Application of IGF2BP2 agonist in preparation of medicine for inhibiting inflammatory response after spinal cord injury

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an IGF2BP2 agonist in the preparation of a drug for inhibiting inflammatory response after spinal cord injury. Background Art

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

[0003] SCI mainly includes two pathological processes: primary injury and secondary injury. At present, the clinical treatment of secondary injury of SCI is mainly short-term high-dose hormone shock therapy after surgery to slow down harmful pathological processes such as inflammatory storm and edema in the spinal cord microenvironment after injury. After SCI, the tissues in the injured area and the surrounding area have a high inflammatory response state, and the inflammatory response plays a huge role in functional recovery and will cause serious secondary reactions to a certain extent. The characteristics of the inflammatory response are mainly the recruitment of immune cells, the secretion of proinflammatory 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. After SCI, astrocytes around the injured area are activated and proliferated. Activated astrocytes are also called reactive astrocytes.

[0004] In recent years, the regulatory role of epigenetic modification in the 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 the most abundant dynamic chemical modification in RNA. It is dynamically regulated by methylation by methyltransferases, recognition and processing by reader proteins, and demethylation by demethylases.

[0005] Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) is an important reader protein of m6A methylation modification. It 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] β-Arrestin-1 (ARRB1) is a ubiquitous multifunctional adaptor protein that regulates G protein-coupled receptor (GPCR) signaling. It has been identified as a mediator that connects receptors to downstream signaling pathways. It 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] Currently, there are no relevant research reports on IGF2BP2 or ARRB1 in the treatment of spinal cord injury. Summary of the invention

[0008] The present invention provides the use of an IGF2BP2 agonist in the preparation of a drug for inhibiting inflammatory response after spinal cord injury. The present invention has found that IGF2BP2 can inhibit inflammatory response after spinal cord injury, and it targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, thereby inhibiting inflammatory response after spinal cord injury, thereby contributing to functional recovery after spinal cord injury.

[0009] The technical solution of the present invention is as follows: Use of an IGF2BP2 agonist in the preparation of a drug for inhibiting 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 in SEQ ID NO.1.

[0010] 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; the amino acid sequence of ARRB1 is shown in SEQ ID NO.2.

[0011] 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.

[0012] Preferably, the IGF2BP2 agonist targets astrocytes.

[0013] 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.

[0014] Preferably, the spinal cord injury is secondary spinal cord injury.

[0015] Preferably, the inflammatory response includes the expression of pro-inflammatory cytokines and chemokines.

[0016] Further preferably, the proinflammatory cytokines and chemokines include IL-1β, IL-6, CCL2, and CXCL2.

[0017] Preferably, the IGF2BP2 agonist can promote the expression of IGF2BP2, and IGF2BP2 targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway and suppress the inflammatory response after spinal cord injury.

[0018] Use of an ARRB1 agonist in the preparation of a drug for inhibiting inflammatory response after spinal cord injury; the ARRB1 agonist can promote the expression of ARRB1.

[0019] The invention discloses an application of a MAPKs / NF-κB signal transduction pathway inhibitor in the preparation of a drug for inhibiting inflammatory response after spinal cord injury; the MAPKs / NF-κB signal transduction pathway inhibitor can inhibit the activation of P-P38, P-ERK, P-JNK and P-P65 in the pathway.

[0020] Application of IGF2BP2 agonist or ARRB1 agonist or MAPKs / NF-κB signal transduction pathway inhibitor in the preparation of spinal cord injury repair drugs.

[0021] Application of an IGF2BP2 agonist in the preparation of a drug for inhibiting inflammatory response; the IGF2BP2 agonist can promote the expression of IGF2BP2, and IGF2BP2 targets astrocytes.

[0022] Preferably, the inflammatory response includes the expression of proinflammatory cytokines and chemokines, and the proinflammatory cytokines and chemokines include IL-1β, IL-6, CCL2, and CXCL2.

[0023] Beneficial effects: The present invention provides the use of an IGF2BP2 agonist in the preparation of a drug for inhibiting inflammatory response after spinal cord injury. The present invention has found that insulin-like growth factor 2-mRNA binding protein 2 (IGF2BP2) can inhibit inflammatory response after spinal cord injury, promote the expression of IGF2BP2 by an IGF2BP2 agonist, and IGF2BP2 targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, thereby inhibiting the inflammatory response after spinal cord injury, thereby contributing to functional recovery after spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the change in the mRNA expression of IGF2BP2 in primary astrocytes within 24 hours after TNF-α stimulation in Example 1.

[0025] Figure 2 This is Example 1, which shows the changes in the mRNA expression level of IGF2BP2 in spinal cord injury tissue after spinal cord injury.

[0026] Figure 3 This is the change in protein expression of IGF2BP2 in primary astrocytes within 24 hours after TNF-α stimulation in Example 1.

[0027] Figure 4 This is Example 1, which shows the changes in the protein expression of IGF2BP2 in spinal cord injury tissue after spinal cord injury.

[0028] Figure 5 This is the plasmid map used to knock out IGF2BP2 in astrocytes in Example 2.

[0029] Figure 6 This is a verification of the knockout results of IGF2BP2 in astrocytes in Example 2.

[0030] Figure 7 This is a verification of the overexpression results of IGF2BP2 in astrocytes in Example 2.

[0031] Figure 8 This is the effect of knocking out IGF2BP2 in Example 2 on the inflammatory response of astrocytes.

[0032] Fig. 9 This is the effect of overexpression of IGF2BP2 on the inflammatory response of astrocytes in Example 2.

[0033] Fig.10 This is the effect of knocking out or overexpressing IGF2BP2 in Example 3 on the MAPKs / NF-κB signal transduction pathway in astrocytes.

[0034] Fig.11 This is the result of the joint analysis of RIP-seq, RNA-seq, and mouse spinal cord injury tissue m6A data in Example 4.

[0035] Fig.12 This is the effect of overexpression of IGF2BP2 on the expression level of ARRB1 in astrocytes.

[0036] Fig.13 This is the small interference result of ARRB1 in Example 4.

[0037] Fig.14 This is the effect of knocking down ARRB1 on the inflammatory response of astrocytes in Example 4.

[0038] Fig.15 This is the effect of knocking down ARRB1 in Example 4 on the MAPKs / NF-κB signal transduction pathway in astrocytes.

[0039] Fig.16 This is to verify the knockdown effect of IGF2BP2 targeting astrocytes in mice in Example 5.

[0040] Fig.17 This is the BMS score result of the IGF2BP2 knockdown spinal cord injury model mice in Example 5.

[0041] Fig.18 This is the repair result of the IGF2BP2 knockdown spinal cord injury model mice after spinal cord injury in Example 5. DETAILED DESCRIPTION

[0042] The following is explained in conjunction with specific embodiments: Source of experimental materials or description of preparation methods: Experimental mice were purchased from Jinan Pengyue Animal Breeding Co., Ltd.

[0043] HBSS buffer: purchased from Wuhan Prosai Life Science Technology Co., Ltd., catalog number PB180321.

[0044] Complete culture medium: DMEM medium (purchased from gibico) + FBS (10 mL / 100 mL, purchased from gibico) + double antibody (1 mL / 100 mL, purchased from Solebol, product number P1400).

[0045] TNF-α: purchased from Ibotek Biotechnology, catalog number RP01071.

[0046] Gencefe TransExp transfection reagent: purchased from Research Cloud Biotechnology.

[0047] Opti-MEM culture medium: purchased from Gibco, catalog number 31985-07.

[0048] Example 1: Expression of IGF2BP2 in astrocytes and spinal cord injury tissue 1. Extraction and culture of primary astrocytes The T25 cell culture flask was coated with a 0.1 mg / mL PDL (poly-D-lysine) solution at 37°C for 30 min, and then the PDL solution was aspirated, washed three times with HBSS buffer, and placed in a 37°C incubator for use.

[0049] Add an appropriate amount of HBSS buffer to a 10 cm culture dish and place it on an ice plate for precooling; disinfect the suckling mice (1 day old) with 75% (v / v) ethanol solution, decapitate, and use fine scissors to cut the skin and skull along the midline, taking care not to damage the cortex; then use fine forceps to peel off the skull on both sides, and then remove the entire brain below the olfactory bulb (remove the meninges and blood vessels), and place the removed brain in the above-mentioned precooled 10 cm culture dish; after all the brains have been removed, aspirate the HBSS buffer and use scissors to cut the brain tissue as much as possible; add 10 mL of 0.25% trypsin solution (taking 10 brains as an example), blow and beat to promote tissue digestion, and obtain a semi-suspension; add the semi-suspension to a conical flask, put in a stirrer, seal it with tin foil, and stir and digest it with a magnetic stirrer at 37°C for 15 minutes (at this time there will be undigested tissue clumps and jelly-like clumps); add Add 2 times the volume of trypsin solution (20 mL) of complete culture medium and appropriate amount of DNA enzyme to terminate digestion, blow and sieve through a 200-mesh sieve. During the sieving process, grind the undigested tissue masses and jelly-like masses on the filter for 25 minutes. Add appropriate amount of HBSS buffer to promote sieving to obtain filtrate; transfer the filtrate to a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, add 10 mL of complete culture medium to the precipitate for resuspending, and transfer the resuspended liquid to a 15 mL centrifuge tube; centrifuge at 1000 rpm for 5 minutes, remove the supernatant, add 2 mL of complete culture medium to the precipitate for resuspending, add the resuspended liquid to a 10 cm culture dish containing 10 mL of complete culture medium, and allow differential attachment for 1 hour. After 1 hour, transfer the liquid in the 10 cm culture dish to the T25 cell culture flask prepared above for cell culture and passaging.

[0050] After 2 days of cell culture, the cells were subcultured for the first generation to a new T25 cell culture flask, and the subculture was performed at the ratio of 1 flask to 3 flasks; after another 2 days of culture, the cells were subcultured for the second generation to a new T25 cell culture flask, and the subculture was performed at the ratio of 3 flasks to 9 flasks; before subculture, the culture flasks were placed in a 37°C shaker at 200 rpm for 24 hours; after another 2 days of culture, the cells were subcultured for the third generation to a 6-well plate, and the plates were plated at the ratio of 9 flasks to 9 plates. The attachment time in the 6-well plate was 48 hours to obtain primary astrocytes.

[0051] 2. Construction of a Spinal Cord Contusion Injury Mouse Model Mice (6-8 weeks) were fasted 8 h before surgery to avoid vomiting or aspiration during surgery.

[0052] A 3% pentobarbital solution was prepared with distilled water as the solvent and anesthetized into the mice by intraperitoneal injection at a dose of 30 mg / kg. No response after clamping the limbs indicated complete anesthesia.

[0053] After complete anesthesia, the hair on the back of the mouse was shaved to expose the surgical area; the mouse was fixed in a prone position on the operating table to ensure the stability of the mouse's position during the operation; the surgical area was disinfected with iodine cotton balls to reduce the risk of contamination during the operation; a disposable sterile surgical drape was laid, and a 1.5 cm incision was made at the T9 segment of the spine along the midline of the mouse's back, and the muscles and fascia were bluntly separated layer by layer until the spinous process was exposed; T9 laminectomy was performed using fiber forceps under a stereomicroscope to expose the spinal cord.

[0054] The mice were divided into Sham group and SCI group. The mice in Sham group were not given spinal cord injury treatment after laminectomy, and the injured area was cleaned with sterile saline, the surgical incision was sutured layer by layer with sterile sutures, and disinfected with iodine cotton balls; the mice in SCI group were struck on the spinal cord with a pneumatic spinal cord injury impactor at a speed of 1m / s, a depth of 2mm, and a dwell time of 1s. The cleaning, suturing, and disinfection steps and operations were the same as those in the Sham group, and a spinal cord contusion injury mouse model was established.

[0055] Each group of mice was placed on a heating blanket for 2-3 hours. After the mice woke up, they were placed in a cage (the mice had free access to food and water in the cage). To prevent urinary tract infection in the mice, artificial bladder compression was performed twice a day to help the mice urinate.

[0056] 3. Expression of IGF2BP2 in astrocytes and spinal cord injury tissues (1) Primary astrocytes were seeded into 6-well plates and cultured in an incubator at 37°C, 5% CO2 and saturated humidity until the cells reached 80% confluence. Then, TNF-α (tumor necrosis factor α) was stimulated at a concentration of 1 μg / mL to stimulate an inflammatory response and simulate the inflammatory state after spinal cord injury in vivo. The dose was 10 ng / mL; then, total RNA of the cells was extracted using Trizol reagent after 0 h, 2 h, 4 h, 6 h, 12 h, and 24 h; total RNA of the cells was reverse transcribed into cDNA using a reverse transcription kit, and then qPCR quantitative analysis of IGF2BP2 in astrocytes was performed using an RT-qPCR kit. The qPCR primers used to identify IGF2BP2 were Mus-Igf2bp2-F and Mus-Igf2bp2-R, and their sequences are shown in SEQ ID NOs.3~4, respectively.

[0057] The expression level of IGF2BP2 relative to GAPDH is as shown in Figure 1 As shown in the figure, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; Figure 1It can be seen that within 24 hours after TNF-α stimulation, the mRNA expression level of IGF2BP2 in primary astrocytes showed a gradual increasing trend.

[0058] (2) After the construction of the spinal cord contusion mouse model, the spinal cord injury tissues of the spinal cord contusion mouse model were removed on the 3rd, 7th and 14th days respectively, ground with liquid nitrogen, and 1 mL of Trizol reagent was added to every 50-100 mg of tissue. The tissues were broken up with a homogenizer and total cellular RNA was extracted. The same method as above was used to perform qPCR quantitative analysis of IGF2BP2 in the spinal cord injury tissues.

[0059] The expression level of IGF2BP2 relative to GAPDH is as shown in Figure 2 As shown in the figure, **** P < 0.0001; Figure 2 It can be seen that on the 3rd and 7th days after spinal cord injury, the mRNA expression of IGF2BP2 in the spinal cord injury tissue showed an upward trend, and then showed a downward trend on the 14th day.

[0060] 4. Western Blot Protein Identification (1) Primary astrocytes were seeded into 6-well plates and cultured in an incubator at 37°C, 5% CO2 and saturated humidity until the cells reached a confluence of 80%. TNF-α stimulation was given according to the same method as above, and then protein was extracted from the cell culture medium using cell lysis buffer after 2h, 4h, 6h, 12h and 24h to obtain protein solutions; the concentration of each group of protein solutions was adjusted using the BCA method. Each group of protein samples was subjected to SDS-PAGE electrophoresis and color development using the Western Blot method to observe the protein expression of IGF2BP2 after inflammatory response stimulation.

[0061] The experimental parameters of Western Blot were as follows: SDS-PAGE separation gel concentration 10%; protein loading amount 20 μg; color development system was ECL method combined with X-ray film color development.

[0062] The results are as follows Figure 3 As shown by Figure 3 It can be seen that within 24 hours after TNF-α stimulation, the protein expression of IGF2BP2 in primary astrocytes showed a gradually increasing trend, which was consistent with the qPCR results.

[0063] (2) After the construction of the spinal cord contusion mouse model, the spinal cord injury tissue of the spinal cord contusion mouse model was removed on the 3rd, 7th and 14th days respectively, and the tissue was quickly placed in a 4°C pre-cooled PBS buffer to wash off the blood on the surface; the tissue was weighed and placed in a centrifuge tube containing PBS buffer, cut into smaller tissue blocks, centrifuged, the discolored PBS buffer was removed, and the new PBS buffer was replaced and centrifuged again. The above process was repeated until the PBS buffer did not change color, centrifuged, and the tissue pellet was retained; a 4°C pre-cooled protein extraction reagent containing inhibitors (1 mL of protein extraction reagent was added for every 250 mg of tissue) was added to the tissue pellet. When the tissue became soft, the tissue was continuously blown with a 1 mL pipette tip; the tissue was homogenized multiple times at a low speed using a homogenizer (each homogenization was 30 seconds, and the tissue was ice-bathed for 1 minute between homogenizations) until the tissue was completely lysed to obtain a protein solution; the concentration of each group of protein solutions was adjusted to a level using the BCA method. Each group of protein samples was subjected to SDS-PAGE electrophoresis and color development by Western Blot method to observe the protein expression of IGF2BP2. The Western Blot experimental parameters were the same as above.

[0064] The results are as follows Figure 4 As shown by Figure 4 It can be seen that the protein expression of IGF2BP2 in the spinal cord injury tissue showed an upward trend on the 3rd and 7th days after spinal cord injury, and then showed a downward trend on the 14th day, which was consistent with the qPCR results.

[0065] Example 2: Anti-inflammatory effect of IGF2BP2 in astrocytes I. Knockout of IGF2BP2 CRISPR / Cas9 gene editing technology is a technology that can accurately edit specific sites in the genome of any species. This technology can be used to knock out single or multiple genes at the cellular level.

[0066] This experiment used CRISPR / Cas9 gene editing technology to knock out the IGF2BP2 coding gene in primary mouse astrocytes. The specific steps are as follows: (1) Add 200 μL Opti-MEM medium and 7.5 μL Gencefe TransExp transfection reagent to sterile tube A, mix gently, and let stand at room temperature for 5 min to obtain mixture A.

[0067] (2) Add 200 μL of Opti-MEM medium and 2.5 μg of recombinant plasmid containing sgRNA to sterile tube B, mix gently, and let stand at room temperature for 5 min to obtain mixture B.

[0068] (3) Gently mix mixture A and mixture B and let stand at room temperature for 15 minutes to obtain a transfection complex.

[0069] (4) 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 50%. 400 μL of transfection complex was added to each well of cells and gently shaken. After continuing to culture for 48 h, the knockout of the IGF2BP2 encoding gene was verified by Western Blot. The protein extraction method and Western Blot identification steps in astrocytes were the same as those in Example 1.

[0070] Among them, the recombinant plasmid containing sgRNA is four-in-one (the four sgRNAs are Igf2bp2-sgRNA1, Igf2bp2-sgRNA2, Igf2bp2-sgRNA3, and Igf2bp2-sgRNA4, and the nucleotide sequences are shown in SEQ ID NOs.5~8, respectively), which can guide the Cas9 protein to accurately identify and cut the target gene to obtain higher gene activation efficiency. The plasmid map is shown in Figure 5 The construction of the above recombinant plasmid was entrusted to Guangzhou Ruibo Biotechnology Co., Ltd.

[0071] At the same time, the empty plasmid vector was introduced into astrocytes according to the same method as above to obtain Vector, which was used as control group 1 for subsequent experiments.

[0072] The knockout results of IGF2BP2 encoding gene are as follows Figure 6 As shown in the figure, "KO1, KO2" represent "knockout group 1, knockout group 2", respectively. Knockout group 1 and knockout group 2 are repeated experiments. Figure 6 It was found that after genetic interference with the IGF2BP2 encoding gene in astrocytes, the expression level of IGF2BP2 protein in astrocytes was significantly reduced, indicating that IGF2BP2 knockout primary astrocytes (KO) were successfully obtained.

[0073] 2. Overexpression of IGF2BP2 Overexpression is the most commonly used method to upregulate gene expression. Its basic principle is to construct the target gene into a plasmid or viral vector, and then introduce it into the cell to increase the expression level of the target gene.

[0074] In this experiment, IGF2BP2 was overexpressed in astrocytes by plasmid transfection technology. One day before transfection, astrocytes were digested with 0.25% trypsin solution, counted, and adjusted to an appropriate density, then inoculated into a 24-well plate. Cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity until the confluence reached 80%, and then transfection was performed; 2 hours before transfection, the complete medium with serum was replaced with a complete medium without serum. The specific steps are as follows: (1) Add 200 μL Opti-MEM medium and 7.5 μL Gencefe TransExp transfection reagent to sterile tube C, mix gently, and let stand at room temperature for 5 min to obtain mixture C.

[0075] (2) Add 200 μL of Opti-MEM medium and 2.5 μg of overexpression recombinant plasmid to sterile tube D, mix gently, and let stand at room temperature for 5 min to obtain mixture D.

[0076] (3) Gently mix mixture C and mixture D and let stand at room temperature for 15 minutes to obtain a transfection complex.

[0077] (4) 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 50%. 400 µL of transfection complex was added to each well of cells and gently shaken. After further culture for 48 h, the overexpression of the IGF2BP2 encoding gene was verified by Western Blot. The protein extraction method and Western Blot identification steps in astrocytes were the same as those in Example 1.

[0078] Wherein, the nucleotide sequence of the overexpression recombinant plasmid is shown as SEQ ID NO.9.

[0079] At the same time, the empty plasmid vector was transfected into astrocytes according to the same method as above to obtain NC, which was used as control group 2 in subsequent experiments.

[0080] The results of overexpression of IGF2BP2 encoding gene are as follows Figure 7 As shown in the figure, "OE" means "overexpression group". Figure 7 It was found that after plasmid transfection, IGF2BP2 protein was expressed at a high level in primary astrocytes, indicating that IGF2BP2-overexpressing primary astrocytes (OE) were successfully obtained.

[0081] 3. Anti-inflammatory effect of IGF2BP2 in astrocytes Four types of cells, including IGF2BP2 knockout primary astrocytes (KO), control group 1 (Vector), IGF2BP2 overexpression primary astrocytes (OE), and control group 2 (NC), were seeded into 6-well plates, and the cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity until the confluence reached 80%. TNF-α stimulation was given in the same manner as in Example 1, and total cell RNA was extracted 24 hours later; RT-PCR kits were used to perform qPCR quantitative analysis of cytokines and chemokines (IL-1β, IL-6, CCL2, CXCL2) in astrocytes before and after TNF-α stimulation. Among them, the qPCR primer sequences used to identify IL-1β, IL-6, CCL2, and CXCL2 are shown in SEQ ID NOs. 10 to 17, respectively.

[0082] The qPCR analysis results of the Vector group and KO group are shown in Figure 8 As shown in Figures a to d, the expression levels of IL-1β, IL-6, CCL2, and CXCL2 in the Vector group and the KO group before and after TNF-α stimulation, respectively. Figure 8 It was found that compared with the Vector group, the expression levels of IL-1β, IL-6, CCL2, and CXCL2 in primary astrocytes after IGF2BP2 knockout were significantly increased after TNF-α stimulation, showing a higher level of inflammation.

[0083] The qPCR analysis results of the NC and OE groups are shown in Fig. 9 As shown in Figures a to d, the expression levels of IL-1β, IL-6, CCL2, and CXCL2 in the NC group and the OE group before and after TNF-α stimulation, respectively. Fig. 9 It was found that compared with the NC group, the expression levels of IL-1β, IL-6, CCL2, and CXCL2 in primary astrocytes overexpressing IGF2BP2 were significantly reduced after TNF-α stimulation, showing a lower level of inflammation.

[0084] The above results indicate that IGF2BP2 can play an inhibitory role in the inflammatory response of primary astrocytes.

[0085] Example 3: IGF2BP2 exerts anti-inflammatory effects by inhibiting the MAPKs / NF-κB signal transduction pathway Four types of cells, including IGF2BP2 knockout primary astrocytes (KO), control group 1 (Vector), IGF2BP2 overexpressing primary astrocytes (OE), and control group 2 (NC), were seeded into 6-well plates, respectively, and cultured in an incubator at 37°C, 5% CO2, and saturated humidity until the cell confluence reached 80%, then starved in the incubator for 6 hours, stimulated with TNF-α in the same manner as in Example 1, and continued to be cultured in the incubator, and then the cell proteins at 0 min, 15 min, 30 min, and 60 min of culture were respectively extracted and identified by Western Blot in the same manner as in Example 1.

[0086] Western Blot results Fig.10 shown by Fig.10 We can get: Compared with the Vector group, the expression levels of P-P38, P-ERK, P-JNK, and P-P65 in the MAPKs / NF-κB signaling pathway in primary astrocytes after IGF2BP2 knockout were increased at 15min, 30min, and 60min, indicating that the MAPKs / NF-κB signaling pathway was activated after IGF2BP2 knockout.

[0087] Compared with the NC group, the expression levels of P-P38, P-ERK, P-JNK, and P-P65 in the MAPKs / NF-κB signaling pathway in primary astrocytes overexpressing IGF2BP2 were reduced at 15min, 30min, and 60min, indicating that the MAPKs / NF-κB signaling pathway was inhibited after overexpression of IGF2BP2.

[0088] Example 4: Downstream targets of IGF2BP2 1. RIP-seq to find downstream binding targets of IGF2BP2: RIP-seq is a technology that combines RNA immunoprecipitation (RIP) with next-generation sequencing (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 after enrichment and purification, the RNA bound to the complex can be sequenced and analyzed. RIP-seq uses high-throughput sequencing of the enriched RNA fragments to analyze the binding of RNA and protein in cells across the entire transcriptome, 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 the regulatory targets of miRNA.

[0089] RIP-seq sample delivery: primary astrocytes were seeded into 6-well plates, and the cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity until the confluence reached 80%, the culture 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, the cells were scraped off with a cell scraper, transferred to a 15mL centrifuge tube, and centrifuged at 4°C and 1000g for 5min to collect the cells; the cells were resuspended in 1mL 4°C pre-cooled PBS buffer, transferred to a 1.5mL centrifuge tube, and centrifuged at 4°C and 1000g for 5min to collect the cells; the cells were frozen in liquid nitrogen and then sent to Wuhan Kangce Technology Co., Ltd. for loading.

[0090] The results of the combined analysis of RIP-seq, RNA-seq, and sequencing data of N6-methyladenosine (m6A) in mouse spinal cord injury tissue are shown in Figure 2. Fig.11 shown by Fig.11 Analysis showed that ARRB1 may be a downstream target of IGF2BP2 to inhibit inflammation.

[0091] To further prove that ARRB1 may be a downstream target of IGF2BP2, we performed Western Blot analysis on the expression of IGF2BP2 and ARRB1 after overexpression of IGF2BP2. Fig.12 As shown. Fig.12 It can be seen that when IGF2BP2 is overexpressed, the expression level of ARRB1 increases, which further proves that ARRB1 may be the downstream target of IGF2BP2 to inhibit inflammation.

[0092] 2. Anti-inflammatory effect of ARRB1 in primary astrocytes (1) Knockdown of ARRB1 Small interference is to reduce the expression level of the target gene through methods such as RNA interference (RNAi). RNAi is usually achieved by synthesizing artificial post-transcriptional small interfering RNA (siRNA) to mediate targeted degradation of mRNA. Since the effect of RNAi is usually to partially inhibit the expression of the target gene, it is called gene knockdown.

[0093] In this experiment, ARRB1 expression was knocked down in IGF2BP2-overexpressing primary astrocytes (OE) by small interference. The specific steps are as follows: (1) Add 200 μL Opti-MEM medium and 7.5 μL Gencefe TransExp transfection reagent to sterile tube E, mix gently, and let stand at room temperature for 5 min to obtain mixture E.

[0094] (2) Add 200 μL of Opti-MEM medium and 150 pmol of siRNA small interfering sequence into sterile tube F, mix gently, and let stand at room temperature for 5 min to obtain mixture F.

[0095] (3) Gently mix mixture E and mixture F and let stand at room temperature for 15 minutes to obtain a transfection complex.

[0096] (4) IGF2BP2-overexpressing primary astrocytes (OE) were seeded into 6-well plates, and the cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity until the confluence reached 80%. 400 μL of transfection complex was added to each well of cells and gently shaken. After continuing to culture for 48 h, the knockdown of ARRB1 was verified by Western Blot. The protein extraction method and Western Blot identification steps in astrocytes were the same as those in Example 1.

[0097] Among them, the siRNA small interfering sequence is four-protect-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 to 21. The construction of the above siRNA small interfering sequence was entrusted to Shanghai Jima Gene.

[0098] The results of small perturbations of ARRB1 are as follows Fig.13 As shown in the figure, "NC, si1ARRB1, si2ARRB1" represent "non-interference group, interference group 1, interference group 2", respectively. Interference group 1 and interference group 2 are repeated experiments. Fig.13 It can be seen that after small interference, the protein expression level of ARRB1 was significantly reduced, indicating that ARRB1 knockdown primary astrocytes (OE+siARRB1) were successfully obtained.

[0099] (2) Anti-inflammatory effect of ARRB1 knockdown in primary astrocytes IGF2BP2 overexpressing primary astrocytes (OE) and ARRB1 knockdown primary astrocytes (OE+siARRB1) were seeded into 6-well plates, respectively, and the cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity until the confluence reached 80%. TNF-α stimulation was given according to the same method as Example 1, and total cell RNA was extracted 24 hours later; RT-qPCR kits were used to perform qPCR quantitative analysis of cytokines and chemokines (IL-1β, IL-6, CCL2, CXCL2) in astrocytes before and after TNF-α stimulation.

[0100] The results are as follows Fig.14As shown in Figures a to d, 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. Fig.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, showing a higher level of inflammation.

[0101] (3) Changes in MAPKs / NF-κB signaling pathways in primary astrocytes after ARRB1 knockdown The primary astrocytes in the non-interference group (NC), the primary astrocytes in the interference group 1 (si1ARRB1), and the primary astrocytes in the interference group 2 (si2ARRB1) were respectively inoculated into 6-well plates, and the cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity until the confluence reached 80%, and then starved in the incubator for 6 hours, and stimulated with TNF-α in the same manner as in Example 1, and continued to be cultured in the incubator, and then the cell proteins at 0 min and 15 min of culture were respectively extracted and identified by Western Blot in the same manner as in Example 1.

[0102] The results are as follows Fig.15 shown by Fig.15 It was found that compared with NC group, 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 increased at 15 minutes after TNF-α stimulation, indicating that compared with NC group, the MAPKs / NF-κB signaling pathway in si1ARRB1 group and si2ARRB1 group was activated.

[0103] The above results indicate that IGF2BP2 targets ARRB1 to inhibit the MAPKs / NF-κB signaling pathway, thereby inhibiting the inflammatory response after spinal cord injury.

[0104] Example 5: Animal experiments to verify the effect of knocking down IGF2BP2 on functional recovery after spinal cord injury 1. Knockdown of IGF2BP2 in astrocytes at the site of spinal cord injury in mice by adeno-associated virus (AAV) This example provides an adeno-associated virus vector AAV9-GFAP-GFP-mir30-shRNA (Mouse Igf2bp2) expressing the IGF2BP2 gene (purchased from Weizhen Biotechnology, wherein the promoter is GFAP, which can target astrocytes to knock down the target gene), the vector resistance is Amp, and the resistance concentration is 100 μg / mL.

[0105] The mice were anesthetized and fixed on a stereotaxic apparatus. A laminectomy was performed at T9 under a stereomicroscope using fiber forceps to expose the spinal cord. Taking the center of T9 as the origin, a 5 μL microsyringe was used to inject 2.5 μL of 1×10-1% ... 13 gc / mL (gc represents AAV virus titer) of the above adeno-associated virus vector was injected at a rate of 500nL / min. The needle remained in place for 5 minutes to allow for adequate penetration before moving to the next injection site.

[0106] At the same time, a negative control group (AAV+shNC) was set up: u6-shRNA (scramble) with a nonsense sequence inserted.

[0107] The knockdown effect was verified by Western Blot method, and the results were as follows Fig.16 shown by Fig.16 It was found that the expression level of IGF2BP2 protein was decreased, which indicated that IGF2BP2 in astrocytes at the spinal cord injury site of mice was successfully knocked down, and IGF2BP2 knockdown spinal cord injury model mice were obtained.

[0108] 2. Effect of knockdown of IGF2BP2 on functional recovery after spinal cord injury in mice (1) BMS score The neurological function of IGF2BP2 knockdown spinal cord injury model mice at specific time points after SCI was evaluated by BMS scoring. The mice were placed in an open field every day one week before surgery to familiarize themselves with the environment. BMS scoring began on the first day after surgery: the mice were placed in an open field and the hind limb movement of the mice was observed. The observation time for each mouse was 5 minutes. The BMS score of the mouse motor function was scored by an experimenter who was familiar with the scoring rules and did not know the grouping situation according to the international scoring standards. The BMS scores of the mice were recorded on the 0th, 3rd, 7th, 14th, 21st, and 28th days after surgery.

[0109] The results are as follows Fig.17 shown by Fig.17 It was found that compared with the control group, the recovery of motor function in IGF2BP2 knockdown spinal cord injury model mice after spinal cord injury was significantly hindered.

[0110] (2) MRI (magnetic resonance imaging) On the 28th day after surgery, the mice underwent magnetic resonance imaging using a small animal magnetic resonance imaging scanner (9.4T Biospec), and then sagittal T2-weighted images were scanned using the ParaVision 6.0 system. The scanning parameters were set as follows: T2-weighted, 320×320 matrix, slice thickness 0.3mm, echo time / repetition time = 24 / 1200ms, and rotation angle 90°.

[0111] The imaging results are as follows Fig.18 As shown in the figure, the red box indicates the size of the hematoma in the injured spinal cord area. Fig.18 It was found that compared with the control group, the hematoma area of ​​IGF2BP2 knockdown spinal cord injury model mice after spinal cord injury was larger, and the spinal cord repair process was significantly hindered, which was consistent with the BMS score results.

[0112] The above results indicate that IGF2BP2 can promote 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 drug for inhibiting 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 in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that 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; the amino acid sequence of ARRB1 is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that 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.

4. The use according to claim 1, characterized in that The IGF2BP2 agonist targets astrocytes.

5. The use according to claim 1, characterized in that The IGF2BP2 agonist includes one or more of compounds, proteins, polypeptides, polysaccharides, glycoproteins, glycopeptides, and nucleic acids.

6. The use according to claim 1, characterized in that The spinal cord injury is secondary spinal cord injury.

7. The use according to claim 1, characterized in that The inflammatory response includes the expression of pro-inflammatory cytokines and chemokines.

8. The use according to claim 7, characterized in that The proinflammatory cytokines and chemokines include IL-1β, IL-6, CCL2, and CXCL2.

9. Use of an ARRB1 agonist in the preparation of a drug for inhibiting inflammatory response after spinal cord injury; the ARRB1 agonist can promote the expression of ARRB1.

10. Use of MAPKs / NF-κB signal transduction pathway inhibitors in the preparation of drugs for inhibiting inflammatory response after spinal cord injury; the MAPKs / NF-κB signal transduction pathway inhibitors can inhibit the activation of P-P38, P-ERK, P-JNK, and P-P65 in the pathway.

11. Application of IGF2BP2 agonist or ARRB1 agonist or MAPKs / NF-κB signal transduction pathway inhibitor in the preparation of spinal cord injury repair drugs.

12. Use of an IGF2BP2 agonist in the preparation of a drug for inhibiting inflammatory response; the IGF2BP2 agonist can promote the expression of IGF2BP2, and IGF2BP2 targets astrocytes.

13. The use according to claim 12, characterized in that 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.

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