Use of igf2bp2 agonists in the preparation of a medicament for inhibiting the inflammatory response following spinal cord injury

Abstract

The application provides an application of an IGF2BP2 agonist in preparation of a medicine for inhibiting an inflammatory response after spinal cord injury, and belongs to the technical field of biological medicines. The application research finds that IGF2BP2 can inhibit the inflammatory response after spinal cord injury, high-level expression of IGF2BP2 is promoted through an IGF2BP2 agonist, IGF2BP2 targets ARRB1 to inhibit a MAPKs / NF-kappa B signal pathway, and then the inflammatory response after spinal cord injury is inhibited, so as to help functional recovery after spinal cord injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly to application of IGF2BP2 agonist in preparation of a medicine for inhibiting inflammatory reaction after spinal cord injury. BACKGROUND

[0002] Traumatic spinal cord injury (SCI) is a serious central nervous system (CNS) disease, which can cause severe motor dysfunction, somatosensory dysfunction, autonomic nervous dysfunction and multiple nervous system dysfunction.

[0003] SCI mainly includes two pathological processes of primary injury and secondary injury. At present, the clinical treatment of SCI secondary injury is mainly short-term high-dose hormone shock therapy after operation to slow down the inflammatory storm, edema and other harmful pathological processes in the microenvironment of the spinal cord after injury. After SCI, the tissues in the injury area and the surrounding tissues have a high inflammatory reaction state, and the inflammatory reaction plays a huge hindering role in functional recovery and will cause serious secondary reactions to a certain extent. The characteristics of inflammatory reaction mainly include the recruitment of immune cells, the secretion of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6) and chemokines (such as CCL2, CXCL2), and the activation of resident glial cells (astrocytes and microglia); among them, astrocytes are the most common resident cells in the central nervous system, and after SCI, the astrocytes around the injury area are activated and proliferated, and the activated astrocytes are also called reactive astrocytes.

[0004] In recent years, the regulatory role of epigenetic modification in the occurrence and development of spinal cord injury has attracted much attention. RNA epigenetics is an important way to regulate gene expression at the RNA level. N6-methyladenosine (m6A) is a ubiquitous epigenetic modification in mammals and is the most abundant dynamic chemical modification in RNA, which is dynamically regulated by methylation of methyltransferase, recognition and processing of reading protein, and demethylation of demethylase.

[0005] Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) is an important reading protein of m6A methylation modification, which is involved in RNA localization, stability and translation, and plays a significant role in the progression of diseases such as osteoarthritis, breast cancer, ovarian cancer and colorectal cancer.

[0006] Beta-arrestin-1 (ARRB1) is a ubiquitous multifunctional adaptor protein that regulates G protein-coupled receptor (GPCR) signaling, has been identified as a mediator that links receptors to downstream signaling pathways, and is involved in pathophysiological processes such as proliferation, apoptosis and differentiation, and plays an important role in the onset and progression of various diseases such as sepsis, cerebral ischemia and asthma.

[0007] At present, there is no relevant research report on IGF2BP2 or ARRB1 in the treatment of spinal cord injury. SUMMARY

[0008] The application provides an application of an IGF2BP2 agonist in the preparation of a medicine for inhibiting an inflammatory response after spinal cord injury. The application research finds that IGF2BP2 can inhibit the inflammatory response after spinal cord injury, and targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, thereby inhibiting the inflammatory response after spinal cord injury, and thus helping the functional recovery after spinal cord injury.

[0009] The technical scheme of the application is as follows:

[0010] The application of the IGF2BP2 agonist in the preparation of the medicine for inhibiting the inflammatory response after spinal cord injury; the IGF2BP2 agonist can promote the expression of IGF2BP2, and the amino acid sequence of the IGF2BP2 is shown as SEQ ID NO. 1.

[0011] Preferably, the IGF2BP2 agonist can increase the expression level of ARRB1 by promoting the expression of IGF2BP2, thereby inhibiting the inflammatory response after spinal cord injury; and the amino acid sequence of the ARRB1 is shown as SEQ ID NO. 2.

[0012] Preferably, the IGF2BP2 agonist can inhibit the MAPKs / NF-κB signaling pathway by promoting the expression of IGF2BP2, thereby inhibiting the inflammatory response after spinal cord injury.

[0013] Preferably, the IGF2BP2 agonist targets the astrocytes.

[0014] Further preferably, the IGF2BP2 agonist comprises one or more of a compound, a protein, a polypeptide, a polysaccharide, a glycoprotein, a glycopeptide, and a nucleic acid.

[0015] Preferably, the spinal cord injury is secondary spinal cord injury.

[0016] Preferably, the inflammatory response comprises the expression of pro-inflammatory cytokines and chemotactic factors.

[0017] Further preferably, the pro-inflammatory cytokines and chemokines include IL-1β, IL-6, CCL2, CXCL2.

[0018] Preferably, the IGF2BP2 agonist can promote the expression of IGF2BP2, IGF2BP2 targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, and inhibit the inflammatory response after spinal cord injury.

[0019] The application of the ARRB1 agonist in the preparation of a drug for inhibiting the inflammatory response after spinal cord injury; the ARRB1 agonist can promote the expression of ARRB1.

[0020] The application of the MAPKs / NF-κB signaling pathway inhibitor in the preparation of a drug for inhibiting the inflammatory response after spinal cord injury; the MAPKs / NF-κB signaling pathway inhibitor can inhibit the activation of P-P38, P-ERK, P-JNK and P-P65 in the pathway.

[0021] The application of the IGF2BP2 agonist or the ARRB1 agonist or the MAPKs / NF-κB signaling pathway inhibitor in the preparation of a drug for repairing spinal cord injury.

[0022] The application of the IGF2BP2 agonist in the preparation of a drug for inhibiting the inflammatory response; the IGF2BP2 agonist can promote the expression of IGF2BP2, and IGF2BP2 targets star-shaped glial cells.

[0023] Preferably, the inflammatory response includes the expression of pro-inflammatory cytokines and chemokines, and the pro-inflammatory cytokines and chemokines include IL-1β, IL-6, CCL2 and CXCL2.

[0024] Beneficial effects:

[0025] The application provides the application of the IGF2BP2 agonist in the preparation of a drug for inhibiting the inflammatory response after spinal cord injury. The application research finds that insulin-like growth factor 2-mRNA binding protein 2 (IGF2BP2) can inhibit the inflammatory response after spinal cord injury, the expression of IGF2BP2 is promoted by the IGF2BP2 agonist, IGF2BP2 targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, and then the inflammatory response after spinal cord injury is inhibited, thereby helping the functional recovery after spinal cord injury. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The change of the mRNA expression amount of IGF2BP2 in the primary star-shaped glial cells within 24 hours after TNF-α stimulation in Example 1.

[0027] Figure 2Figure 1. Changes of IGF2BP2 mRNA expression in spinal cord injury tissues after spinal cord injury.

[0028] Figure 3 Figure 1. Changes of IGF2BP2 protein expression in primary astrocytes within 24 h after TNF-a stimulation.

[0029] Figure 4 Figure 1. Changes of IGF2BP2 protein expression in spinal cord injury tissues after spinal cord injury.

[0030] Figure 5 Figure 2. Plasmid map for knocking out IGF2BP2 in astrocytes.

[0031] Figure 6 Figure 2. Verification of the results of knocking out IGF2BP2 in astrocytes.

[0032] Figure 7 Figure 2. Verification of the results of overexpressing IGF2BP2 in astrocytes.

[0033] Figure 8 Figure 2. Effects of knocking out IGF2BP2 on the inflammatory response of astrocytes.

[0034] Figure 9 Figure 2. Effects of overexpressing IGF2BP2 on the inflammatory response of astrocytes.

[0035] Figure 10 Figure 3. Effects of knocking out or overexpressing IGF2BP2 on the MAPKs / NF-κB signaling pathway in astrocytes.

[0036] Figure 11 Figure 4. Results of joint analysis of RIP-seq, RNA-seq, and m6A data of mouse spinal cord injury tissues.

[0037] Figure 12 Figure 4. Effects of overexpressing IGF2BP2 on the expression level of ARRB1 in astrocytes.

[0038] Figure 13 Figure 4. Small interference results of ARRB1.

[0039] Figure 14 Figure 4. Effects of knocking down ARRB1 on the inflammatory response of astrocytes.

[0040] Figure 15 Figure 4. Effects of knocking down ARRB1 on the MAPKs / NF-κB signaling pathway in astrocytes.

[0041] Figure 16 To verify the knockdown effect of IGF2BP2 targeting astrocytes in vivo in mice in Example 5.

[0042] Figure 17 BMS score results of IGF2BP2 knockdown spinal cord injury model mice in Example 5.

[0043] Figure 18 Repair results of IGF2BP2 knockdown spinal cord injury model mice after spinal cord injury in Example 5. DETAILED DESCRIPTION

[0044] The following will be described in conjunction with specific embodiments:

[0045] The source of experimental materials or the preparation method is explained as follows:

[0046] Experimental mice: purchased from Jinan Pengyue Animal Breeding Co., Ltd.

[0047] HBSS buffer: purchased from Wuhan Punuo Life Science and Technology Co., Ltd., item number PB180321.

[0048] Complete culture medium: DMEM culture medium (purchased from gibico) + FBS (10 mL / 100 mL, purchased from gibico) + double antibody (1 mL / 100 mL, purchased from Solaybao, item number P1400).

[0049] TNF-α: purchased from Aiboteke Biology, item number RP01071.

[0050] Gencefe TransExp transfection reagent: purchased from Kexiansyun Biology.

[0051] Opti-MEM medium: purchased from gibco, item number 31985-07.

[0052] Example 1: Expression of IGF2BP2 in astrocytes and spinal cord injury tissues

[0053] I. Extraction and culture of primary astrocytes

[0054] The T25 cell culture bottle was coated with a PDL (poly-D-lysine) solution with a concentration of 0.1 mg / mL at 37°C for 30 min, then the PDL solution was aspirated, washed with HBSS buffer for 3 times, and placed in a 37°C incubator for standby.

[0055] Pre-cool 10 cm culture dish with HBSS buffer solution; sterilize the newborn mice (1 day old) with 75% (v / v) ethanol solution, decapitate, and cut the skin and skull along the midline with fine scissors, taking care not to damage the skin; then use fine tweezers to peel off the skull on both sides, and remove the whole brain below the olfactory bulb (remove the dura mater and blood vessels); place the removed brain into the pre-cooled 10 cm culture dish; after all the brains are removed, aspirate the HBSS buffer solution, and use scissors to cut the brain tissue as much as possible; add 10 mL of 0.25% trypsin solution (for 10 brains), and blow to promote tissue digestion; add the semi-suspension into a conical flask, add a stirring bar, seal with tin foil, and stir at 37°C for 15 min (at this time, there are undigested tissue clumps and jelly-like clumps); add 20 mL of complete medium and an appropriate amount of DNase to stop the digestion, blow, and sieve through a 200-mesh sieve; during the sieving process, grind the undigested tissue clumps and jelly-like clumps on the filter screen for 25 min, and add an appropriate amount of HBSS buffer solution to promote sieving; obtain the filtrate; transfer the filtrate to a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend the precipitate with 10 mL of complete medium, and transfer the resuspension to a 15 mL centrifuge tube; centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend the precipitate with 2 mL of complete medium, and add the resuspension to a 10 cm culture dish containing 10 mL of complete medium, and perform differential adhesion for 1 h; after 1 h, transfer the liquid in the 10 cm culture dish to the prepared T25 cell culture bottle, and perform cell culture and subculture.

[0056] Subculture the cells to new T25 cell culture bottles after 2 days of culture, and subculture according to 1 bottle to 3 bottles; subculture to new T25 cell culture bottles after another 2 days of culture, and subculture according to 3 bottles to 9 bottles; the culture bottles can be placed in a 37°C shaker for 200 rpm shaking for 24 h before subculture; subculture to 6-well plates after another 2 days of culture, and seed the plates according to the ratio of 9 bottles to 9 plates; the adhesion time in the 6-well plates is 48 h, and the primary astrocytes are obtained.

[0057] II. Construction of spinal cord contusion injury mouse model

[0058] The mice (6-8 weeks old) were subjected to fasting treatment 8 h before the operation to avoid vomiting or aspiration during the operation.

[0059] Prepare a 3% pentobarbital solution using distilled water as the solvent, and intraperitoneally inject the mice at a dose of 30 mg / kg for anesthesia; the limbs do not respond to clamping, indicating complete anesthesia.

[0060] After complete anesthesia, the hair on the back of the mouse was removed to expose the surgical area; the mouse was fixed in a prone position on the operating table to ensure the stability of the mouse position during the operation; the surgical area was disinfected with iodophor cotton ball to reduce the risk of contamination during the operation; a disposable sterile surgical drape was laid, a 1.5 cm incision was made along the midline of the mouse back at the T9 segment of the spine, and the muscles and fascia were bluntly separated layer by layer to expose the spinous process; T9 laminectomy was performed under a body microscope using a fiber forceps to expose the spinal cord.

[0061] The mice were divided into Sham group and SCI group. Among them, the mice in the Sham group were not treated with spinal cord injury after laminectomy, the injury site was cleaned with sterile normal saline, the surgical incision was sutured layer by layer with sterile suture, and the iodophor cotton ball was disinfected; the mice in the SCI group were hit with a pneumatic spinal cord injury striker, the hitting speed was 1 m / s, the hitting depth was 2 mm, the striker stayed for 1 s, and the cleaning, suturing, disinfection steps and operations were the same as those of the Sham group, and the spinal cord contusion injury mouse model was constructed.

[0062] The mice in each group were placed on a heating blanket for 2-3 h, and after the mice woke up, they were placed in a feeding cage (mice could freely obtain food and water in the cage); to prevent urinary system infection in mice, the mice were manually pressed twice a day to help urination.

[0063] III. Expression of IGF2BP2 in astrocytes and spinal cord injury tissues

[0064] (1) The primary astrocytes were inoculated into a 6-well plate, and the cells were cultured in a 37°C, 5% CO2 and saturated humidity incubator until the confluence reached 80%. Then, TNF-α (tumor necrosis factor alpha) with a concentration of 1 μg / mL was used to stimulate the inflammatory response, simulating the inflammatory state after spinal cord injury in vivo. The dose was 10 ng / mL; then the total RNA of the cells was extracted by Trizol reagent at 0 h, 2 h, 4 h, 6 h, 12 h and 24 h; the total RNA of the cells was reversely transcribed into cDNA using a reverse transcription kit, and then the IGF2BP2 in the astrocytes was quantitatively analyzed by qPCR using an RT-qPCR kit. The qPCR primers used to identify IGF2BP2 were Mus-Igf2bp2-F and Mus-Igf2bp2-R, and their sequences were shown in SEQ ID NOs. 3-4.

[0065] The expression level of IGF2BP2 relative to GAPDH is shown in Figure 1 , in which, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001; and Figure 1It was found that within 24 hours after TNF-α stimulation, the mRNA expression level of IGF2BP2 in primary astrocytes showed a gradual upward trend.

[0066] (2) After the spinal cord contusion mouse model was constructed, spinal cord injury tissues were taken from the spinal cord contusion mouse model on the 3rd, 7th and 14th days. The tissues were ground with liquid nitrogen, and 1 mL of Trizol reagent was added for every 50-100 mg of tissue. The tissues were homogenized and the total RNA of the cells was extracted. The same method was used to perform qPCR quantitative analysis of IGF2BP2 in the spinal cord injury tissues.

[0067] The expression level of IGF2BP2 relative to GAPDH is as follows: Figure 2 As shown in the figure, **** P<0.0001; by Figure 2 It was found that the mRNA expression level of IGF2BP2 in the spinal cord injury tissue increased on the 3rd and 7th day after spinal cord injury, and then decreased on the 14th day.

[0068] IV. Western Blot Protein Identification

[0069] (1) Primary astrocytes were seeded into 6-well plates and cultured in an incubator at 37°C, 5% CO2, and saturated humidity until the confluence reached 80%. TNF-α stimulation was administered using the same method as described above. Proteins were extracted from the cell culture medium using cell lysis buffer at 2h, 4h, 6h, 12h, and 24h to obtain protein solutions. The concentration of each protein solution was balanced using the BCA method. The protein samples were subjected to SDS-PAGE electrophoresis using Western blotting to observe the protein expression of IGF2BP2 after inflammatory stimulation.

[0070] The Western Blot experimental parameters are as follows: SDS-PAGE separating gel concentration 10%; protein loading amount 20 μg; color development system is ECL method combined with X-ray film development.

[0071] The results are as follows Figure 3 As shown, by Figure 3 It was found that within 24 hours after TNF-α stimulation, the protein expression level of IGF2BP2 in primary astrocytes showed a gradual increasing trend, consistent with the qPCR results.

[0072] (2) After the spinal cord contusion injury mouse model is constructed, the spinal cord injury tissue of the spinal cord contusion injury mouse model is taken out on the 3rd day, the 7th day and the 14th day, and the tissue is quickly placed in a 4°C pre-cooled PBS buffer to wash off the blood on the surface; the tissue is weighed and placed in a centrifuge tube containing PBS buffer, cut into smaller tissue blocks, centrifuged, and the discolored PBS buffer is removed and replaced with new PBS buffer and centrifuged again. The above process is repeated until the PBS buffer is not discolored. Centrifuge and leave the tissue precipitate. Add 4°C pre-cooled protein extraction reagent containing inhibitors (1 mL of protein extraction reagent per 250 mg of tissue) to the tissue precipitate. When the tissue becomes soft, use a 1 mL pipette gun to continuously blow the tissue. Use a homogenizer to homogenize the tissue at low speed multiple times (30 s each time, with 1 min ice bath between homogenization). Until the tissue is completely lysed, the protein solution is obtained. The concentration of the protein solution in each group is adjusted to be flat using the BCA method. The protein samples in each group are subjected to SDS-PAGE electrophoresis and color development by Western Blot method, and the protein expression of IGF2BP2 is observed. The Western Blot experiment parameters are the same as above.

[0073] The results are shown in Table 1. Figure 4 As can be seen from Table 1, Figure 4 It can be seen that on the 3rd day and the 7th day after spinal cord injury, the protein expression of IGF2BP2 in the spinal cord injury tissue showed an upward trend, and then decreased on the 14th day, which was consistent with the qPCR results.

[0074] Example 2: Anti-inflammatory effect of IGF2BP2 in astrocytes

[0075] I. Knockout of IGF2BP2

[0076] CRISPR / Cas9 gene editing technology is a technology that can accurately edit specific sites in the genome of any species. Using this technology, single gene or multiple gene knockout at the cellular level can be achieved.

[0077] In this experiment, CRISPR / Cas9 gene editing technology was used to knockout the IGF2BP2 coding gene in mouse primary astrocytes. The specific operation steps are as follows:

[0078] (1) Add 200 μL Opti-MEM medium and 7.5 μL Gencefe TransExp transfection reagent to a sterile tube A, mix gently, and let stand at room temperature for 5 min to obtain mixture A.

[0079] (2) Add 200 μL Opti-MEM medium and 2.5 μg sgRNA-containing recombinant plasmid to a sterile tube B, mix gently, and let stand at room temperature for 5 min to obtain mixture B.

[0080] (3) Mix mixture A and mixture B gently, and let stand at room temperature for 15 min to obtain a transfection complex.

[0081] (4) The primary astrocytes are inoculated into a 6-well plate, and the cells are cultured at 37°C, 5% CO2 and saturated humidity in an incubator until the confluence is 50%. Then, 400 μL of the transfection complex is added to each well of the cells, and the cells are gently shaken. After 48 h of continuous culture, the knockout of the IGF2BP2 coding gene is verified by Western Blot, and the extraction method of the protein in the astrocytes is the same as that in Example 1.

[0082] The recombinant plasmid containing sgRNA is four-to-one (4 sgRNAs are Igf2bp2-sgRNA1, Igf2bp2-sgRNA2, Igf2bp2-sgRNA3 and Igf2bp2-sgRNA4, and the nucleotide sequences are shown in SEQ ID NOs. 5-8), which can guide the Cas9 protein to accurately recognize and cut the target gene to obtain higher gene activation efficiency. The plasmid map is shown in Figure 5 The construction of the recombinant plasmid is entrusted to Guangzhou Ruibo Biotechnology Co., Ltd.

[0083] Meanwhile, the empty plasmid vector is introduced into the astrocytes by the same method, and Vector is obtained as a control group 1 for subsequent experiments.

[0084] The knockout results of the IGF2BP2 coding gene are shown in Figure 6 The "KO1, KO2" in the figure respectively represent "knockout group 1 and knockout group 2", and the knockout group 1 and the knockout group 2 are repeated experiments. It can be seen from Figure 6 that after the IGF2BP2 coding gene in the astrocytes is subjected to gene interference, the expression level of the IGF2BP2 protein in the astrocytes is significantly reduced, which indicates that the IGF2BP2 knockout primary astrocytes (KO) are successfully obtained.

[0085] II. Overexpression of IGF2BP2

[0086] Overexpression is the most commonly used method for up-regulating gene expression, and the basic principle is to construct a target gene into a plasmid or a viral vector, and then introduce it into cells to increase the expression amount of the target gene.

[0087] The experiment overexpresses IGF2BP2 in astrocytes by plasmid transfection technology. One day before transfection, the astrocytes were digested with 0.25% trypsin solution, counted, and then inoculated into 24-well plates at an appropriate density. The cells were cultured in a 37°C, 5% CO2, and saturated humidity incubator until the confluence reached 80%, and then transfection was performed. Two hours before transfection, the complete medium with serum was replaced with serum-free complete medium. The specific operation steps are as follows:

[0088] (1) Add 200 μL Opti-MEM medium and 7.5 μL Gencefe TransExp transfection reagent to a sterile tube C, mix gently, and incubate at room temperature for 5 min to obtain mixture C.

[0089] (2) Add 200 μL Opti-MEM medium and 2.5 μg overexpression recombinant plasmid to a sterile tube D, mix gently, and incubate at room temperature for 5 min to obtain mixture D.

[0090] (3) Mix mixture C and mixture D gently, incubate at room temperature for 15 min, and then obtain the transfection complex.

[0091] (4) Seed the primary astrocytes into a 6-well plate and culture in a 37°C, 5% CO2, and saturated humidity incubator until the confluence reaches 50%. Add 400 μL transfection complex to each well of cells and mix gently. Continue to culture for 48 h, then verify the overexpression of IGF2BP2 coding gene by Western Blot method. The protein extraction method of astrocytes and the Western Blot identification steps are the same as those in Example 1.

[0092] The nucleotide sequence of the overexpression recombinant plasmid is shown in SEQ ID NO. 9.

[0093] Meanwhile, the empty plasmid vector is transfected into astrocytes according to the same method as above to obtain NC, which is used as Control Group 2 for subsequent experiments.

[0094] The overexpression results of IGF2BP2 coding gene are shown in Figure 7 The figure "OE" represents "overexpression group". It can be seen from Figure 7 that after plasmid transfection, the primary astrocytes highly express IGF2BP2 protein, which indicates that IGF2BP2 overexpression primary astrocytes (OE) are successfully obtained.

[0095] III. Anti-inflammatory effect of IGF2BP2 in astrocytes

[0096] The four kinds of cells of IGF2BP2 knockout primary astrocytes (KO), control group 1 (Vector), IGF2BP2 overexpression primary astrocytes (OE), control group 2 (NC) were inoculated into 6-well plates, and the cells were cultured in a incubator at 37℃, 5% CO2 and saturated humidity until the confluence was 80%, then TNF-α stimulation was given according to the same method as in Example 1 and total RNA was extracted after 24h; qPCR quantitative analysis of cytokines and chemokines (IL-1β, IL-6, CCL2, CXCL2) in astrocytes before and after TNF-α stimulation was performed using an RT-PCR kit. Among them, the qPCR primer sequences for identifying IL-1β, IL-6, CCL2, CXCL2 are shown in SEQ ID NOs. 10-17.

[0097] The qPCR analysis results of the Vector group and the KO group are shown in Figure 8 Figures a-d are the expression levels of IL-1β, IL-6, CCL2, CXCL2 in the Vector group and the KO group before and after TNF-α stimulation. It can be seen from Figure 8 that compared with the Vector group, the expression levels of IL-1β, IL-6, CCL2, CXCL2 in the primary astrocytes after knocking out IGF2BP2 all increased significantly after TNF-α stimulation, showing a higher inflammatory level.

[0098] The qPCR analysis results of the NC group and the OE group are shown in Figure 9 Figures a-d are the expression levels of IL-1β, IL-6, CCL2, CXCL2 in the NC group and the OE group before and after TNF-α stimulation. It can be seen from Figure 9 that compared with the NC group, the expression levels of IL-1β, IL-6, CCL2, CXCL2 in the primary astrocytes after overexpressing IGF2BP2 all decreased significantly after TNF-α stimulation, showing a lower inflammatory level.

[0099] The above results show that IGF2BP2 can play an inhibitory role in the inflammatory response of primary astrocytes.

[0100] Example 3: IGF2BP2 plays an anti-inflammatory role by inhibiting the MAPKs / NF-κB signal transduction pathway

[0101] IGF2BP2 knockout primary astrocytes (KO), control group 1 (Vector), IGF2BP2 overexpression primary astrocytes (OE), control group 2 (NC) were inoculated into 6-well plates, and the cells were cultured in a incubator at 37℃, 5% CO2 and saturated humidity until the confluence was 80%. After 6 hours of starvation in the incubator, TNF-α stimulation was given according to the same method as in Example 1, and the cells were cultured in the incubator. Then, the cell proteins at 0 min, 15 min, 30 min and 60 min were extracted and identified by Western Blot according to the same method as in Example 1.

[0102] The Western Blot identification results are shown in Figure 10 Figure 10 It can be seen that:

[0103] Compared with the Vector group, the expression levels of P-P38, P-ERK, P-JNK and P-P65 in the MAPKs / NF-κB signal transduction pathway in the primary astrocytes after knocking out IGF2BP2 were increased at 15 min, 30 min and 60 min, which indicated that the MAPKs / NF-κB signal transduction pathway was activated after knocking out IGF2BP2.

[0104] Compared with the NC group, the expression levels of P-P38, P-ERK, P-JNK and P-P65 in the MAPKs / NF-κB signal transduction pathway in the primary astrocytes after overexpressing IGF2BP2 were decreased at 15 min, 30 min and 60 min, which indicated that the MAPKs / NF-κB signal transduction pathway was inhibited after overexpressing IGF2BP2.

[0105] Example 4: Downstream target points of IGF2BP2

[0106] I. RIP-seq to find downstream binding target points of IGF2BP2:

[0107] RIP-seq is a technology that combines RNA immunoprecipitation (RIP) with next-generation sequencing technology (NGS) to study the binding of RNA and protein in cells. RIP uses target protein antibodies to precipitate the corresponding RNA-protein complex (RBP), and then the RNA bound to the complex can be sequenced and analyzed after enrichment and purification. RIP-seq analyzes the binding of RNA and protein in cells within the whole transcriptome through high-throughput sequencing, revealing the interaction between RNA molecules and RBP. RIP-seq is a powerful tool for understanding the dynamic process of post-transcriptional regulatory networks and can more effectively discover miRNA regulatory targets.​

[0108] RIP-seq sample submission: The primary astrocytes were inoculated into a 6-well plate and cultured in a incubator at 37°C, 5% CO2 and saturated humidity until the confluence reached 80%. The medium was aspirated and the cells were washed three times with 4°C pre-cooled PBS buffer. An appropriate amount of 4°C pre-cooled PBS buffer was added and the cells were scraped with a cell scraper and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 4°C, 1000g for 5 min and collected. The cells were resuspended in 1 mL of 4°C pre-cooled PBS buffer and transferred to a 1.5 mL centrifuge tube. The cells were centrifuged at 4°C, 1000g for 5 min and collected. The cells were stored in liquid nitrogen and then sent to Wuhan Kangmei Technology Co., Ltd. for machine operation.

[0109] The combined analysis results of RIP-seq, RNA-seq and sequencing data of N6-methyladenosine acid (m6A) in mouse spinal cord injury tissue are shown in Figure 11 , and Figure 11 Analysis shows that ARRB1 may be a downstream target of IGF2BP2 for inhibiting inflammation.

[0110] To further prove that ARRB1 may be a downstream target of IGF2BP2, we performed Western Blot identification of the expression levels of IGF2BP2 and ARRB1 after overexpression of IGF2BP2, and the results are shown in Figure 12 . It can be seen from Figure 12 that when IGF2BP2 is overexpressed, the expression level of ARRB1 increases, which further proves that ARRB1 may be a downstream target of IGF2BP2 for inhibiting inflammation.

[0111] II. Anti-inflammatory effect of ARRB1 in primary astrocytes

[0112] (1) Knockdown of ARRB1

[0113] Small interference is to reduce the expression level of target genes by RNA interference (RNAi) and other methods. RNAi usually mediates targeted degradation of mRNA by synthesizing artificial post-transcriptional small interfering RNA (siRNA). Since the effect of RNAi is usually partial inhibition of the expression of target genes, it is called gene knockdown.

[0114] In this experiment, small interference was used to knock down the expression of ARRB1 in IGF2BP2 overexpressed primary astrocytes (OE), and the specific operation steps were as follows:

[0115] (1) Add 200 μL Opti-MEM medium and 7.5 μL Gencefe TransExp transfection reagent to a sterile tube E, mix gently, and stand at room temperature for 5 min to obtain mixture E.

[0116] (2) In the sterile tube F, add 200 μL Opti-MEM medium and 150 pmol siRNA small interference sequence, mix gently, and stand at room temperature for 5 min to obtain mixture F.

[0117] (3) Mix mixture E and mixture F gently, stand at room temperature for 15 min to obtain the transfection complex.

[0118] (4) The IGF2BP2 overexpressed primary astrocytes (OE) are inoculated into a 6-well plate, and the cells are cultured in a culture box at 37°C, 5% CO2 and saturated humidity until the confluence is 80%. Then, 400 μL of the transfection complex is added to each well of the cells, and the cells are gently shaken. After 48 h of continuous culture, the knockdown of ARRB1 is verified by Western Blot method, and the extraction method of the protein in the astrocytes and the identification steps of Western Blot are the same as those in Example 1.

[0119] The siRNA small interference sequence is four-to-one, and the four siRNAs are Arrb1-Mus-305, Arrb1-Mus-1238, Arrb1-Mus-923 and Arrb1-Mus-1085, and the nucleotide sequences are shown in SEQ ID NOs. 18-21. The construction of the above siRNA small interference sequence is entrusted to Shanghai Jimar Gene.

[0120] The small interference results of ARRB1 are shown in Figure 13 The "NC, si1ARRB1, si2ARRB1" in the figure respectively represent "non-interference group, interference group 1, interference group 2", and the interference group 1 and the interference group 2 are repeated experiments. Figure 13 It can be seen that after small interference, the protein expression amount of ARRB1 is obviously reduced, which indicates that the ARRB1 knockdown primary astrocyte (OE+siARRB1) is successfully obtained.

[0121] (2) Anti-inflammatory effect of ARRB1 knockdown primary astrocyte

[0122] The IGF2BP2 overexpressed primary astrocytes (OE) and the ARRB1 knockdown primary astrocytes (OE+siARRB1) are inoculated into a 6-well plate, and the cells are cultured in a culture box at 37°C, 5% CO2 and saturated humidity until the confluence is 80%. Then, the cells are stimulated according to the same method as in Example 1, and the total RNA of the cells is extracted after 24 h. The cytokines and chemokines (IL-1β, IL-6, CCL2 and CXCL2) in the astrocytes before and after TNF-α stimulation are quantitatively analyzed by RT-qPCR kit.

[0123] The results are as follows Figure 14 As shown in the figures, figures a-d represent the expression levels of IL-1β, IL-6, CCL2, and CXCL2 in the OE group and the OE+siARRB1 group before and after TNF-α stimulation, respectively. Figure 14 It was found that, compared with the OE group, the expression levels of IL-1β, IL-6, CCL2 and CXCL2 in primary astrocytes after ARRB1 knockdown were significantly increased after TNF-α stimulation, indicating a higher level of inflammation.

[0124] (3) Changes in the MAPKs / NF-κB signaling pathway in primary astrocytes due to ARRB1 knockdown

[0125] Primary astrocytes (NC) from the non-interference group, primary astrocytes from interference group 1 (si1ARRB1), and primary astrocytes from interference group 2 (si2ARRB1) were seeded into 6-well plates and cultured at 37°C, 5% CO2, and saturated humidity until confluence reached 80%. The cells were then starved in the incubator for 6 hours, stimulated with TNF-α in the same manner as in Example 1, and cultured in the incubator for another 6 hours. Cell proteins were then extracted at 0 min and 15 min of culture time and identified by Western blotting in the same manner as in Example 1.

[0126] The results are as follows Figure 15 As shown; by Figure 15 It was found that, compared with NC, the expression levels of P-P38, P-ERK, P-JNK, and P-P65P65 in the MAPKs / NF-κB signaling pathway in primary astrocytes after ARRB1 knockdown were all increased at 15 min of TNF-α stimulation, indicating that the MAPKs / NF-κB signaling pathway was activated in the si1ARRB1 group and the si2ARRB1 group compared with the NC group.

[0127] The above results indicate that IGF2BP2 targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, thereby suppressing the inflammatory response following spinal cord injury.

[0128] Example 5: Animal experiments to verify the effect of knockdown of IGF2BP2 on functional recovery after spinal cord injury.

[0129] I. Knockdown of IGF2BP2 in astrocytes at the site of spinal cord injury in mice using adeno-associated virus (AAV)

[0130] This embodiment provides an adeno-associated virus vector AAV9-GFAP-GFP-mir30-shRNA (Mouse Igf2bp2) expressing IGF2BP2 gene (purchased from ViraBio, with GFAP promoter, targeting astrocyte to knock down target gene), vector resistance is Amp, and the resistance concentration is 100 μg / mL.

[0131] The mice were anesthetized and then fixed on a stereotaxic instrument, and T9 laminectomy was performed to expose the spinal cord under a body microscope using a fiber tweezers. Using a 5 μL microsyringe, 2.5 μL of the above adeno-associated virus vector with a concentration of 1×10 13 gc / mL (gc represents AAV virus titer) was injected at 1 mm (depth 0.8 mm) on both sides of the beak and tail of the T9 center, and the injection speed was 500 nL / min. The needle was left in place for 5 minutes before being transferred to the next injection site to allow sufficient penetration.

[0132] A negative control group (AAV+shNC) was also set up: u6-shRNA (scramble) with a nonsense sequence was inserted.

[0133] The knockdown effect was verified by Western Blot method, and the results are shown in Figure 16 ; it can be seen from Figure 16 that the expression level of IGF2BP2 protein is decreased, which indicates that IGF2BP2 in the astrocytes of the injured site of the mouse spinal cord is successfully knocked down, and an IGF2BP2 knockdown spinal cord injury model mouse is obtained.

[0134] II. The effect of knocking down IGF2BP2 on the functional repair of mice after spinal cord injury

[0135] (1) BMS score

[0136] The BMS score was used to evaluate the neural function of the IGF2BP2 knockdown spinal cord injury model mouse at a specific time point after SCI. One week before the operation, the mice were placed in an open field every day to familiarize themselves with the environment. BMS scoring started on the first day after the operation: the mice were placed in an open field, and the movement of the hind limbs of the mice was observed. Each mouse was observed for 5 minutes. The experimenters who were familiar with the scoring rules and did not know the grouping situation referred to the international scoring standard to score the movement function of the mice. The BMS scores of the mice at 0d, 3d, 7d, 14d, 21d, and 28d after the operation were recorded.

[0137] The results are shown in Figure 17 ; it can be seen from Figure 17 that compared with the control group, the motor function recovery of the IGF2BP2 knockdown spinal cord injury model mouse after spinal cord injury was significantly hindered.

[0138] (2) MRI (magnetic resonance imaging)

[0139] On the 28th day after the operation, the mice were subjected to magnetic resonance imaging using a small animal nuclear magnetic resonance imaging scanner (9.4T Biospec), and then sagittal T2 weighted images were scanned using a ParaVision 6.0 system, with the following scanning parameter settings: T2 weighted, 320x320 matrix, layer thickness 0.3mm, echo time / repetition time = 24 / 1200ms, flip angle 90°.

[0140] The imaging results are shown in FIG. 6, wherein the red square represents the size of the hematoma area in the damaged spinal cord region. As can be seen from FIG. 6, compared with the control group, the IGF2BP2 knockdown spinal cord injury model mice have a larger hematoma area after spinal cord injury, and the spinal cord repair process is obviously hindered, which is consistent with the BMS score results. Figure 18 Figure 18 The above results show that IGF2BP2 can promote the functional recovery after spinal cord injury by inhibiting the inflammatory response after spinal cord injury.

[0141] The above results show that IGF2BP2 can promote the functional recovery after spinal cord injury by inhibiting the inflammatory response after spinal cord injury.​

Claims

1. Use of an IGF2BP2 agonist in the preparation of a medicament for inhibiting inflammatory response after spinal cord injury, wherein the IGF2BP2 agonist is capable of promoting expression of IGF2BP2, the amino acid sequence of IGF2BP2 is shown as SEQ ID NO. 1, the IGF2BP2 agonist targets astrocytes, and the IGF2BP2 agonist is an IGF2BP2 gene.

2. Use according to claim 1, wherein The IGF2BP2 agonist is capable of increasing the expression level of ARRB1 by promoting expression of IGF2BP2, the amino acid sequence of ARRB1 is shown as SEQ ID NO. 2, thereby inhibiting inflammatory response after spinal cord injury.

3. The use according to claim 1, wherein The IGF2BP2 agonist is capable of inhibiting MAPKs / NF-κB signaling pathway by promoting expression of IGF2BP2, thereby inhibiting inflammatory response after spinal cord injury.

4. The use according to claim 1, wherein The spinal cord injury is secondary spinal cord injury.

5. The use according to claim 1, wherein the compound is ###0002### The inflammatory response includes expression of pro-inflammatory cytokines and chemokines.

6. Use according to claim 5, wherein The pro-inflammatory cytokines and chemokines include IL-1β, IL-6, CCL2, CXCL2.

7. Use of an IGF2BP2 agonist in the preparation of a medicament for restoring motor function after spinal cord injury, wherein the IGF2BP2 agonist is an IGF2BP2 gene.

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