Substance for targeted inhibition of IRF1 gene expression and application thereof

Abstract

The invention discloses a substance for targeted inhibition of IRF1 gene expression and application thereof. The substance comprises nucleic acid for specifically silencing the IRF1 gene. The nucleic acid is siRNA (small interfering Ribonucleic Acid) containing the following sequence or a cloning vector for transcriptional coding of the following sequence: 5 '-TCACCAAGAACCAGGAA-3'. The siRNA is applied to preparation of drugs for regulating differentiation and apoptosis of oligodendrocyte precursor cells and drugs for promoting remyelination and repairing spinal cord injury. According to the invention, IRF1 is taken as a molecular intervention target, and IRF1 expression is reduced or inhibited, so that apoptosis of oligodendrocyte precursor cells is significantly inhibited, differentiation and maturation of the oligodendrocyte precursor cells are promoted, and the purpose of promoting myelin sheath recovery of spinal cord injury is achieved.

Description

Technical Field

[0001] The present invention relates to a drug for treating nerve damage, and in particular to a substance for inhibiting IRF1 gene expression in a targeted manner and application thereof in repairing spinal cord damage. Background Art

[0002] Spinal cord injury is a devastating neurological disease that can lead to cell loss and damage to descending motor and ascending sensory tracts, ultimately causing permanent neurological deficits. Although some progress has been made in the treatment of spinal cord injury, there is still no effective treatment that can fully restore spinal cord function.

[0003] Under normal circumstances, the myelin sheath of the spinal cord forms a dense, multi-layered insulating membrane around axons, providing nutritional support, maintaining axonal survival and integrity, and promoting the propagation of axonal electrical signals. Oligodendrocytes are the primary cells in the central nervous system responsible for axonal myelination. After spinal cord injury, oligodendrocytes are destroyed and undergo necrosis and apoptosis, leading to demyelination of axons at the injured site. Simultaneously, endogenous oligodendrocyte precursor cells are activated after spinal cord injury, proliferating and migrating to the injured site, where they differentiate into new oligodendrocytes. Newly formed oligodendrocytes continue to form mature myelin sheaths and remyelinate residual or regenerating axons, promoting the recovery of neurological function. Studying the regulation and molecular mechanisms of oligodendrocytes after spinal cord injury will open new avenues for the treatment of spinal cord injury and is of great significance and value.

[0004] IRF1 (Interferon Regulatory Factor 1) is the first member of the interferon regulatory factor family and was originally identified as a key regulator of type I interferons (α / β). Its expression is upregulated after spinal cord injury and is closely associated with neuronal apoptosis and inflammatory responses. Modulating IRF1 gene expression or activity may inhibit neuronal apoptosis and alleviate inflammatory damage. Currently, there are no reports linking IRF1 to myelin regeneration in nerve injury. Summary of the Invention

[0005] Purpose of the Invention: The purpose of the present invention is to provide a substance that targets and inhibits IRF1 gene expression, thereby addressing the problem of how to reduce IRF1 gene expression. Another purpose of the present invention is to propose the use of a substance that targets and inhibits IRF1 gene expression in the preparation of a drug, thereby addressing the problem of how to promote the differentiation of dendrite progenitor cells, inhibit oligodendrocyte progenitor cell apoptosis, promote myelin regeneration, and thus treat spinal cord injury.

[0006] Technical solution: The present invention provides a substance for targeted inhibition of IRF1 gene expression, comprising a nucleic acid for specific silencing of the IRF1 gene.

[0007] The present invention discloses for the first time that the interferon regulatory factor 1 encoding gene (IRF1) is upregulated after spinal cord injury in rats and is closely related to the differentiation and apoptosis of oligodendrocyte precursor cells, and can become a new target for the repair of spinal cord nerve injury.

[0008] Preferably, the nucleic acid is an siRNA containing any of the following sequences or a cloning vector encoding any of the following sequences:

[0009] 5'-TCACCAAGAACCAGAGGAA-3'

[0010] 5′-GGGATACTTGCTCAATGAA-3′.

[0011] Preferably, the cloning vector comprises a plasmid or a virus.

[0012] The second aspect of the present invention discloses the use of the above-mentioned substance for targeted inhibition of IRF1 gene expression in any of the following:

[0013] preparing a drug for promoting differentiation of oligodendrocyte precursor cells;

[0014] preparing a drug for inhibiting apoptosis of oligodendrocyte precursor cells;

[0015] Preparation of a drug for promoting myelin regeneration;

[0016] Preparation of a drug for repairing spinal cord injuries.

[0017] Preferably, the drug contains siRNA of any of the following sequences:

[0018] 5'-TCACCAAGAACCAGAGGAA-3'

[0019] 5′-GGGATACTTGCTCAATGAA-3′.

[0020] In some embodiments, the final concentration of siRNA in the medicament is 0.05-1 nmol / μL.

[0021] Preferably, the drug further comprises a carrier for loading and delivering siRNA.

[0022] Furthermore, the carrier for loading and delivering siRNA includes at least one of liposome nanoparticles, non-viral nanocarriers, and siRNA expression vectors.

[0023] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0024] Preferably, the dosage form of the drug includes at least one of tablets, capsules, granules, pills, suspensions, injections, powder injections, aerosols, ointments, and emulsions.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] The present invention uses IRF1 as a molecular intervention target to downregulate or inhibit IRF1 expression, thereby significantly inhibiting the apoptosis of oligodendrocyte precursor cells and promoting their differentiation and maturation, thereby achieving the purpose of promoting myelin recovery in spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The expression changes of IRF1 in the spinal cord tissue at different time points after spinal cord injury in rats;

[0028] Figure 2 The effect of different IRF1 siRNA transfection on IRF1 expression in oligodendrocyte precursor cells 48 hours later;

[0029] Figure 3 The effect of siRNA-IRF1-1 on oligodendrocyte precursor cell differentiation;

[0030] Figure 4 The effect of siRNA-IRF1-1 on apoptosis of oligodendrocyte precursor cells;

[0031] Figure 5 The effect of siRNA-IRF1-1 on the recovery of motor function in rats with spinal cord impact injury;

[0032] Figure 6 This is the effect of siRNA-IRF1-1 on myelin regeneration after spinal cord impact injury in rats. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] Example 1: Investigating the expression changes of IRF1 at different time points after spinal cord injury in adult rats

[0035] 1. Rat spinal cord impact injury model

[0036] Forty female SPF Sprague-Dawley rats weighing approximately 250 g were injected intraperitoneally with a combination anesthetic (85 mg / kg chloral hydrate, 42 mg / kg magnesium sulfate, and 17 mg / kg sodium pentobarbital, Shanghai Lingfeng Chemical Reagent Co., Ltd., China). After successful anesthesia, the rats were placed supine on a laboratory bench and disinfected with iodine. After locating the T10 spinal cord segment, a longitudinal incision was made through the skin, fascia, and muscle. The rats were then secured with a spreader and the vertebral lamina above the T9 segment was removed with rongeurs to expose the spinal cord. After setting the parameters for the PSI-IH spinal cord impactor (impact force 150 kilodynes, dwell time 5 seconds), the rats were secured to the injury frame and impacted with the PSI-IH spinal cord impactor. Following the impact, the muscles, fascia, and skin of the rats were sutured, disinfected with alcohol cotton balls, and the ears were marked with ear tags. The rats were then placed on a 37°C heated electric blanket and allowed to awaken. After SD rats regain consciousness, they are transferred to cages and given feed and water on schedule. They are allowed to urinate twice a day, morning and evening, until they can urinate independently.

[0037] 2. RNA extraction and qRT-PCR of spinal cord tissue

[0038] After the rats were fully anesthetized by intraperitoneal injection of compound anesthetics, the spinal cord tissues of the rats were collected from the uninjured rats and the rats 1 day, 3 days, 7 days, and 14 days after injury (distal spinal cord tissue within 5 mm of the injury site). Total RNA was extracted from the spinal cord tissues using TRIzol reagent (Gibco, USA). The DNA was reverse transcribed into complementary DNA using the III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, China). qRT-PCR was performed using the StepOne Real-Time PCR System (Applied Biosystems, USA). qPCR was performed using the SYBR Green Master Mix (High ROX Premixed) kit (Vazyme, China) with GAPDH as the internal reference. The primer sequences for IRF1 and GAPDH are as follows:

[0039] IRF1-F: 5'-TCA CTG TCT GAG CCA AAC CGA-3'

[0040] IRF1-R: 5'-GAT AGG CAT GGT GGC TTT GCT G-3'

[0041] GAPDH-F: 5'-TGG AGT CTACTG GCG TCT T-3'

[0042] GAPDH-R: 5'-TGT CAT ATT TCT CGT GGT TCA-3'.

[0043] qRT-PCR results are as follows Figure 1 As shown, the results showed that compared with day 0, the expression of IRF1 in spinal cord nerve tissue increased significantly 1 day, 3 days, 7 days, and 14 days after injury, with the highest expression level 14 days after injury.

[0044] Example 2: Effect of downregulating IRF1 expression on oligodendrocyte precursor cell differentiation and apoptosis

[0045] 1. Culture of primary oligodendrocyte precursor cells

[0046] Coat the medium dish with poly-lysine (Poly-l-lysine, PLL) (Sigma-Aldrich, USA), place it in the refrigerator at 4°C overnight, and discard the PLL. Take an appropriate amount of Milli-Q and wash it repeatedly three times, then blow dry it under ultraviolet irradiation on the clean bench. Spray 75% alcohol to disinfect the SD rats one day after birth, remove the cerebral cortex under a microscope in dissecting fluid, and operate on ice. Use a 1ml pipette tip to blow away the cerebral cortex, digest it with 2ml 0.25% trypsin for 30 minutes, and shake it once every 15 minutes. After the digestion is terminated, filter the cell suspension with a 30μm filter. Then use A2B5 MACS magnetic beads (Miltenyi Biotec, Germany) to separate primary oligodendrocyte precursor cells. After the cells are resuspended, they are evenly added to the culture dish coated in advance and placed in a 5% CO2 constant temperature cell culture incubator (37°C) for culture. Observe the growth and change the medium the next day. Wait until the oligodendrocyte precursor cells grow to an appropriate density for subsequent experiments.

[0047] 2. Oligodendrocyte Precursor Cell Transfection

[0048] Based on the full-length sequence of the IRF1 gene, siRNA was designed for siRNA interference experiments. The siRNA sequences used for subsequent transfection are as follows:

[0049] siRNA-IRF1-1: 5'-TCACCAAGAACCAGAGGAA-3'

[0050] siRNA-IRF1-2: 5'-GGGATACTTGCTCAATGAA-3'

[0051] siRNA-IRF1-3: 5'-TAGCCCTGACACCTTATCT-3'

[0052] siRNA-NC: Random siRNA sequence was purchased from Guangzhou Ruibo, China, catalog number: siN0000001-1-5.

[0053] The cells were digested with 0.25% trypsin, and the cell suspension was collected after the digestion was terminated; Opti-MEM, Lipofectamine TM RNAiMAX and siRNA mixture (24-well culture plate: 100 μl Opti-MEM + 1 μl Lipofectamine TM Mix RNAiMAX + 1.25 μl siRNA (50 nM) thoroughly, add to the culture plate, pipette the cell suspension, count and evenly add the target number of cells to the culture plate, top up with transfection medium, gently shake, and place in a cell culture incubator. After 12 hours, replace the transfection medium with normal culture medium and continue culturing for 24-36 hours before subsequent experiments.

[0054] 3. Oligodendrocyte Precursor Cell RNA Extraction, Reverse Transcription, and qRT-PCR

[0055] Oligodendrocyte precursor cell line OLN93 was transfected with control siRNA Negative Control (siRNA-NC) and various IRF1 siRNAs for 48 h, the culture medium was discarded, the cells were washed twice with PBS, an appropriate amount of pre-cooled Trizol was added, and the cells were repeatedly pipetted and evenly mixed. Cell RNA was extracted, reverse transcribed, and qRT-PCR was performed using the same method as described in Example 1, with GAPDH as the internal control.

[0056] The results are as follows Figure 2 As shown, after 48 h of siRNA treatment, siRNA-IRF1-1 had the best knockdown effect, and siRNA-IRF1-1 was selected for subsequent experiments.

[0057] 4. Primary Oligodendrocyte Precursor Cell Differentiation Experiment

[0058] Primary oligodendrocyte precursor cells were transfected with control siRNA-NC and siRNA-IRF1-1, and the cells were cultured at 1×10 5 Cells were seeded at a density of 100 μg / well in 24-well plates coated with PLL. After 2 days of culture in proliferation medium (1× B27, 1× N2, 20 ng / mL bFGF, 10 ng / mL IGF, and 1× penicillin / streptomycin), the proliferation medium was replaced with differentiation medium containing CNTF and T3 and cultured for another 4 days for subsequent experiments. Oligodendrocytes were labeled with MBP (Abcam, USA), and oligodendrocyte precursor cells were labeled with PDGFRα (Abcam, USA). Immunofluorescence staining was performed, and the cells were photographed and counted.

[0059] The results of differentiation experiments were as follows Figure 3 As shown, Figure 3Figure A in the middle is a representative image of immunofluorescence of oligodendrocyte differentiation experiments after IRF1 interference in primary oligodendrocyte precursor cells, PDGFRα (green) and MBP (red) are co-labeled, and DAPI (blue) marks the cell nucleus; Figure 3 Figure B in the middle shows the quantitative analysis of MBP area in the cell immunofluorescence experiment results; Figure 3 Figure C in the middle shows the qRT-PCR results of the oligodendrocyte differentiation experiment after interfering with IRF1 in primary oligodendrocyte precursor cells to detect the mRNA expression levels of mature oligodendrocyte markers Mog and Ndrg1 (GAPDH was used as an internal reference).

[0060] Depend on Figure 3 As shown, the differentiation area of ​​oligodendrocytes after IRF1 knockdown was significantly higher than that of the control group, and the mRNA expression of mature myelin markers Mog and Ndrg1 in oligodendrocytes after IRF1 knockdown was significantly higher than that of the control group. In vitro differentiation experiments showed that siRNA transfection significantly increased the differentiation of oligodendrocyte precursor cells, indicating that knockdown of IRF1 expression can significantly promote the differentiation of oligodendrocyte precursor cells into mature oligodendrocytes.

[0061] 5. TUNEL cell apoptosis assay

[0062] Oligodendrocyte precursor cell line OLN93 was transfected with siRNA-NC and siRNA-IRF1-1, and the cells were cultured at 1×10 5 Cells were seeded at a density of 100 μg / well into a 24-well plate coated with PLL. Twelve hours after transfection, the culture medium was replaced with proliferation medium containing 100 ng / ml LPS, and apoptosis was induced for 24 hours. TUNEL BrightGreen Apoptosis Detection Kit (Vazyme, China) was then used for staining, and images were taken and counted.

[0063] The results of apoptosis experiments were as follows Figure 4 As shown, Figure 4 Figure A in the middle is a representative image of TUNEL staining after IRF1 interference in OLN93 cells, TUNEL (red) marks apoptotic cells, and DAPI (blue) marks cell nuclei; Figure 4 Figure B is the quantitative analysis of the proportion of apoptotic cells to total cells in TUNEL staining results. Figure 4 It can be seen that interfering with IRF1 expression can significantly inhibit the apoptosis of oligodendrocyte precursor cells.

[0064] Example 3: Effect of IRF1 on the repair of motor function in spinal cord impact injury

[0065] 1. Functional repair experiment of rat spinal cord impact injury

[0066] Sixty female SPF SD rats weighing approximately 250 g were randomly divided into sham group, SCI group, siRNA-NC group and siRNA-IRF1-1 group, with 15 rats in each group. Spinal cord impact surgery was performed on rats according to the method in Example 1. The sham group was a sham operation group, in which the wound was directly sutured without impact after excision of the lamina; the SCI group was not injected with drugs after impact. After the impact, the SD rats in the siRNA-NC group and siRNA-IRF1-1 group were fixed on a rotating digital stereotaxic instrument, the injury site was observed under a microscope, and siRNA-NC or siRNA-IRF1-1 was injected at four points symmetrically along the injury site (diluted with 6 μL PBS for each 1 nmol), with three injection depths at each point (0.6 mm, 0.9 mm, and 1.2 mm below the dura mater, and 0.5 μL was injected at each depth). After each injection, the injection was left for 3 minutes. After the operation, the rats were placed on a 37°C constant temperature electric blanket. After SD rats woke up, they were transferred to cages and given feed and water on time. They were allowed to urinate twice a day, morning and evening, until they could urinate independently.

[0067] 2. Basso Beattie Bresnahan (BBB) ​​Score of Rat Hindlimb Movement

[0068] After establishing the spinal cord impact injury model in SD rats, the rats were placed on an open platform and allowed to crawl freely. Two individuals performed a double-blind BBB score. A hindlimb BBB score of 0 one day after injury was considered a successful procedure. Scoring was performed weekly for eight weeks, and the average score was used for statistical analysis.

[0069] The results are as follows Figure 5 As shown, the BBB scores of rat hindlimbs at different time points after spinal cord injury showed that the BBB scores of rats in the siRNA-IRF1-1 group were higher than those in the siRNA-NC and SCI groups starting two weeks after spinal cord injury, indicating that IRF1 interference can promote the recovery of hindlimb motor function in rats and could be used for the treatment of spinal cord injury. In vivo injection of siRNA-IRF1-1 can promote the recovery of motor and sensory function in rats after spinal cord impact injury.

[0070] Example 4: Effect of downregulating IRF1 expression on myelin regeneration after spinal cord impact injury

[0071] Eight weeks after spinal cord injury, the spinal cord at the injured T9 segment was removed by perfusion, and the white matter tissue distal to the spinal cord injury was cut into small pieces of 1 mm*1 mm*3 mm and immersed in 2.5% glutaraldehyde (Solarbio, China). The pieces were further washed with PBS, fixed with osmium phosphate, dehydrated in gradients, treated with propylene oxide, immersed in embedding agent and polymerized, sectioned, and double electron stained. Finally, they were photographed and observed using a transmission electron microscope.

[0072] The results are as follows Figure 6 As shown, Figure 6 Figure A in the middle shows a representative transmission electron microscopy image of myelin sheath 8 weeks after spinal cord injury (upper panel), and an enlarged view of the white framed area (lower panel); Figure 6 Middle panel B shows the quantification of the proportion of myelinated axons; Figure 6 Middle C shows the G ratio of myelinated axons; Figure 6 Middle panel D is a dot plot of the relationship between G ratio and axon diameter.

[0073] Depend on Figure 6 It can be seen that 8 weeks after spinal cord injury, the siRNA-IRF1-1 group had more axons wrapped by myelin sheaths and the myelin sheath was thicker.

Claims

1. A substance that targets and inhibits IRF1 gene expression, characterized in that: The substance includes a nucleic acid for specifically silencing the IRF1 gene.

2. The substance for targeted inhibition of IRF1 gene expression according to claim 1, characterized in that: The nucleic acid is an siRNA containing any of the following sequences or a cloning vector encoding any of the following sequences: 5'-TCACCAAGAACCAGAGGAA-3' 5′-GGGATACTTGCTCAATGAA-3′.

3. The substance for targeted inhibition of IRF1 gene expression according to claim 2, characterized in that: The cloning vector includes a plasmid or a virus.

4. Use of the substance for targeted inhibition of IRF1 gene expression according to any one of claims 1 to 3 in any of the following: preparing a drug for promoting differentiation of oligodendrocyte precursor cells; preparing a drug for inhibiting apoptosis of oligodendrocyte precursor cells; Preparation of a drug for promoting myelin regeneration; Preparation of a drug for repairing spinal cord injuries.

5. The use according to claim 4, characterized in that The drug contains siRNA of any of the following sequences: 5'-TCACCAAGAACCAGAGGAA-3' 5′-GGGATACTTGCTCAATGAA -3′.

6. The use according to claim 5, characterized in that The medicine also includes a carrier for loading and delivering siRNA.

7. The use according to claim 6, characterized in that The carrier used for loading and delivering siRNA includes at least one of liposome nanoparticles, non-viral nanocarriers, and siRNA expression vectors.

8. The use according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable excipients.

9. The use according to claim 4, characterized in that The dosage form of the drug includes at least one of tablets, capsules, granules, pills, suspensions, injections, powder injections, aerosols, ointments, and emulsions.

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