosis, efficacy evaluation, or drug screening of Parkinson's disease.

[0005] Therefore, providing a new use for FBXW7 for the intervention, detection, or drug development of Parkinson's disease-related lesions has significant theoretical and practical value. Summary of the Invention

[0006] In view of this, the present invention provides the application of FBXW7 modulators in the preparation of drugs for the prevention or improvement of Parkinson's disease. Based on the regulation of abnormal mitochondrial dynamics and apoptosis by the FBXW7 / p-STAT3 signaling axis, this invention offers a novel application for the prevention, auxiliary diagnosis, efficacy evaluation, or treatment of Parkinson's disease.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of FBXW7 expression upregulators in the preparation of drugs for the prevention, delay or improvement of Parkinson's disease.

[0009] Preferred: FBXW7 expression upregulators are preparations that enhance intracellular FBXW7 expression or activity, including FBXW7 overexpression vectors, recombinant viral vectors, nucleic acid constructs, mRNA preparations, gene delivery systems, or combinations thereof.

[0010] Preferred: The FBXW7 expression upregulator is a eukaryotic overexpression plasmid carrying the Fbxw7 coding sequence. The eukaryotic expression plasmid can mediate Fbxw7 overexpression in nerve cells.

[0011] Preferred: The Fbxw7 overexpression plasmid contains a mammalian cell promoter, an Fbxw7 coding sequence, a transcription termination sequence, and a selection marker sequence, wherein the Fbxw7 coding sequence is located downstream of the promoter.

[0012] Preferred: Parkinson's disease is Parkinson's disease associated with mitochondrial dysfunction.

[0013] Preferred: Improving mitochondrial function includes at least one of the following: improving mitochondrial membrane potential, reducing reactive oxygen species levels, increasing ATP production, improving mitochondrial morphology, and restoring mitochondrial division / fusion balance.

[0014] The present invention also provides a pharmaceutical composition for preventing, delaying or improving Parkinson's disease, comprising an FBXW7 expression upregulator and a pharmaceutically acceptable carrier or excipient.

[0015] This invention also provides a method for screening candidate substances for the prevention, delay, or improvement of Parkinson's disease, wherein at least one of the following criteria is met: increasing FBXW7 expression, reducing abnormal STAT3 activation, and improving mitochondrial dysfunction.

[0016] Preferred: Abnormal activation of STAT3 is characterized by an increase in STAT3 phosphorylation levels.

[0017] Preferred: Increased STAT3 phosphorylation level: Increased p-STAT3 Tyr705 site level.

[0018] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of FBXW7 modulators in the preparation of drugs for the prevention or improvement of Parkinson's disease. The technical effects achieved are as follows: in animal and cell models of Parkinson's disease, FBXW7 expression is reduced, accompanied by abnormal activation of p-STAT3 and mitochondrial dysfunction; this invention, by upregulating FBXW7 expression, can inhibit the increase of p-STAT3 levels and improve mitochondrial dysfunction. This indicates that FBXW7 can serve as a new target for Parkinson's disease intervention, and preparations or compositions based on the upregulation of FBXW7 expression can be used to prevent, delay, or improve Parkinson's disease, providing a new technical basis for the screening of related candidate drugs. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The attached figure is a schematic diagram showing the detection results of behavioral abnormalities, decreased FBXW7 expression, p-STAT3 activation, changes in mitochondrial dynamics-related proteins, and changes in apoptosis-related proteins in the MPTP-induced Parkinson's disease animal model provided by this invention. In the figure, A represents the rotarod experiment results; B represents the pole climbing experiment results; C and D represent the detection results of FBXW7 and p-STAT3 protein expression; E and F represent the detection results of mitochondrial dynamics-related proteins; and G and H represent the detection results of apoptosis-related proteins.

[0021] Figure 2 The attached figure shows the MPP provided by the present invention. + The diagram shows the experimental design of FBXW7 overexpression and the results of apoptosis-related proteins in the cell damage model. In the diagram, A shows the validation results of FBXW7 overexpression; B shows the p-STAT3 protein band obtained by Western blot detection; C shows the quantitative analysis results of the corresponding band gray values; and D and E show the detection results of apoptosis-related proteins.

[0022] Figure 3 The attached figure shows the MPP provided by the present invention. + A schematic diagram of mitochondrial damage results in the treated cell damage model, where A and B are the results of reactive oxygen species detected by the DCFH-DA probe; C and D are the results of JC-1 staining; and E and F are the results of mitochondrial dynamics-related protein detection. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention discloses the application of FBXW7 modulators in the preparation of drugs for the prevention or improvement of Parkinson's disease.

[0025] All raw materials and reagents not mentioned in the examples are commercially available, and all methods not mentioned are conventional experimental methods, which will not be described in detail here.

[0026] In this invention, "FBXW7 expression upregulator" refers to a substance, formulation, or construct that can increase the expression level and / or activity of FBXW7 in cells or tissues, including but not limited to FBXW7 overexpression plasmids, recombinant viral vectors, nucleic acid constructs, mRNA formulations, liposome delivery systems, or combinations thereof.

[0027] In this invention, “improving Parkinson’s disease” includes, but is not limited to, improving motor dysfunction, reducing neuronal damage, inhibiting the activation of abnormal signaling pathways, improving mitochondrial dysfunction, reducing cell apoptosis, delaying disease progression, or improving one or more of the following pathobiological indicators:

[0028] In this invention, "mitochondrial dysfunction" includes, but is not limited to, decreased mitochondrial membrane potential, increased reactive oxygen species levels, abnormal mitochondrial dynamics, and disordered expression of related proteins.

[0029] In this invention, "mitochondrial dynamics-related proteins" include proteins associated with mitochondrial division and fusion, preferably one or more of Drp1, Fis1, Mfn1, Mfn2, and OPA1.

[0030] In this invention, "apoptosis-related proteins" include proteins that reflect the state of cell apoptosis, preferably one or more of Bax, Bcl-2, cleaved caspase-3, and caspase-3.

[0031] In this invention, "STAT3 aberrant activation" preferably refers to an increase in p-STAT3 levels. p-STAT3 is the phosphorylated form of Tyr705.

[0032] The animal model used in this invention is an MPTP-induced Parkinson's disease mouse model. Behavioral tests included the rotarod test and the pole climbing test. Western blot was used to detect FBXW7 and p-STAT3. Mitochondrial dynamics and apoptosis in the animal samples were assessed by evaluating the expression of relevant proteins.

[0033] The cell experiments used in this invention employ an MPP+-treated cell damage model, and based on this model, FBXW7 overexpression intervention is performed. Mitochondrial function assays in the cellular portion include JC-1 staining to detect mitochondrial membrane potential, DCFH-DA probe detection of reactive oxygen species levels, and detection of mitochondrial dynamics-related proteins; apoptosis is assessed through the detection of apoptosis-related proteins.

[0034] Example 1 In an MPTP-induced animal model of Parkinson's disease, behavioral abnormalities, decreased FBXW7 expression, p-STAT3 activation, abnormalities in mitochondrial dynamics-related proteins, and alterations in apoptosis-related proteins were observed.

[0035] 1. Establishment of animal models After one week of acclimatization feeding, the MPTP group was intraperitoneally injected with MPTP (30 mg / kg), while the control group was intraperitoneally injected with an equal volume of physiological saline, once daily for 7 consecutive days. Behavioral and protein assays were performed after modeling was completed.

[0036] 2. Behavioral testing The motor function of each group of animals was evaluated using the rotarod test and pole climbing test. The results showed that, compared with the control group, the MPTP model group mice had a shorter rotarod dwell time and a longer pole climbing completion time, such as... Figure 1 The values ​​in A and B indicate that the model group showed significant motor dysfunction.

[0037] 3. FBXW7 and p-STAT3 detection Total protein was extracted from the substantia nigra of mice, and the protein expression levels of FBXW7 and p-STAT3 were detected by Western blot. The results showed that, compared with the control group, FBXW7 protein expression was decreased and p-STAT3 protein expression was increased in the MPTP model group. Figure 1 C and D in the middle.

[0038] 4. Detection of mitochondrial dynamics-related proteins Western blot was used to detect changes in the expression of mitochondrial dynamics-related proteins. The results showed that, compared with the control group, the expression of DRP1 protein was significantly increased and the expression of Mfn2 protein was significantly decreased in the brain tissue of the MPTP model group mice. Figure 1E and F. This indicates that the model mice exhibit abnormal mitochondrial dynamics.

[0039] 5. Detection of apoptosis-related proteins Western blot was used to detect the expression of apoptosis-related proteins. The results showed that, compared with the control group, the pro-apoptotic protein Bax was increased and the anti-apoptotic protein Bcl-2 was decreased in the MPTP model group. Figure 1 The presence of G and H in the PD mouse model indicates increased apoptosis.

[0040] Example 2 MPP + The establishment of an experimental system for FBXW7 overexpression in a cell damage model was demonstrated, and FBXW7 overexpression was observed in MPP. + Reduce p-STAT3 activation and alleviate apoptosis in the context of cell damage.

[0041] Cell grouping and processing Take MN9D cells that are in good culture condition and in the logarithmic growth phase, and carry out subsequent processing when the cell confluence reaches 70%-80%.

[0042] Grouping: (1) Normal group: No MPP performed + (2) Model group: MPP was used. + MN9D cells were treated to establish a Parkinson's disease-related cell damage model; (3) Model + empty vector group: after transfection with the blank expression vector, MPP was used. + Treatment; (4) Model + FBXW7 overexpression group: After transfecting the FBXW7 overexpression plasmid, MPP was then used. + deal with.

[0043] Primers were designed based on the coding sequence of the mouse Fbxw7 gene to amplify the Fbxw7 open reading frame using cDNA containing the Fbxw7 coding sequence as a template. The upstream primer was 5′-GAGACCCAAGCTGGCTAGTTGAATTCGCCACCATGAATCAGGAACTGCT-3′, and the downstream primer was 5′-CACTTAAGCTTGGTACCGAGGATCCTTTCATGTCCACATCAAAGTCCAG-3′.

[0044] The PCR amplification system is shown in Table 1:

[0045] The PCR procedure is shown in Table 2:

[0046] After identification by agarose gel electrophoresis, the PCR amplification products were excised from the specific bands of the expected size and purified using a gel extraction kit to obtain the Fbxw7 target fragment. The purified Fbxw7 target fragment was then recombinantly ligated into the linearized pcDNA3.1-CMV-MCS-3Flag-EF1-ZsGreen-T2A-Puro vector. The vector was linearized by double digestion with EcoRI and BamHI, and the linearized vector fragment was recovered and purified. Subsequently, the HB infusion™ one-step cloning and ligation system was used to prepare a reaction system containing the linearized vector, the Fbxw7 target fragment, HB infusion™ reaction solution, and nuclease-free water in an ice-water bath to obtain the FBXW7 overexpression plasmid. In this plasmid, the Fbxw7 coding sequence is located downstream of the CMV promoter and maintains the same open reading frame as the 3Flag tag; ZsGreen was used to observe transfection efficiency, and the Puro resistance gene was used to screen for positive cells. The ligation products were transformed, screened for positive clones, extracted plasmids, and sequenced for verification. After confirming the correct Fbxw7 insert sequence and orientation, they were used in subsequent experiments. The empty vector group used a homologous blank vector without the Fbxw7 coding sequence. The pcDNA3.1-CMV-MCS-3Flag-EF1-ZsGreen-T2A-Puro vector was a commercially available expression vector purchased from Hanheng Biotechnology.

[0047] When the MN9D cells reached 70%-80% confluence, the FBXW7 overexpression plasmid was transfected using liposome transfection. For example, in a 6-well plate, 2 μg of plasmid was transfected into each well; the model + empty vector group was transfected with an equal amount of blank vector. After 6-8 h of transfection, the medium was replaced with fresh complete medium, and the cells were cultured for another 24 h. Subsequently, MPP was added to the model group, the model + empty vector group, and the model + FBXW7 overexpression group. + Processing, MPP + The final concentration was 1.6 mmol / L, and the treatment lasted for 24 h to establish a Parkinson's disease-associated MN9D cell damage model. An equal volume of culture medium was added to the normal group as a control.

[0048] FBXW7 overexpression validation Cells from each group were collected, total protein was extracted, and the expression level of FBXW7 protein was detected by qPCR and Western blot.

[0049] The results showed that, compared with the model group and the model + empty vector group, the expression of FBXW7 mRNA and protein was significantly increased in the model + FBXW7 overexpression group, indicating that the overexpression system was successfully constructed. Figure 2 As shown in Figures A through C.

[0050] p-STAT3 detection Cells were collected from the normal group, model group, model + empty vector group, and model + FBXW7 overexpression group. Total protein was extracted, and p-STAT3 protein expression level was detected by Western blot. Results showed that compared with the model group and / or model + empty vector group, the p-STAT3 level was decreased in the model + FBXW7 overexpression group, suggesting that FBXW7 overexpression can inhibit abnormal STAT3 activation under MPP+ injury background. Figure 2 As shown in B and C.

[0051] Apoptosis-related protein detection Western blot analysis was used to detect the expression of apoptosis-related proteins. Results showed that, compared with the model group and / or the model + empty vector group, the abnormal expression of apoptosis-related proteins was improved in the model + FBXW7 overexpression group, suggesting that FBXW7 overexpression can alleviate cell apoptosis. Figure 2 As shown in D and E.

[0052] Example 3 FBXW7 overexpression can improve mitochondrial function in the context of MPP+ cell damage (see results). Figure 3 ) 1. DCFH-DA probe for detecting reactive oxygen species Intracellular reactive oxygen species (ROS) levels in each group were detected using the DCFH-DA probe. Results showed that ROS levels were decreased in the model group and / or the model + empty vector group compared to the model group, suggesting that FBXW7 overexpression can alleviate oxidative stress. Figure 3 As shown in A and B.

[0053] 2. JC-1 staining to detect mitochondrial membrane potential JC-1 staining was used to detect mitochondrial membrane potential in each group of cells. Results showed that compared with the model group and / or the model + empty vector group, the mitochondrial membrane potential was improved in the model + FBXW7 overexpression group, suggesting that FBXW7 overexpression can improve impaired mitochondrial function. Figure 3 As shown in C and D.

[0054] 3. Detection of mitochondrial dynamics-related proteins Western blot was used to detect the expression of mitochondrial dynamics-related proteins. The results showed that, compared with the model group and / or the model + empty vector group, the abnormal expression of mitochondrial dynamics-related proteins was improved in the model + FBXW7 overexpression group, suggesting that FBXW7 overexpression can alleviate mitochondrial damage. Figure 3 As shown in E and F.

[0055] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0056] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of FBXW7 expression upregulators in the preparation of drugs for the prevention, delay or improvement of Parkinson's disease.

2. The application as described in claim 1, characterized in that, The FBXW7 expression upregulator is a preparation that increases the expression or activity of FBXW7 in cells, including FBXW7 overexpression vectors, recombinant viral vectors, nucleic acid constructs, mRNA preparations, gene delivery systems, or combinations thereof.

3. The application as described in claim 2, characterized in that, The FBXW7 expression upregulator is a eukaryotic overexpression plasmid carrying the Fbxw7 coding sequence, which can induce Fbxw7 overexpression in nerve cells.

4. The application as described in claim 3, characterized in that, The Fbxw7 overexpression plasmid contains a mammalian cell promoter, an Fbxw7 coding sequence, a transcription termination sequence, and a selection marker sequence, wherein the Fbxw7 coding sequence is located downstream of the promoter.

5. The application as described in claim 4, characterized in that, The Parkinson's disease mentioned is Parkinson's disease associated with mitochondrial dysfunction.

6. The application as described in claim 5, characterized in that, The improvement of mitochondrial function includes at least one of the following: improving mitochondrial membrane potential, reducing reactive oxygen species levels, increasing ATP production, improving mitochondrial morphology, and restoring mitochondrial division / fusion balance.

7. A pharmaceutical composition for preventing, delaying, or improving Parkinson's disease, characterized in that, It includes FBXW7 expression upregulators and pharmaceutically acceptable carriers or excipients.

8. A method for screening candidate substances for the prevention, delay, or improvement of Parkinson's disease, characterized in that, The screening criteria included at least one of the following: increased FBXW7 expression, reduced abnormal STAT3 activation, and improved mitochondrial dysfunction.

9. The method as described in claim 8, characterized in that, The abnormal activation of STAT3 is characterized by an increase in STAT3 phosphorylation levels.

10. The method as described in claim 9, characterized in that, The STAT3 phosphorylation level was increased: the level at the p-STAT3Tyr705 site was increased.

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