IGF2BP1 targeting reagent and application of IGF2BP1 protein in treatment of acute ischemic stroke

Abstract

The invention relates to the field of physiology and molecular biology, and discloses an IGF2BP1 targeting reagent and application of IGF2BP1 protein in treatment of acute ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the fields of physiology and molecular biology, and specifically to the application of reagents targeting IGF2BP1 and IGF2BP1 protein in the treatment of acute ischemic stroke. Background Art

[0002] Acute ischemic stroke (AIS) is a disease caused by local cerebral blood supply disorders due to various reasons, resulting in ischemic and hypoxic necrosis of brain tissue, and then corresponding neurological deficits. During severe cerebral ischemia, severe shortages of oxygen and nutrients lead to cellular hypoxia depolarization and rapid necrosis, which is defined as the core area. The ischemic penumbra is brain tissue with low perfusion, where the supply of oxygen and glucose is reduced. It is at risk but may be salvageable tissue in focal ischemic stroke, which provides a potential therapeutic target in the field of ischemic stroke.

[0003] In the ischemic core area, energy failure can lead to rapid cell necrosis within minutes. In contrast, in the ischemic penumbra, cell death can occur through apoptosis over a longer period. Some mechanisms have been proposed to promote the transformation of apoptosis to necrosis, such as excitotoxicity, spreading depolarization, free radical production, reperfusion injury, etc. Although the neuroprotective value of treating the penumbra has not been realized clinically, its importance is obvious through clinical trials of endovascular treatment. In these trials, patients with a small infarct core and a relatively large penumbra tissue have the highest chance of achieving good functional outcomes. Restoring blood supply through emergency reperfusion or intervening in the process of cells entering the death pathway presents an opportunity to rescue the penumbral brain tissue. However, the neuroprotection of this salvageable tissue has proven elusive. A large number of experimental studies have been conducted to achieve neuroprotection by targeting the occurrence of the ischemic penumbra in ischemic stroke. However, so far, no experimental study has achieved translational success. Therefore, research is needed to target the penumbra region, determine the timing of intervening in the above pathological mechanisms, extend the apoptosis time of the penumbra to reverse cell death, and precisely interfere with the cell death mechanism.

[0004] RNA m6A modification is one of the most common RNA methylation modifications, which is the methylation of the 6th nitrogen (N) atom of adenosine (A) in RNA. m6A is installed by a methyltransferase complex consisting of METTL3, METTL14, WTAP, KIAA1429, METTL16, RBM15, and ZC3H13, removed by demethylases such as FTO and ALKBH, and m6A-binding proteins can recognize m6A-modified RNA. m6A can regulate a variety of physiological processes and affect disease progression. Transient focal ischemia in mice can alter the brain m6A epitranscriptome, and m6A modification increases under conditions of OGD / R or MCAO / R. In addition, in the MCAO model, m6A demethylase can selectively cause demethylation of Bcl2 mRNA, increasing Bcl2 protein expression and reducing neuronal damage. METTL3 can promote the maturation of miR-335, increase the formation of stress granules in the cytoplasm, and thus reduce neuronal apoptosis. These studies demonstrate that m6A modification plays an important role in ischemic brain injury. By altering m6A modification, the expression of stroke-related proteins can be regulated, affecting the corresponding signaling pathways.

[0005] IGF2BP1 is an m6A-binding protein that can recognize m6A modification and promote mRNA stability and translation by protecting m6A-containing mRNA from degradation, thus affecting gene expression. Studies have shown that IGF2BP1 can promote cancer progression by regulating the potential of gene stability through recognizing m6A modification of target genes. Unfortunately, as an important m6A-binding protein, changes in IGF2BP1 expression have been observed in the mouse MCAO model, but its role in the occurrence and development of ischemic stroke, especially its expression and specific mechanism of action in the ischemic penumbra, has not been investigated. Summary of the Invention

[0006] In view of the fact that the prior art has not systematically evaluated the effects of IGF2BP1 on neurites, cell survival, and neuronal structure in vitro and in vivo and their mechanisms, and the prior art is limited to the exploration of the role of IGF2BP1 in tumor diseases, and has failed to explore the neuroprotective effect of IGF2BP1 protein in regulating the ischemic penumbra in ischemic stroke, and no research on therapeutic targets for acute ischemic stroke has been carried out on this basis. To overcome the deficiencies in the prior art, this application comprehensively describes the expression of IGF2BP1 protein in neurons of MCAO mice, studies the neuroprotective effect of knocking down IGF2BP1 in mouse MCAO injury, and accordingly proposes a promising strategy for the effective treatment of ischemic stroke to guide the research on rescuing the ischemic penumbra.

[0007] This application provides the use of a reagent targeting IGF2BP1 in the preparation of a drug for treating acute ischemic stroke.

[0008] The present application also provides the use of a reagent for rescuing synaptic damage and apoptosis of neurons in the ischemic penumbra by changing the level of IGF2BP1 protein in the development of guiding the treatment of acute ischemic stroke.

[0009] In some embodiments, the reagent inhibits and / or reduces the expression level of IGF2BP gene and / or protein.

[0010] In some embodiments, the reagent is selected from an antisense nucleic acid molecule complementary to the IGF2BP1 gene sequence, an interfering RNA for knocking down the expression level of IGF2BP1 protein, an antagonist antibody against the function of IGF2BP1 protein, and a small molecule compound selectively inhibiting IGF2BP1 protein.

[0011] In some embodiments, the interfering RNA for knocking down the expression level of IGF2BP1 protein is selected from siRNA, shRNA, single-stranded interfering RNA, and microRNA.

[0012] The present application also provides the use of IGF2BP1 protein as a therapeutic target in the preparation of a drug for treating acute ischemic stroke.

[0013] In a preferred embodiment of the present invention, inhibiting and / or reducing the expression level of IGF2BP1 protein is achieved by knocking down the expression level of IGF2BP1 protein with interfering RNA. When introduced in vivo, the interfering RNA forms an RNA-induced silencing complex ("RISC") with other proteins and initiates a process called RNA interference. During RNA interference, RISC incorporates one strand of single-stranded interfering RNA or double-stranded interfering RNA. The incorporated strand serves as a template for RISC to recognize the complementary mRNA transcript. Once the complementary mRNA is identified, the protein components in RISC activate and cleave the mRNA, resulting in the knockdown of target gene expression. Non-limiting examples of interfering RNA molecules for knocking down target gene expression include small interfering RNA (siRNA), short hairpin RNA (shRNA), single-stranded interfering RNA, and microRNA (miRNA). The methods of using these interfering RNAs are well known to those skilled in the art.

[0014] In the present application, interfering RNA refers to an RNA nucleic acid molecule that is double-stranded or single-stranded and is capable of inducing the RNA interference mechanism for knocking down the expression of a target gene.

[0015] In the present application, siRNA is double-stranded RNA, and its length is usually less than 30 nucleotides. Gene silencing by siRNA begins with one strand of the siRNA incorporating into a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). The strand incorporated into RISC recognizes mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then RISC cleaves these target mRNAs or inhibits their translation.

[0016] In the present application, miRNA is a small non-coding RNA molecule that can hybridize with complementary sequences within mRNA molecules, resulting in cleavage of the mRNA or destabilizing the mRNA by shortening its poly(A) tail.

[0017] In the present application, single-stranded interfering RNA can achieve mRNA silencing in a manner similar to double-stranded siRNA, although with lower efficiency than double-stranded siRNA. Single-stranded interfering RNA usually has a length of about 19 to about 49 nucleotides.

[0018] In the present application, shRNA is an artificial RNA molecule with a tight hairpin turn and can be used to silence the expression of target genes through the siRNA it generates in cells. The expression of shRNA in cells is usually achieved through plasmid vectors or through viral or bacterial vectors.

[0019] In the present application, antagonist antibodies are used in the broadest sense and include antibodies that inhibit or reduce the biological activity of the anti-IGF2BP to which the antibody binds. Thus, IGF2BP antagonist antibodies encompass antibodies that bind IGF2BP and block, inhibit, counteract, antagonize, or reduce IGF2BP agonist activity to any significant extent (including significantly).

[0020] Advantages of the present invention: (1) The mouse MCAO model simulates ischemic stroke. There are differences in the expression of IGF2BP1 protein in neurons of the ischemic penumbra and the infarct core region after MCAO injury in mice, and more IGF2BP1 is expressed in neurons of the ischemic penumbra; (2) After knocking down IGF2BP1, the infarct volume of MCAO mice can be reduced; (3) It is determined that knocking down IGF2BP1 under hypoxic conditions can affect the expression of synaptic-related genes, and it is crucial to regulate the protection of nerve synapses in mouse ischemic stroke through IGF2BP1-regulated RNA; (4) After knocking down IGF2BP1, the synaptic damage caused by the MCAO mouse model can be improved, thereby improving neuronal apoptosis; (5) A potential therapeutic method for rescuing synaptic damage and cell apoptosis in neurons of the ischemic penumbra by changing the expression of IGF2BP1 is proposed, which can be used to guide the treatment of AIS. Brief Description of the Drawings

[0021] Figure 1. Fluorescent expression of IGF2BP1 protein in the ischemic penumbra and infarct core: A. Schematic diagram of the distinction between the ischemic penumbra and infarct core after the mouse MCAO model (left), and fluorescent expression of IGF2BP1 protein in the ischemic penumbra and infarct core (right); B. Western blot assay to detect the expression of IGF2BP1 protein in the ischemic penumbra and infarct core after the mouse MCAO model.

[0022] Figure 2 . Detection of the knockdown of IGF2BP1 protein after lentivirus injection; A. After injecting the lentivirus for knocking down IGF2BP1, Western blot assay was used to detect the expression of IGF2BP1 protein in the ischemic penumbra after the mouse MCAO model; B. After injecting the lentivirus for knocking down IGF2BP1, immunofluorescence was used to detect the expression of IGF2BP1 protein in the ischemic penumbra after the mouse MCAO model.

[0023] Figure 3 . Effects of knocking down IGF2BP1 on infarct volume and neuronal apoptosis in MCAO mice; A / C. The 3D reconstruction software Slicer was used to reconstruct the infarct regions of each MCAO group and evaluate the infarct volume; B / D. Nissl staining combined with TUNEL staining was used to show the ratio of apoptotic positive cells in the ischemic penumbra of mice in the MCAO+sh NC group and MCAO+sh IGF2BP1 group.

[0024] Figure 4 . RNA sequencing of the IGF2BP1 knockdown group and the knockdown control group in SH-SY5Y cells under ischemic and hypoxic conditions: A. RNA-seq volcano plot showing differentially expressed genes (IGF2BP1KD VS NCKD) in the IGF2BP1 knockdown group compared with the control siRNA group after hypoxia treatment of SH-SY5Y cells, N = 3; B. Heat map showing the top 10 genes with low and high differential expression (IGF2BP1KD VS NCKD); C / D. Bubble plot showing the gene enrichment pathways of highly expressed mRNAs after knocking down IGF2BP1 under hypoxia treatment.

[0025] Figure 5 . Effects of knocking down IGF2BP1 on synaptic structure in MCAO mice: A. Schematic diagram of the synaptic structure of intact neurons; B. Western blot analysis of the expression levels of synaptic-related proteins PSD95 and SYN in the ischemic penumbra brain tissue of MCAO mice after injecting sh NC and sh IGF2BP1 lentiviruses and statistics. N = 4.

[0026] Figure 6 . A. Electron microscopy showed the ultrastructure of synapses in each group and counted the number of intact synapses and the width of synaptic clefts, scale bar = 1μm. N = 14 synapses / group.

[0027] Figure 7.A. Western blot analysis of the expression levels of apoptosis-related proteins Cleaved caspase3 and BAX / Bcl2 in the ischemic penumbra brain tissue of MCAO mice injected with sh NC and sh IGF2BP1 lentiviruses.

[0028] Figure 8 .A. Immunofluorescence staining showed the expression of apoptosis-related protein Cleaved Caspase 3 (CC3) in the ischemic penumbra of mice in the SHAM+sh NC group, MCAO+sh NC group, and MCAO+sh IGF2BP1 group. Detailed implementation

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0030] Example 1: Animal and MCAO model construction

[0031] Animals Sixty adult male C57BL / J6 mice, weighing 22.0 - 25.5 g, 5 - 7 weeks old, were randomly assigned and raised in a strictly constant temperature and humidity environment. The mice were anesthetized with isoflurane gas (4% induction anesthesia, 1.5% maintenance anesthesia) to construct a mouse MCAO ischemic brain injury model. After anesthesia, the mice were fixed in the supine position, and the neck surgical field of the mice was fully exposed. The skin on the left side of the neck was cut open, the left carotid sheath of the mice was fully exposed, the common carotid artery was ligated to block blood flow, and then the external carotid artery was ligated to block external carotid blood flow. A hemostatic clip was clamped on the internal carotid artery to temporarily stop blood supply to the area where the wire embolization was to be inserted to avoid bleeding. A small incision was made in the external carotid artery with a fine ophthalmic scissors, and the wire embolization was slowly inserted to the position of the left middle cerebral artery, and the time was recorded for 3 hours. In the sham operation group (SHAM group), other operations were the same as above, the neck blood vessels were separated, and the surgical suture was placed, but not tied and not inserted with the wire embolization. All were evaluated for successful model establishment using a 9.4T small animal MRI scanner (Bruker PharmaScan).

[0032] Example 2: SH-SY5Y cell culture and establishment of oxygen-glucose deprivation (OGD) model

[0033] The human neuronal replacement neuroblastoma SH-SY5Y cell line was cultured in DMEM / F-12 containing 10% FBS and 1% penicillin-streptomycin in an incubator at 37°C and 5% CO2. When approaching confluence, passage was carried out using 0.25% trypsin. After the SH-SY5Y cells were cultured to 80% confluence, the medium was discarded, washed twice with Hank's buffered salt, then washed once with sugar-free DMEM, and then replaced with sugar-free DMEM medium and placed in a modeling gas chamber (N2 95%, CO2 5%) for 3 hours of hypoxia.

[0034] 2.1 RNA sequencing of SH-SY5Y cells

[0035] 2.1.1 After the SH-SY5Y cells reached 80% confluence, they were grouped as follows:

[0036] (1) Ischemia-hypoxia group (OGD group): Incubated in a three-gas incubator with sugar-free DMEM medium for 3 hours. The conditions of the three-gas incubator were: N2 95%, CO2 5%.

[0037] (2) Ischemia-hypoxia + knockdown of IGF2BP1 group (OGD + KD IGF2BP1 group): Transfected with siRNA of IGF2BP1 2 days before modeling and incubated in a three-gas incubator with sugar-free DMEM medium for 3 hours. The conditions of the three-gas incubator were: N2 95%, CO2 5%.

[0038] 2.1.2 RNA sequencing: The cells after the above grouping treatments were washed twice with PBS, and Trizol was added to collect cell RNA. The RNA library construction and sequencing were entrusted to Guangzhou Epigenetic Biotechnology Co., Ltd. Then, the sequencing results were analyzed and it was found that in the knockdown of IGF2BP1 group compared with the control siRNA group, there were 1,696 down-regulated genes and 664 up-regulated genes. Then the heat map showed the top 20 differentially expressed genes ( Figure 4 A and 4B). Next, enrichment analysis was performed on these highly expressed genes. Through Gene Ontology analysis, it was found that these highly expressed genes were mainly enriched in synaptic-related functions and mainly located in axons, neuronal cell bodies, synaptic membranes, etc. ( Figure 4 C and 4D). It shows that knockdown of IGF2BP1 under hypoxic conditions can affect the expression of synaptic-related genes, and regulating RNA through IGF2BP1 may be crucial for the protective effect of nerve synapses in murine ischemic stroke.

[0039] Example 3: Administration of lentivirus with knockdown of IGF2BP1 in mice

[0040] To detect the neuroprotective effect of knocking down IGF2BP1 in vivo, lentiviruses specific for knocking down IGF2BP1 (which can produce shRNA targeting IGF2BP1) and control lentiviruses (sh NC) were constructed. Thirty mice were divided into three groups: SHAM, MCAO+shNC, and MCAO+sh IGF2BP1 groups. The mice were respectively microinjected with 4 μL of control lentivirus sh NC and lentivirus sh IGF2BP1 for knocking down IGF2BP1 by stereotactic injection at the following positions in the left cortex near the infarction boundary (relative to the Bregma point: 1): 1) 0.2 mm posterior, 2 mm lateral to the left, depth 1.5 mm; 2) 0.2 mm posterior, 3.0 mm lateral to the left, depth 2.0 mm). Seven days after the injection, an MCAO model was constructed.

[0041] Example 4: Mouse MRI and three-dimensional reconstruction

[0042] Mice were subjected to MRI using a 9.4T small animal MRI scanner (Bruker PharmaScan). The mice were anesthetized via nasal inhalation with 2% isoflurane, and their body temperature and respiratory rate were monitored. T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI) were performed 3 hours after MCAO modeling. The success of MCAO modeling in various mice was determined based on T2 Flair and DWI ( Figure 3 A). Then, at the same MCAO mouse image scale and brain section, the T2WI images were scanned and quantified using 3D Slicer software. Three-dimensional images were reconstructed based on the T2WI images using 3D Slicer, the infarcted area and non-infarcted area were identified by threshold adjustment, and the infarct volume was determined by the software. Then, the infarct volume was calculated, and it was found that the infarct volume in the MCAO+sh IGF2BP1 group was smaller than that in the MCAO+sh NC group ( Figure 3 A and 3C). This indicates that knocking down IGF2BP1 can reduce the infarct volume in MCAO mice.

[0043] Example 5: Immunofluorescence of brain tissue

[0044] (1) The frozen brain tissue sections after the above lentivirus injection were taken out and rewarmed, and washed 3 times with PBS for 5 minutes each time.

[0045] (2) Use an immunohistochemical pen to carefully draw a circle around the brain tissue.

[0046] (3) Incubate with 0.1% Triton X-100 prepared with PBS at room temperature for 10 minutes to permeabilize the membrane to facilitate antibody entry into the cells.

[0047] (4) Wash the cells 3 times with PBS for 5 minutes each time.

[0048] (5) Block with 1% BSA solution at room temperature for 30 minutes to remove non-specific binding.

[0049] (6) The stock solutions of the antibodies against IGF2BP1, NeuN, and CC3 were diluted 1:200 with the prepared 5% BSA.

[0050] (7) Aspirate the blocking solution, add the prepared primary antibody dilution, and incubate overnight at 4°C.

[0051] (8) Take out the confocal dish the next day, wash it 3 times with PBS for 5 minutes each time.

[0052] (9) The stock solution of the secondary antibody corresponding to the primary antibody was diluted 1:200 with the prepared 5% BSA.

[0053] (10) Aspirate the PBS, add the prepared secondary antibody dilution, and incubate for 1 hour at room temperature in the dark.

[0054] (11) Wash the cells 3 times with PBS for 5 minutes each time.

[0055] (12) Incubate with DAPI staining solution for 5 minutes at room temperature to stain the cell nuclei.

[0056] (13) Wash the cells 3 times with PBS for 5 minutes each time, and then mount the slides with 50% glycerol. (14) Take pictures with a Zeiss confocal microscope 880 and perform statistical analysis.

[0057] To detect the expression of IGF2BP1 after MCAO injury, immunofluorescence staining was performed on the expression of IGF2BP1 protein in the brain tissue of the MCAO mouse model. It was found that IGF2BP1 protein was co-localized with the neuronal marker NeuN, indicating that IGF2BP1 protein was mainly expressed in neurons. Moreover, compared with the infarct core area, the IGF2BP1 protein in the ischemic penumbra area was significantly more than that in the infarct core area ( Figure 1 A). It shows that IGF2BP1 protein is mainly expressed in the ischemic penumbra area of MCAO mice.

[0058] To detect whether the lentivirus knocking down IGF2BP1 successfully knocked down the expression of IGF2BP1 protein, immunofluorescence staining was performed on the MCAO mice injected with the lentivirus knocking down IGF2BP1 to detect the expression of IGF2BP1 protein. The cell nuclei were visualized with DAPI, and the fluorescence intensity was compared. It was found that compared with the injection of the control virus, the injection of the lentivirus knocking down IGF2BP1 could reduce the expression of IGF2BP1 protein in the penumbra area ( Figure 2 B). It shows that the lentivirus knocking down IGF2BP1 can successfully reduce the expression of IGF2BP1 protein in the ischemic penumbra.

[0059] Example 6: TUNEL staining of frozen sections of mouse brain tissue

[0060] (1) Take out the frozen brain tissue sections and let them warm up. Wash them 3 times with PBS for 5 minutes each time.

[0061] (2) Carefully draw a circle around the brain tissue using an immunohistochemistry pen.

[0062] (3) Incubate the brain tissue with 0.1% Triton X-100 prepared with PBS on ice for 2 minutes to facilitate the entry of the TdT labeling solution into the cells.

[0063] (4) Wash 3 times with PBS for 5 minutes each time.

[0064] (5) Add 50 μl of the prepared TUNEL detection solution to each brain section and incubate at 37°C in the dark for 1 hour.

[0065] (6) Wash 3 times with PBS for 5 minutes each time.

[0066] (7) Prepare Nissl fluorescent staining solution by diluting it with PBS at a ratio of 1:200 and incubate at room temperature for 20 minutes.

[0067] (8) Wash 3 times with PBS for 5 minutes each time.

[0068] (9) Incubate with DAPI staining solution at room temperature for 5 minutes to label the cell nuclei.

[0069] (10) Wash 3 times with PBS for 5 minutes each time.

[0070] (11) Mount the slides with 50% glycerol, observe the red positive cells under an inverted fluorescence microscope, and take pictures for statistical analysis.

[0071] To evaluate the effect of the lentivirus knocking down IGF2BP1 on the apoptosis level in MCAO mice, the brain sections of the MCAO+sh NC group and the MCAO+sh IGF2BP1 group were subjected to TUNEL staining to label apoptotic cells, Nissl staining to label neurons, and DAPI to label cell nuclei. The ratio of TUNEL-positive cells to Nissl neurons was statistically analyzed. The results showed that compared with the MCAO+sh NC group, after knocking down IGF2BP1, the apoptotic positive labeling in the infarcted area of MCAO mice was significantly reduced ( Figure 3 B and 3D). Then, immunofluorescence staining was used to detect the expression of the apoptotic protein CC3 in the brain sections of each group, and the ratio of CC3 protein to DAPI in the same area was statistically analyzed (%). The results showed that compared with the SHAM+sh NC group, the MCAO+sh NC group showed positive labeling of the apoptotic protein in the infarcted area; while knocking down IGF2BP1 could reduce the expression of the apoptotic protein CC3 in the infarcted area of MCAO mice ( Figure 8 A and 8B). This indicates that knocking down IGF2BP1 can reduce the neuronal apoptosis level in MCAO mice.

[0072] Example 7: Western blot analysis of mouse MCAO tissues

[0073] Add an appropriate amount of protein lysate to the harvested brain tissues of each group. Place the EP tubes on ice for 30 minutes for lysis. Then centrifuge the cell lysate at 12,000 g at 4°C for 15 minutes, and take the supernatant as the protein sample. Refer to the BCA protein quantification method to calculate the concentration of the protein to be measured using the standard curve. Prepare the samples into the same concentration, add 1 / 4 of the sample volume of 5×loading buffer and mix well. Denature the protein in a metal bath at 100°C for 5 minutes. Load 10 μl per group, with a total of 20 μg of protein, and separate it by 10% SDS-polyacrylamide gel electrophoresis. First, run the stacking gel at a constant voltage of 60 mV until completion, and then change the voltage to 100 mV and continue running until the end. Then transfer the protein molecules to the PVDF membrane by wet transfer at a constant current. After blocking the PVDF membrane with milk blocking solution at room temperature for 1 hour, rinse it with TBST, and then add the corresponding antibody and incubate overnight (more than 16 hours) on a shaker at 4°C. After rinsing the PVDF membrane with TBST, add the corresponding secondary antibody (diluted with 5% skim milk at 1:5000) and incubate at room temperature for 1 hour. After rinsing the PVDF membrane with TBST, drip the enhanced chemiluminescence (ECL) solution and expose it on a Tanon 2500 gel imaging system.

[0074] To detect the expression of IGF2BP1 after MCAO injury, Western blot analysis was performed to detect the expression of IGF2BP1. It was found that after MCAO treatment, compared with the infarct core area, the protein expression of IGF2BP1 in the ischemic penumbra area was significantly increased ( Figure 1 B). This indicates that the IGF2BP1 protein is mainly expressed in the ischemic penumbra area of MCAO mice.

[0075] To detect whether the lentivirus knocking down IGF2BP1 successfully knocked down the expression of the IGF2BP1 protein, Western blot analysis was performed to detect the expression of IGF2BP1. The experimental results showed that compared with the SHAM+sh NC group, the expression of the IGF2BP1 protein in the infarcted brain tissues of the MCAO+sh NC group of mice was up-regulated. And compared with the MCAO+sh NC group, the expression of the IGF2BP1 protein in the infarcted brain tissues of the MCAO+sh IGF2BP1 group of mice was decreased ( Figure 2 B), indicating that the lentivirus knocking down IGF2BP1 can successfully reduce the expression of the IGF2BP1 protein.

[0076] To evaluate whether knockdown of IGF2BP1 can improve synaptic damage induced by the mouse MCAO model, Western blot analysis was performed to detect the expression of synaptophysin (SYN), a marker of presynaptic membrane integrity, and postsynaptic density protein 95 (PSD95), a marker of postsynaptic membrane integrity. Analysis of the brain tissue in the infarct border zone (penumbra) of mice in the MCAO+sh NC group and the MCAO+sh IGF2BP1 group revealed that: compared with the SHAM group, the expression of synaptic-related proteins PSD95 and SYN in the MCAO group decreased, indicating that MCAO caused impaired synaptic function. Compared with the MCAO+sh NC group, after knockdown of IGF2BP1, the expression of synaptic-related proteins PSD95 and SYN increased significantly( Figure 5 A and 5B), indicating that the maintenance of the intact synaptic structure was better after knockdown of IGF2BP1.

[0077] To evaluate the effect of lentivirus with knockdown of IGF2BP1 on the apoptosis level in MCAO mice, Western blot analysis was performed to detect the expression of apoptotic proteins CC3, BAX, and anti-apoptotic protein Bcl2. The results showed that: compared with the SHAM+sh NC group, the expression of apoptotic protein CC3 increased in the MCAO+sh_NC group, and the BAX / Bcl2 ratio tended to increase. Compared with the MCAO+sh NC group, the expression of apoptotic protein CC3 decreased in the MCAO+sh IGF2BP1 group, and the BAX / Bcl2 ratio tended to decrease( Figure 7 A). This indicates that knockdown of IGF2BP1 can reduce the neuronal apoptosis level in MCAO mice.

[0078] Example 8: Transmission electron microscopy of brain tissue synapses

[0079] (1) Sampling for transmission electron microscopy of synapses: Perfusion was not required. Mice in the MCAO+sh NC group and the MCAO+sh IGF2BP1 group were euthanized, and the brain tissue was quickly removed. The tissue blocks in the infarct border zone were placed in the electron microscopy fixative. (2) Entrust Wuhan Baiqiandu Biotechnology Co., Ltd. to perform staining and photography.

[0080] (3) Use ImageJ to perform relevant analysis on the neuronal structure.

[0081] The brain tissues of the infarction border zone (penumbra) of the mice in the MCAO+sh NC group and the MCAO+sh IGF2BP1 group were fixed and processed by electron microscopy. Then, the ultrastructure of synapses in the local brain tissue area was observed by scanning electron microscopy, and the number of intact synapses and the width of the synaptic cleft in each group were counted. The results showed that compared with the MCAO+sh NC group, the number of intact synaptic structures in the MCAO+sh IGF2BP1 group was larger. At the same time, the changes in the damaged synaptic clefts of neuronal synapses after MCAO were detected. Compared with the MCAO+sh NC group, in the MCAO+sh IGF2BP1 group, the width of the synaptic cleft was narrower ( Figure 6 A), which was more conducive to information transmission between neurons. It was indicated that knocking down IGF2BP1 could improve synaptic damage caused by the MCAO mouse model and thus improve neuronal apoptosis.

Claims

1. Use of a reagent targeting IGF2BP1 in the preparation of a medicament for treating acute ischemic stroke.

2. Use of the reagent targeting IGF2BP1 according to claim 1 in the preparation of a medicament for treating acute ischemic stroke, characterized in that: The reagent inhibits and / or reduces the expression level of IGF2BP gene and / or protein.

3. Use of the reagent targeting IGF2BP1 according to claim 2 in the preparation of a medicament for treating acute ischemic stroke, characterized in that: The reagent is selected from an antisense nucleic acid molecule complementary to the IGF2BP1 gene sequence, an interfering RNA that knocks down the expression level of IGF2BP1 protein, an antagonist antibody that antagonizes the function of IGF2BP1 protein, and a small molecule compound that selectively inhibits IGF2BP1 protein.

4. Use of the reagent targeting IGF2BP1 according to claim 3 in the preparation of a medicament for treating acute ischemic stroke, characterized in that: The interfering RNA that knocks down the expression level of IGF2BP1 protein is double-stranded or single-stranded and can induce an RNA interference mechanism for knocking down the expression of the target gene.

5. Use of the reagent targeting IGF2BP1 according to claim 4 in the preparation of a medicament for treating acute ischemic stroke, characterized in that, The interfering RNA that knocks down the expression level of IGF2BP1 protein is selected from siRNA, shRNA, single-stranded interfering RNA, and microRNA.

6. Application of IGF2BP1 protein as a therapeutic target in the preparation of a medicament for treating acute ischemic stroke.

7. Application of a reagent that rescues synaptic injury and apoptosis of neurons in the ischemic penumbra by altering the level of IGF2BP1 protein in guiding the development of treatment for acute ischemic stroke.

8. Use of the reagent for rescuing synaptic damage and apoptosis of neurons in the ischemic penumbra by changing the level of IGF2BP1 protein in the development of guiding the treatment of acute ischemic stroke, characterized in that: The reagent is selected from an antisense nucleic acid molecule complementary to the IGF2BP1 gene sequence, an interfering RNA that knocks down the expression level of IGF2BP1 protein, an antagonist antibody that antagonizes the function of IGF2BP1 protein, and a small molecule compound that selectively inhibits IGF2BP1 protein.

9. Use of the reagent for rescuing synaptic damage and apoptosis of neurons in the ischemic penumbra by changing the level of IGF2BP1 protein in the development of guiding the treatment of acute ischemic stroke, characterized in that: The interfering RNA that knocks down the expression level of IGF2BP1 protein is selected from siRNA, shRNA, single-stranded interfering RNA, and microRNA.

Images