Use of ssrp1 gene as a target in preparation of a drug for preventing or treating nmosd

Abstract

This invention belongs to the field of biomedical technology and discloses the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD. These drugs contain components that target and interfere with the SSRP1 gene or its expression. This invention creatively discovers the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD. Knockdown of SSRP1 can alleviate motor balance impairment in NMOSD, improve the severity of demyelination damage in NMOSD, improve the myelin regeneration microenvironment in NMOSD, alleviate microglial activation caused by NMOSD, alleviate microglial-mediated neuroinflammation in NMOSD, and alleviate microglial ferroptosis in NMOSD. Therefore, it can be applied to the preparation of drugs for the treatment of NMOSD and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD. Background Technology

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune demyelinating disease that primarily affects the optic nerve and spinal cord. It mainly manifests as symptoms associated with optic neuritis and acute transverse myelitis, such as decreased vision, limb numbness and weakness, motor dysfunction, and autonomic dysfunction. Pathologically, it presents with white matter demyelination, inflammatory cell infiltration, and astrocyte lesions. NMOSD is a highly relapsing and disabling disease; over 90% of patients experience a multiphase disease course; approximately 60% of patients relapse within one year, and 90% relapse within three years, placing a significant economic burden on families and society.

[0003] Structure-specific recognition protein 1 (SSRP1) is a core component of the highly conserved Promote Chromatin Transcription Complex (FACT), serving as an important histone chaperone involved in key nuclear processes such as transcriptional regulation, DNA replication, and damage repair. Besides its synergistic effect with SPT16, SSRP1 can also independently perform transcriptional regulation. Existing research indicates that under cellular stress conditions such as oxidative stress, the FACT complex can exert cytoprotective effects by regulating pathways such as NRF2. However, current research on the related mechanisms is largely limited to tumor cell models, and the biological function and clinical significance of the SSRP1 gene in NMOSD remain unknown.

[0004] Therefore, it is crucial to explore the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD. Summary of the Invention

[0005] This invention discloses the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD, which has good clinical application value.

[0006] To achieve the above objectives, this application adopts the following technical solution: This invention provides the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD.

[0007] In the above technical solutions, the drugs for preventing or treating NMOSD contain components that target and interfere with the SSRP1 gene or its expression.

[0008] In the above technical solutions, the methods of targeting and interfering with the SSRP1 gene or its expression include gene knockdown, transcriptional inhibition, translational inhibition, and post-translational protein modification inhibition.

[0009] In the above technical solution, the gene knockdown method is to knock down the SSRP1 gene using recombinant adenovirus AAV-SSRP1. The recombinant adenovirus AAV-SSRP1 is constructed by inserting mir30 shRNA into the genome of adenovirus AAV. The nucleotide sequence of mir30 shRNA is shown in SEQ ID No. 1, which is GCAGAGGAGTTTGACAGCAAT.

[0010] In the above technical solutions, knocking down SSRP1 alleviates the impairment of motor balance in NMOSD.

[0011] In the above technical solutions, knocking down SSRP1 improves the severity of demyelination damage in NMOSD.

[0012] In the above technical solutions, knocking down SSRP1 improves the myelin regeneration microenvironment in NMOSD.

[0013] In the above technical solutions, knocking down SSRP1 alleviates microglial activation caused by NMOSD.

[0014] In the above technical solutions, knocking down SSRP1 alleviates microglia-mediated neuroinflammation in NMOSD.

[0015] In the above technical solutions, knocking down SSRP1 alleviates ferroptosis in microglia in NMOSD.

[0016] The beneficial effects of this invention are as follows: This invention creatively discovers the application of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD. Knocking down SSRP1 can alleviate motor balance impairment in NMOSD, improve the severity of demyelinating damage in NMOSD, improve the myelin regeneration microenvironment in NMOSD, alleviate microglial activation caused by NMOSD, alleviate microglial-mediated neuroinflammation in NMOSD, and alleviate microglial ferroptosis in NMOSD. Therefore, it can be applied to the preparation of drugs for the treatment of NMOSD and has good clinical application value. Attached Figure Description

[0017] Figure 1 This is a statistical graph of SSRP1 expression in microglia; Figure 2 This is a statistical graph of the fluorescence intensity of SSRP1; Figure 3 This is a statistical chart showing the number of times a mouse's paw slips off the balance beam; Figure 4 It is a statistical chart of myelin loss; Figure 5 This is a statistical graph showing the density of total oligodendrocyte cell lines and mature oligodendrocytes; Figure 6 It is a statistical graph of microglia density, cell body area, firmness, and roundness; Figure 7 This is a statistical chart showing the percentage of CD16 / 32 and Arg1 positive microglia; Figure 8 This is a statistical graph of the fluorescence intensity of SLC7A11 and 4-HNE. Detailed Implementation

[0018] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims. This invention provides the use of the SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD, wherein the drugs for the prevention or treatment of NMOSD contain components that target and interfere with the SSRP1 gene or its expression.

[0019] Targeting interference with the SSRP1 gene or its expression can be achieved through gene knockdown, transcriptional repression, translational repression, and post-translational protein modification.

[0020] The specific method of gene knockdown is as follows: the SSRP1 gene is knocked down using recombinant adenovirus AAV-SSRP1. The recombinant adenovirus AAV-SSRP1 is constructed by inserting mir30 shRNA into the genome of adenovirus AAV. The nucleotide sequence of mir30 shRNA is shown in SEQ ID No. 1, which is GCAGAGGAGTTTGACAGCAAT.

[0021] Specifically, the method for constructing recombinant adenovirus AAV-SSRP1 includes the following steps: Step 1, Interference Target Design and Sequence Synthesis: Based on the general principles of mir30 shRNA design and the transcript of the SSRP1 gene, target was designed and the sequence shown in SEQ ID No. 1 was synthesized: GCAGAGGAGTTTGACAGCAAT.

[0022] Step 2, Preparation of linearized expression vector: The expression vector pAAV-CBG-DIO-EGFP-miR30shRNA-WPRE was digested with restriction endonucleases. The digestion products were analyzed by agarose gel electrophoresis to detect the digestion efficiency.

[0023] Step 3: The target fragment is ligated into the expression vector and transformed into DH5α competent cells.

[0024] Step 4: Pick the transformants that have grown on the plate and resuspend them in 10µl of LB medium. Take 1µl as a template for colony PCR identification.

[0025] Step 5: Sequencing is performed on the positive clones obtained from colony identification. Once the positive clones are verified, high-purity plasmid extraction is performed to obtain recombinant adenovirus AAV-SSRP1.

[0026] In this invention, the inventors discovered that knocking down SSRP1 can alleviate motor balance impairment in NMOSD, improve the severity of demyelination damage in NMOSD, improve the myelin regeneration microenvironment in NMOSD, alleviate microglial activation caused by NMOSD, alleviate microglial-mediated neuroinflammation in NMOSD, and alleviate ferroptosis of microglia in NMOSD.

[0027] Example 1: Upregulation of SSRP1 expression in microglia of NMOSD mouse model This invention establishes a NMOSD mouse model using stereotactic injection of a mixture of complement (HC) and AQP4-IgG antibody. This is a classic and internationally recognized NMOSD mouse model: through stereotactic injection, human AQP4-IgG antibody and complement are directly injected into the striatum of mice, successfully inducing the characteristic pathological changes of NMOSD in the mouse model. This model is consistent with the pathogenesis and pathophysiology of NMOSD patients and has good representativeness. The injected AQP4-IgG antibody is directly derived from NMOSD patients, accurately simulating the pathological autoimmune response in patients. This construction method avoids the heterogeneity problems that may exist in traditional animal models, making the mouse model closer to the true pathological state of NMOSD patients. Furthermore, using the combination of patient-derived antibodies and complement, it not only effectively induces demyelination, microglial activation, and neuroinflammation in the central nervous system, but also reproduces the key pathological features of the acute phase of NMOSD. Therefore, the mouse model used in this invention is not only an internationally recognized classic construction method, but is also widely used in NMOSD-related research.

[0028] Purification of serum IgG: Serum samples were collected from four AQP4-IgG positive NMOSD patients for purification. The procedure was as follows: After thawing, the serum samples were centrifuged to remove impurities and oils. The column was washed three times with equilibration buffer, and the flow-through was collected after loading. The column was then washed three more times with equilibration buffer, followed by elution with elution buffer, and the IgG was collected. The purified IgG was separated by G-agarose protein separation and prepared into lyophilized powder. The lyophilized powder was dissolved in PBS at pH 7.4 and filtered for sterilization. The concentration of IgG was adjusted to 20 mg / mL, designated as "AQP4-IgG". Each mouse was injected with a mixture containing 6 μL of AQP4-IgG and 4 μL of HC.

[0029] Specifically, after anesthetizing mice with isoflurane, the preauricular region of both ears was fixed using a stereotactic frame. An incision was made along the midline of the scalp, and a cranial hole approximately 1 mm in diameter was drilled 2 mm to the right of the anterior fontanelle. A 33G injection needle connected to a 25 μL microsyringe was vertically inserted into the brain to a depth of 3 mm, and the above-mentioned mixture was slowly injected at a rate of 0.5 μL / min. After injection, the needle was left in place for 10 minutes, then slowly withdrawn, and the scalp incision was sutured. Seven days were selected as the observation time point for NMOSD.

[0030] Microglia were labeled using CD45-APC and CD11b-FITC double-labeled flow cytometry. Specifically, 7 days after NMOSD modeling in mice, 2-3 hours before sampling, mice were intraperitoneally injected with 500 μL of 1% neutral red solution to differentiate demyelinating lesions from surrounding tissue. Mice were anesthetized with isoflurane. Brain cells were isolated from the lesion area of ​​the mice. The extracted brain cells were blocked using Fc-Block and double-labeled with CD45-APC / CD11b-FITC flow cytometry antibodies, incubated at 4°C for 30 minutes, and the resulting cell suspension was filtered through a 40µm filter into flow cytometry tubes for analysis. Then, RNA was extracted from microglia for qPCR detection. Specifically, total RNA was extracted using TRIzol reagent according to the manufacturer's instructions, and the concentration and purity were determined. 1 μg of total RNA was reverse transcribed in a 20 μL reaction system using PrimeScript RT Master Mix. Real-time quantitative PCR (qRT-PCR) was performed using HieffqPCR SYBR Green Master Mix. The expression levels of the target gene were normalized to β-actin, and the relative gene expression was determined using the 2^-ΔΔCt method.

[0031] Figure 1The results showed that SSRP1 expression was increased on CD45+CD11b+ microglia in the lesion area, while the expression of genes inhibiting ferroptosis was decreased, the expression of pro-inflammatory and phagocytic genes was increased, and the expression of anti-inflammatory genes was decreased. This suggests that SSRP1 may be involved in microglial ferroptosis and mediated neuroinflammation in NMOSD.

[0032] Example 2: Establishment of an NMOSD mouse model with SSRP1 knockdown We further established and validated an NMOSD mouse model with SSRP1 microglia-specific knockdown by stereotactic injection of AAV into the brain.

[0033] Specifically, two weeks before NMOSD modeling, in Cx3cr1 CreER 1 μL of AAV was injected into the right striatum of mice (coordinates: 2.0 mm to the right of the midline; 0.0 mm anterior to the anterior fontanelle; 3 mm depth). The final viral load was 1.0E+10 vg / mouse. Specifically: NMOSD-AAV-NC group: Mice were injected with the negative control AAV-NC.

[0034] The preparation method of AAV-NC includes the following steps: Step 1, Interference Target Design and Sequence Synthesis: Based on the general principles of mir30 shRNA design, target sites were designed and sequences as shown in SEQ ID No. 2 were synthesized: GAAGTCGTGAGAAGTAGAA.

[0035] Step 2, Preparation of linearized expression vector: The expression vector pAAV-CBG-DIO-EGFP-miR30shRNA-WPRE was digested with restriction endonucleases. The digestion products were analyzed by agarose gel electrophoresis to detect the digestion efficiency.

[0036] Step 3: The target fragment is ligated into the expression vector and transformed into DH5α competent cells.

[0037] Step 4: Pick the transformants that have grown on the plate and resuspend them in 10µl of LB medium. Take 1µl as a template for colony PCR identification.

[0038] Step 5: Sequencing is performed on the positive clones obtained from colony identification. Once the positive clones are verified, high-purity plasmid extraction is performed to obtain recombinant adenovirus AAV-NC.

[0039] NMOSD-AAV-SSRP1 group: Mice were injected with AAV-SSRP1 to knock down the SSRP1 gene. The preparation method of recombinant adenovirus AAV-SSRP1 is as described above.

[0040] Two weeks after AAV injection, an NMOSD mouse model was established using the aforementioned method. Immunofluorescence staining with SSRP1 antibody was used to assess the expression of SSRP1 in microglia in the damaged white matter region of the mice.

[0041] Specifically, frozen sections were thawed at room temperature and then washed with phosphate-buffered saline (PFS) for 5 min. Next, the membrane was perforated with Triton X-100 immunofluorescence membrane perforation buffer at room temperature for 15 min. Subsequently, the specimens were blocked with immunofluorescence rapid blocking buffer at room temperature for 15 min. The antibody was diluted with primary antibody dilution buffer, and 10 μL of dilution buffer was added to each sample. The samples were incubated at 4°C for 12 hours, then incubated with secondary antibody at room temperature in the dark for one hour. Finally, the slides were mounted, and the fluorescence intensity of SSRP1 was observed and statistically analyzed.

[0042] Figure 2 The results showed that the fluorescence intensity of SSRP1 in the NMOSD-AAV-NC group was significantly reduced compared with that in the NMOSD-AAV-SSRP1 group. These results indicate that the SSRP1 level in microglia was significantly knocked down after administration of AAV-SSRP1.

[0043] Example 3: Knockdown of SSRP1 alleviates motor balance impairment in NMOSD mice To evaluate the effect of SSRP1 knockdown on fine motor coordination and balance in NMOSD mice, a balance beam test was used to measure the time it took for mice to cross the balance beam and the number of paw slips during the test, thereby evaluating their motor coordination, balance, and limb control functions.

[0044] Specifically, a wooden strip (100 cm long and 1.2 cm wide) was fixed to a 50 cm high support, with a sponge pad placed underneath. A black frame was placed at the endpoint of the balance beam as a guide, and a light source was placed at the starting point as a stimulus. For two days prior to the test, the mice were trained daily to successfully cross the entire beam. On the day of the test, the time required for the mice to cross the balance beam and the number of times they slipped were recorded.

[0045] Figure 3 The results showed that compared with the NMOSD-AAV-NC group mice, the NMOSD-AAV-SSRP1 group mice had fewer paw slips, suggesting that knocking down SSRP1 can improve the motor coordination and balance ability of NMOSD mice and alleviate their motor balance function impairment.

[0046] Example 4: Knockdown of SSRP1 improves the severity of demyelinating injury in NMOSD mice NMOSD can lead to damage to neurons or axons in the central nervous system, as well as demyelination. Mouse brain tissue sections were stained with Luxol Fast Blue (LFB). LFB staining is a staining method that reveals the morphology and pathological changes of the myelin sheath, and can reflect the severity of white matter damage by assessing the degree of white matter demyelination.

[0047] Specifically, frozen sections were thawed at room temperature and then washed with phosphate buffer, tap water, and ultrapure water for 5 min each. They were then dehydrated in a gradient of 75%–95%–100% ethanol and stained with 0.1% LFB dye at 60°C for 6–8 h. After staining, the sections were removed and placed at room temperature. Under a microscope, the sections were repeatedly separated by color using 0.05% lithium carbonate differentiation solution and 75% ethanol until the myelin sheath was stained blue and the background was nearly colorless. The area of ​​white matter myelin loss was then calculated using ImageJ software.

[0048] Figure 4 The results showed that compared with the NMOSD-AAV-NC group mice, the demyelination area of ​​the lesion region in the NMOSD-AAV-SSRP1 group mice was significantly reduced, indicating that knocking down SSRP1 can significantly improve demyelination caused by NMOSD.

[0049] Example 5: Knockdown of SSRP1 improves the myelin regeneration microenvironment in NMOSD Oligodendrocytes are extremely sensitive to oxidative stress and inflammatory damage, and their survival and differentiation capacity are highly dependent on the surrounding cellular microenvironment. Abnormal activation of microglia and release of inflammatory factors in NMOSD create a microenvironment that inhibits regeneration, while post-demyelination repair relies on oligodendrocyte precursor cells (OPCs) differentiating into mature myelin-forming oligodendrocytes (OLs) in a suitable microenvironment. Immunofluorescence staining of mouse brain tissue sections using oligodendrocyte lineage markers (Olig2) and mature oligodendrocyte markers (GST-Pi) was performed to visualize and count the number of oligodendrocyte precursor cells and mature oligodendrocytes, thereby assessing the improvement of the myelin regeneration microenvironment.

[0050] Specifically, frozen sections were thawed at room temperature and then washed with phosphate-buffered saline (PBS) for 5 min. Next, the membranes were perforated with Triton X-100 immunofluorescence perforation buffer at room temperature for 15 min. Subsequently, the specimens were blocked with immunofluorescence rapid blocking buffer at room temperature for 15 min. The antibodies were diluted with primary antibody dilution buffer, and 10 μL of dilution buffer was added to each sample. After incubation at 4°C for 12 hours, the secondary antibody was incubated at room temperature in the dark for one hour. Finally, the slides were mounted and observed, and the densities of Olig2 and GST-Pi positive cells were counted.

[0051] Figure 5The results showed that compared with the NMOSD-AAV-NC group mice, the NMOSD-AAV-SSRP1 group mice had increased total oligodendrocyte lineage and mature oligodendrocyte density in the striatum region, indicating that knocking down SSRP1 can improve the myelin regeneration microenvironment of oligodendrocytes in NMOSD.

[0052] Example 6: Knockdown of SSRP1 alleviates NMOSD-induced microglial activation Microglia are innate immune cells of the central nervous system. They are activated under neuroinflammatory conditions. Activated microglia release pro-inflammatory and chemokines, leading to an inflammatory microenvironment, causing cell damage, immune cell infiltration, increased blood-brain barrier permeability, and inhibition of myelin regeneration and repair. Specific manifestations of microglia activation include increased density, decreased cell area, and increased firmness and roundness.

[0053] Figure 6 The results showed that, compared with the NMOSD-AAV-NC group, the NMOSD-AAV-SSRP1 group exhibited reduced microglia aggregation, increased cell area, and decreased robustness and roundness in the striatum of mice. This indicates that SSRP1 knockdown can effectively reduce microglia activation.

[0054] Example 7: Knockdown of SSRP1 alleviates microglia-mediated neuroinflammation in NMOSD Besides morphological overactivation, the functional polarization of microglia towards a pro-inflammatory state is a core element driving the neuroinflammatory cascade and directly leading to neuronal and myelin damage. FcγRIII / II receptors (CD16 / 32) are key markers of M1-type pro-inflammatory microglia / macrophages, while arginase 1 (Arg1) is a key marker of M2-type anti-inflammatory microglia / macrophages. Immunofluorescence staining of mouse brain tissue sections using CD16 / 32 or Arg1 and Iba-1 was used to reflect the level of microglia-mediated neuroinflammation in NMOSD.

[0055] Figure 7 The results showed that, compared with the NMOSD-AAV-NC group, the positive rate of CD16 / 32 in microglia was significantly reduced in the NMOSD-AAV-SSRP1 group, while the positive rate of Arg1 was significantly increased. This indicates that knocking down the SSRP1 gene helps alleviate microglia-mediated neuroinflammation in NMOSD.

[0056] Example 8: Knockdown of SSRP1 alleviates ferroptosis in microglia in NMOSD SLC7A11, a member of the solute carrier family 7, is a key functional subunit of the cysteine / glutamate reverse transport system on the cell membrane and is one of the key molecules in resisting ferroptosis. 4-Hydroxynonenal (4-HNE) is an important marker of lipid peroxidation in ferroptosis and accumulates in large quantities during the process. Immunofluorescence staining of mouse brain tissue sections using SLC7A11, 4-HNE, and Iba-1 was used to detect the level of ferroptosis in microglia.

[0057] Figure 8 The results showed that, compared with the NMOSD-AAV-NC group, the NMOSD-AAV-SSRP1 group had significantly increased SLC7A11 fluorescence intensity and significantly decreased 4-HNE fluorescence intensity. This indicates that knocking down SSRP1 helps alleviate ferroptosis in microglia during NMOSD.

[0058] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of SSRP1 gene as a target in the preparation of drugs for the prevention or treatment of NMOSD.

2. The application according to claim 1, characterized in that: Drugs for the prevention or treatment of NMOSD contain components that target and interfere with the SSRP1 gene or its expression.

3. The application according to claim 2, characterized in that: Targeting interference with the SSRP1 gene or its expression can be achieved through gene knockdown, transcriptional repression, translational repression, and post-translational protein modification.

4. The application according to claim 3, characterized in that: The gene knockdown method is to knock down the SSRP1 gene using recombinant adenovirus AAV-SSRP1. The recombinant adenovirus AAV-SSRP1 is constructed by inserting mir30 shRNA into the genome of adenovirus AAV. The nucleotide sequence of mir30 shRNA is shown in SEQ ID No. 1, which is GCAGAGGAGTTTGACAGCAAT.

5. The application according to claim 1, characterized in that: Knockdown of SSRP1 alleviates motor balance impairment in NMOSD.

6. The application according to claim 1, characterized in that: Knockdown of SSRP1 improves the severity of demyelinating injury in NMOSD.

7. The application according to claim 1, characterized in that: Knockdown of SSRP1 improves the myelin regeneration microenvironment in NMOSD.

8. The application according to claim 1, characterized in that: Knockdown of SSRP1 alleviates microglial activation induced by NMOSD.

9. The application according to claim 1, characterized in that: Knockdown of SSRP1 alleviates microglia-mediated neuroinflammation in NMOSD.

10. The application according to claim 1, characterized in that: Knockdown of SSRP1 alleviates ferroptosis in microglia in NMOSD.

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