Application of METTL1 inhibitor in preparation of medicine for treating inflammation after central nervous system injury

Abstract

The invention relates to application of an METTL1 inhibitor in preparation of a medicine for treating inflammation after central nervous system injury, and belongs to the technical field of biological medicine. The invention proves that the expression level of METTL1 can be increased due to central nervous system injury; compared with mice of a false operation group, the mice of which the METTL1 is specifically knocked down in the astrocytes have the advantages that inflammatory response caused by central nervous system injury is obviously relieved, nerve regeneration is promoted, and the motor function is obviously improved. The METTL1 plays a key role in inflammation of central nervous system injury, secondary pathological changes of the central nervous system injury are reduced by regulating and controlling the m7G methylation level, a new target is provided for treatment of the central nervous system injury, and the METTL1 has important clinical application potential and wide market prospects.

Description

Technical Field

[0001] The invention relates to application of a METTL1 inhibitor in preparing a medicine for treating inflammation after central nervous system injury, and belongs to the technical field of biomedicine. Background Art

[0002] Central nervous system (CNS) injury refers to damage to the brain and spinal cord, including traumatic brain injury (TBI), spinal cord injury (SCI), subarachnoid hemorrhage (SAH), ischemic or hemorrhagic stroke, and some neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, etc.), which is the main cause of death and disability worldwide. Although many clinical treatment measures have alleviated the loss of neurological function to a certain extent, the long-term prognosis of CNS injury and the recovery of neurological function are still not optimistic.

[0003] CNS injury is characterized by two temporal and spatial developments, including primary injury and secondary injury. Primary injury refers to the initial injury caused directly by external forces (such as car accidents, falls, etc.), including the cutting, tearing or extension of axons. Secondary injury is a secondary injury cascade reaction triggered on the basis of primary injury, which includes inflammation, oxidative stress, apoptosis and activation of glial cells. The most studied central nervous system injury inflammation and regeneration include spinal cord injury and traumatic brain injury. Among them, spinal cord injury (SCI) refers to severe neurological dysfunction caused by the destruction of nerve conduction bundles due to damage to the spinal cord by various factors, which is characterized by high morbidity and high disability; Traumatic Brain Injury (TBI) is a structural and functional damage to the brain caused by external forces (such as car accidents, falls, impacts, etc.), and its pathological characteristics include brain tissue contusion, intracranial hematoma and increased intracranial pressure. The overwhelming inflammatory response in the early stages of CNS injury, accompanied by widespread upregulation of cytokines / chemokines and immune cell infiltration, can lead to further damage to neurons and surrounding tissues. Many studies have also shown that inflammation is a huge obstacle to axonal regeneration and functional recovery. Therefore, how to balance and regulate the inflammatory response to maximize its protective effect and reduce its potential harm is the key to treating CNS injury.

[0004] The classical NF-κB pathway in inflammation mainly recruits adaptor proteins to the cytoplasmic domain of the receptors by stimulating proinflammatory receptors (such as the TNF receptor superfamily, the Toll-like receptor family (TLR)) and interleukin cytokine receptors, and in turn recruits the IKK complex. The IKKβ subunit in the IKK complex phosphorylates IκB (mainly IκBα), leading to its ubiquitination and subsequent degradation by the proteasome. The degradation of IκB releases NF-κB dimers (p65 / p50), enabling them to transfer from the cytoplasm to the nucleus. In the nucleus, p65 / p50 dimers bind to the promoters of genes with κB sites, regulate the transcription of these genes, and thus participate in the regulation of various biological processes. (Morgan MJ, Liu ZG. Crosstalk of reactive oxygen speciesand NF-κB signaling. Cell Res. 2011 Jan;21(1):103-15. doi: 10.1038 / cr.2010.178. Epub 2010 Dec 28. PMID: 21187859; PMCID: PMC3193400.)Among them, p65, as part of the NF-κB dimer, is considered to be a key subunit that enters the cell nucleus and regulates gene transcription. At the same time, the phosphorylation modification of p65 has an important influence on its function and activity, which can enhance its ability to bind to DNA and its transcriptional regulatory activity. Therefore, the changes in the expression of p65 and p-p65 directly affect the NF-κB pathway. By regulating the NF-κB pathway, the damage of inflammation to injured tissues can be reduced, and the prognosis and neurological function recovery can be improved.

[0005] In recent years, the regulatory role of epigenetic modification in the occurrence and development of diseases has attracted much attention. 7 -methylguanosine (m 7 G) is one of the most common RNA modifications and has attracted much attention recently. There is increasing evidence that m 7 G plays a key role in the development of human diseases. Internal m 7 G modification has been shown to play a very important role in many aspects of RNA metabolism, including RNA processing, stability, maturation and translation. 7 G modification regulates biological and pathological processes by affecting the metabolism of various RNA molecules (including messenger RNA, ribosomal RNA, microRNA, and transfer RNA). 7 G modification is dynamically regulated by methyltransferases, the most prominent of which is methyltransferase-like 1 (METTL1), which forms a functional methyltransferase complex with the cofactor WD repeat domain 4 (WDR4) to catalyze m 7G methylation, thereby affecting the production, processing and maturation of mRNA, and ultimately mediating a variety of key biological processes. Recent evidence shows that increased METTL1 expression is associated with the occurrence of several types of cancer. For example, in glioma studies, it was found that METTLl was highly expressed in glioma patients and increased with the increase of glioma grade; at the same time, the expression level of METTL1 was significantly correlated with the clinical risk factors and prognosis of glioma patients. The expression of METTLl may be an independent risk factor for the prognosis of glioma patients. (Zhang Miao, Wu Rui, Liu Yue, et al. Research progress on the involvement of N7-methylguanine epigenetic modification in the occurrence and development of malignant tumors [J]. Journal of Chengdu Medical College, 2024, 19 (3): 547-551. DOI: 10.3969 / j.issn.1674-2257.2024.03.037.) However, METTL1 and its mediated m 7 The role of G modification in neurological inflammation or acute and chronic inflammation remains to be explored.

[0006] There are no reports on the effects of METTL1 on inflammation or the NF-κB pathway, and there is no research on the regulatory role of METTL1 in the inflammatory process after central nervous system injury. Therefore, clarifying the role of METTL1 in the inflammatory process after central nervous system injury is of great clinical significance for providing new targets and experimental basis for the treatment of central nervous system injury and designing simple and efficient drug preparations. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides the use of a METTL1 inhibitor in the preparation of a drug for treating inflammation following central nervous system injury, thereby providing a new molecular target for the treatment of central nervous system injury.

[0008] The technical solution of the present invention is as follows: Use of a METTL1 inhibitor in the preparation of a drug for treating inflammation following central nervous system injury.

[0009] Preferably according to the present invention, the METTL1 inhibitor can inhibit the inflammatory pathway NF-κB pathway by inhibiting the expression of METTL1, inhibit p65 phosphorylation, and reduce the degree of inflammation after central nervous system injury.

[0010] Preferably, according to the present invention, the METTL1 inhibitor reduces m by inhibiting the expression of METTL1. 7 G methylation level, which plays a role in inhibiting inflammation.

[0011] Preferably according to the present invention, the METTL1 inhibitor comprises siRNA interfering with the expression of METTL1, or a virus packaged with shRNA interfering with the expression of METTL1.

[0012] Further preferably, the nucleotide sequence of the siRNA is shown as SEQ ID NO.3.

[0013] Further preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2.

[0014] Further preferably, the virus is adeno-associated virus type 9, and the viral vector is AAV9-gfaABC1D-GFP-mir30-shMETTL1, wherein gfaABC1D is an astrocyte-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shMETTL1 is shRNA that interferes with the expression of METTL1.

[0015] Preferably according to the present invention, the central nervous system injury includes spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, neurodegenerative diseases and the like.

[0016] Use of METTL1 inhibitors in the preparation of drugs for promoting nerve regeneration after central nervous system injury.

[0017] Preferably according to the present invention, the METTL1 inhibitor can promote the expression of neurotrophic factors by inhibiting the expression of METTL1, thereby promoting nerve regeneration.

[0018] Preferably according to the present invention, the central nervous system injury includes spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, neurodegenerative diseases and the like.

[0019] A drug for treating inflammation after central nervous system injury or a drug for promoting nerve regeneration after central nervous system injury, comprising an inhibitor of METTL1.

[0020] Preferably according to the present invention, the METTL1 inhibitor comprises siRNA interfering with the expression of METTL1, or a virus packaged with shRNA interfering with the expression of METTL1.

[0021] Further preferably, the nucleotide sequence of the siRNA is as shown in SEQ ID NO.3.

[0022] Further preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.2.

[0023] Further preferably, the virus is adeno-associated virus type 9, and the viral vector is AAV9-gfaABC1D-GFP-mir30-shMETTL1, wherein gfaABC1D is an astrocyte-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shMETTL1 is shRNA that interferes with the expression of METTL1.

[0024] Preferably according to the present invention, the central nervous system injury includes spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, neurodegenerative diseases and the like.

[0025] Beneficial effects: 1. This paper uses spinal cord injury and traumatic brain injury as examples to confirm that central nervous system injury can lead to increased expression of METTL1. The therapeutic effect of METTL1 inhibitors on central nervous system injury was further studied. Compared with the mice in the sham operation group, mice with specific knockdown of METTL1 in astrocytes significantly reduced the inflammatory response caused by central nervous system injury and promoted nerve regeneration, and significantly improved motor function. In terms of mechanism, knockdown of METTL1 leads to m 7 The methylation level of G is reduced, and it can inhibit the phosphorylation of p65 and the conduction of the NF-κB pathway, thereby inhibiting the inflammatory response after central nervous system injury. No researchers in this field have studied the relationship between METTL1 and inflammatory factors, the NF-κB inflammatory pathway, and central nervous system injury. The inventor's research on METTL1 and the central nervous system injury model is pioneering.

[0026] 2. The present invention constructs an adeno-associated virus vector for astrocyte-specific knockdown of METTL1. The vector is appropriately selected in terms of the basic vector, target fragment and insertion position. Animal experiments have confirmed that it can significantly inhibit the inflammatory process, enhance axon regeneration, and promote motor function recovery, and has good application prospects.

[0027] 3. The present invention found that METTL1 plays a key role in inflammation of central nervous system injury and regulates m 7 The discovery that the level of G methylation can reduce the secondary pathological changes of central nervous system injury provides a new target for the treatment of central nervous system injury, which has important clinical application potential and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a bar graph of the mRNA expression level of METTL1 in spinal cord tissue.

[0029] Figure 2This is a Western Blot detection image of METTL1 protein in spinal cord tissue.

[0030] Figure 3 It is a bar graph of the mRNA expression level of METTL1 in brain tissue.

[0031] Figure 4 This is a bar graph showing the mRNA expression level of IL-6, an inflammatory indicator, after knocking down METTL1 in spinal cord tissue.

[0032] Figure 5 This is a bar graph showing the mRNA expression level of IL-1β, an inflammatory indicator, after knocking down METTL1 in spinal cord tissue.

[0033] Figure 6 This is a bar graph showing the mRNA expression level of CCL2, an inflammatory marker, after knocking down METTL1 in spinal cord tissue.

[0034] Figure 7 This is a bar graph showing the mRNA expression level of CXCL2, an inflammatory marker, after knocking down METTL1 in spinal cord tissue.

[0035] Figure 8 This is the ELISA test result of IL-1β after knocking down METTL1 in spinal cord tissue.

[0036] Fig. 9 This is the ELISA test result of IL-6 after knocking down METTL1 in spinal cord tissue.

[0037] Fig.10 This is the ELISA detection result of CCL2 after knocking down METTL1 in spinal cord tissue.

[0038] Fig.11 This is a bar graph showing the mRNA expression level of NGF, an axon regeneration indicator, after knocking down METTL1 in spinal cord tissue.

[0039] Fig.12 This is a bar graph showing the mRNA expression level of BDNF, an axon regeneration indicator, after knocking down METTL1 in spinal cord tissue.

[0040] Fig.13 This is the BMS evaluation score curve of mice after knocking down METTL1 in spinal cord tissue.

[0041] Fig.14 This is the ELISA detection result of IL-1β after knocking down METTL1 in brain tissue.

[0042] Fig.15 This is the ELISA detection result of IL-6 after knocking down METTL1 in brain tissue.

[0043] Fig.16 This is a bar graph showing the mRNA expression level of NGF, an axon regeneration indicator, after knocking down METTL1 in brain tissue.

[0044] Fig.17 This is a bar graph showing the mRNA expression level of BDNF, an axon regeneration indicator, after knocking down METTL1 in brain tissue.

[0045] Fig.18 This is a bar graph showing the mRNA expression level of the inflammatory indicator IL-1β after knockdown of METTL1 in primary astrocytes.

[0046] Fig.19 This is a bar graph showing the mRNA expression level of the inflammatory indicator CXCL2 after knockdown of METTL1 in primary astrocytes.

[0047] Fig. 20 This is a bar graph showing the mRNA expression level of IL-6, an inflammatory indicator, after knocking down METTL1 in primary astrocytes.

[0048] Fig.21 This is a bar graph showing the mRNA expression level of the inflammatory indicator CCL2 after knockdown of METTL1 in primary astrocytes.

[0049] Fig. 22 This is a Western Blot detection of p-p65 and p65 proteins in primary astrocytes after knockdown of METTL1 and stimulation with TNF-α.

[0050] Fig.23 After knockdown of METTL1 in primary astrocytes 7 G Methylation Dot Blot detection diagram.

[0051] Fig.24 This is a bar graph showing the mRNA expression level of METTL1 in primary astrocytes after stimulation with TNF-α over a time gradient.

[0052] Fig.25 m is the time gradient of primary astrocytes stimulated by TNF-α 7 G Methylation Dot Blot detection diagram.

[0053] Fig.26 m 7 G Methylation Dot Blot detection diagram.

[0054] Significance analysis in the above figures: P>0.05 is ns, P<0.05 is *, P<0.01 is **, P<0.001 is ***, and P<0.0001 is ****. DETAILED DESCRIPTION

[0055] The technical scheme of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and consumables involved in the embodiments, unless otherwise specified, are all common commercially available products; the steps and experimental operations involved in the embodiments, unless otherwise specified, are all conventional technical operations in the art.

[0056] This example provides a target that can significantly inhibit the inflammatory process after central nervous system injury and improve neurological function recovery, and is specifically illustrated using the C57BL / 6J mouse spinal cord injury (SCI) and traumatic brain injury (TBI) models as representatives.

[0057] Example 1: Increased expression of METTL1 in astrocytes after central nervous system injury 1.1. Construction of spinal cord injury model First, clean the operating table and sterilize the required surgical instruments in advance by high-pressure steam sterilization. Prepare sterile surgical drapes, sutures, suture needles, and iodine. Fast the mice 8 hours before surgery to avoid vomiting or aspiration during surgery. Prepare 3% pentobarbital solution with distilled water and inject the mice intraperitoneally at a dose of 30 mg / kg for anesthesia. No response to clamping the limbs indicates complete anesthesia. Remove the hair on the back of the mouse to expose the surgical area. Fix the mouse on the operating table in a prone position to ensure the stability of the mouse during surgery. Use iodine cotton balls to disinfect the surgical area to reduce the risk of contamination during surgery. Spread a disposable sterile surgical drape, make a 1.5 cm incision along the midline of the back at the T9 segment of the spine, and bluntly separate the muscles and fascia layer by layer until the spinous process is exposed. Perform T9 laminectomy under a stereomicroscope using fiber forceps to expose the spinal cord. The mice in the sham group were not treated for spinal cord injury after laminectomy. 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 spinal cord of the mice in the SCI group was struck by a pneumatic spinal cord injury striker (68100, RWD, Shenzhen, China) with a strike speed of 1m / s, a strike depth of 2mm, and a striker residence time of 1s. The cleaning, suturing, and disinfection steps and operations were the same as those in the sham group. The mice were placed on a heating blanket for 2-3 hours and placed in a cage after they woke up (the mice had free access to food and water in the cage). To prevent urinary tract infection in mice, the mice were artificially squeezed twice a day to help urinate until they were killed.

[0058] 1.2 Preliminary experimental validation in a spinal cord injury model The mice in the sham group and SCI group were anesthetized by intraperitoneal injection of 3% pentobarbital solution on the 3rd and 7th days after spinal cord injury, respectively, and the spinal cord tissues at the injured site were collected for qPCR and Western Blot to detect the expression levels of METTL1 mRNA and protein.

[0059] The qPCR results showed that Figure 1 Compared with the sham operation group (Sham group), the mRNA level of METTL1 in the SCI group mice gradually increased on the 3rd and 7th days after spinal cord injury.

[0060] Western Blot results showed that Figure 2 , which confirmed that the protein expression level of METTL1 was increased on days 3 and 7 after SCI.

[0061] 1.3 Construction of traumatic brain injury model First, the operating table was cleaned and the required surgical instruments were sterilized by high-pressure steam sterilization in advance. Sterile surgical drapes, sutures, suture needles, and iodine were prepared. The mice were fasted for 8 hours before surgery to avoid vomiting or aspiration during surgery. A 3% pentobarbital solution was prepared with distilled water and anesthetized by intraperitoneal injection at a dose of 30 mg / kg. No response to clamping the limbs indicated complete anesthesia. Then the mice were fixed on the trauma model device, the head of the mouse was depilated and disinfected, and the mouse was positioned 2.0 mm behind the anterior bregma and 2.0 mm outside the midline. The scalp was incised to expose the parietal bone, and the brain tissue was exposed with a micro-drill (RWD, Shenzhen, China). The bone window was expanded to a diameter of 3.5 mm while keeping the dura mater intact. The mice in the sham group were not treated with traumatic brain injury after the bone window was opened. The surgical incision was sutured layer by layer with sterile sutures and disinfected with iodine cotton balls. The mice in the TBI group were struck with a craniocerebral impactor (RWD, 68099, Shenzhen, China) according to the manufacturer's instructions (parameters: impact depth 2 mm, speed 5.0 m / s, dwell time: 200 ms), and the cleaning, suturing, and disinfection steps and operations were the same as those in the Sham group. The mice were placed on a heating blanket for 2-3 hours and placed in a cage after they woke up (the mice had free access to food and water in the cage).

[0062] 1.4 Preliminary experimental validation in a traumatic brain injury model The mice in the sham group and TBI group were anesthetized by intraperitoneal injection of 3% pentobarbital solution on the 3rd and 7th days after traumatic brain injury, respectively, and the brain tissues at the injured site were used to detect the expression of METTL1 mRNA by qPCR.

[0063] The qPCR results showed that Figure 3 Compared with the sham operation group (Sham group), the mRNA level of METTL1 in the TBI group mice gradually increased on the 3rd and 7th days after traumatic brain injury.

[0064] Example 2: In vivo functional experiments after knockdown of METTL1 The construction of cell-specific METTL1 knockdown adeno-associated virus type 9 (AAV9) was completed by Weizhen Biotechnology Co., Ltd. sh-METTL1 group: vector AAV9-gfaABC1D-GFP-mir30-shMETTL1, where gfaABC1D is an astrocyte-specific promoter, sh-METTL1 is shRNA for knockdown of METTL1, and the virus titer is 5.1×10 13 viral genomes / mL; sh-NC group: vector AAV9-gfaABC1D-GFP-mir30 with nonsense sequence inserted, virus titer was 4.6×10 13 viralgenomes / mL.

[0065] Wherein, the nucleotide sequence of gfaABC1D is shown in SEQ ID NO.1; The nucleotide sequence of sh-METTL1 is shown in SEQ ID NO.2.

[0066] 2.1 In vivo functional experiments of the spinal cord injury model after METTL1 knockdown AAV9 of sh-METTL1 group and sh-NC group was injected intrathecally into mice with a microsyringe at a dose of 2 μL / mouse. Spinal cord injury model was established 3 weeks later. The mice were then divided into 4 groups: sh-METTL1+SCI group, sh-METTL1+sham group, sh-NC+SCI group, and sh-NC+sham group. The BMS score was used to evaluate the recovery of hindlimb motor function on days 0, 1, 3, 7, 14, 21, 28, and 35 after spinal cord injury. The mice in the four groups were killed after intraperitoneal injection of 3% pentobarbital solution on day 7, and the spinal cord tissues at the injury site were used for qPCR evaluation of inflammatory indicators (IL-1β, IL-6, CCL2, CXCL2) and ELISA detection (IL-6, IL-1β and CCL2). The mice were killed after intraperitoneal injection of 3% pentobarbital solution on day 35, and the spinal cord tissues at the injury site were used for qPCR evaluation of neural regeneration indicators (NGF, BDNF).

[0067] BMS score: It is suitable for evaluating the recovery of motor function of mice after spinal cord injury. This method was improved and modified by Basso in 2006 based on the BBB score according to the motor characteristics of mice. It is a scoring method specifically for the hind limb function of mice, including the main scoring system and the secondary scoring system. Compared with the BBB score, the BMS scoring system is more sensitive and reliable.

[0068] The test was performed by two experienced researchers. The BMS main score ranged from 0 (no ankle movement) to 9 (completely normal motor ability), and the specific scoring details are shown in Table 1. BMS behavioral assessment was performed on days 0, 1, 3, 7, 14, 21, 28, and 35 after spinal cord injury.

[0069] The specific scoring steps are as follows: ① One day before surgery, place normal mice in an open field to familiarize them with the environment; ② BMS scoring began on the first day after surgery, and the observation period for BMS scoring of mice was 5 minutes; ③ Record the video with a video recorder and analyze it on a computer, or give the score immediately after mastering the scoring rules; ④Result analysis: The BMS score represents the state of recovery of hind limb motor function after spinal cord injury in mice. The higher the score, the better the recovery. A score of 9 is equivalent to that of normal animals.

[0070] Table 1. BMS scoring criteria

[0071] annotation: Mild: less than half activity; Extensive: more than half of the activities; Normal and powerful placement of the lower limbs: actively placing the paw on the ground, with the big toe and the last toe both in contact with the ground; Supporting Weight: The hind legs must be raised high enough so that the base of the tail is off the ground and the knees do not touch the ground during walking; Stepping: The hind legs are able to support the body weight when they initially leave the ground, step forward, and then regain the body weight when they contact the ground; Good coordination: Each forelimb step is accompanied by a hindlimb step, and the hindlimbs step alternately. In order to be able to evaluate, the mouse must step continuously and at a constant speed and at least 3 times its body length. Pauses or hesitations during walking are not included. To test coordination, at least 3 times are required. If one of the 3 times is unsuccessful, it is considered uncoordinated; Paw position: The paw is placed parallel to the body. If it is outward away from the body, it is external rotation, and if it is inward towards the body, it is internal rotation; Severe trunk instability: The hind limbs show obvious postural instability during monitoring, such as extreme leaning, obvious swaying, and near falls.

[0072] The qPCR results showed that Figure 4 , Figure 5 , Figure 6 and Figure 7In the acute phase of spinal cord injury (7 days), the inflammatory-related indicators in the spinal cord tissue of the experimental group (sh-METTL1+SCI group) were significantly reduced compared with those in the control group (sh-NC+SCI group). The ELISA results were as follows: Figure 8 , Fig. 9 and Fig.10 As shown in Figure 2, the inflammatory-related indicators in the experimental group were significantly reduced, and in the chronic stage of spinal cord injury (35 days), Fig.11 and Fig.12 The qPCR results showed that the levels of various neurotrophic factors were significantly increased, and the BMS score results also showed that Fig.13 As shown in the figure, the hind limb motor function of the mice in the experimental group improved more significantly. The above results suggest that specific knockdown of METTL1 in mouse astrocytes significantly reduced the inflammatory response caused by spinal cord injury and promoted nerve regeneration.

[0073] 2.2 In vivo functional experiments in traumatic brain injury models after METTL1 knockdown AAV9 of sh-METTL1 group and sh-NC group was injected intrathecally into mice with a microsyringe at a dose of 2 μL / mouse. Traumatic brain injury model was established 3 weeks later. The mice were then divided into 4 groups: sh-METTL1+TBI group, sh-METTL1+sham group, sh-NC+TBI group, and sh-NC+sham group. The mice in the above 4 groups were killed after intraperitoneal injection of 3% pentobarbital solution on the 7th day, and brain tissues of the injured part were taken for ELISA detection (IL-6, IL-1β); the mice were killed after intraperitoneal injection of 3% pentobarbital solution on the 35th day, and brain tissues of the injured part were taken for qPCR evaluation of neural regeneration indicators (NGF, BDNF).

[0074] ELISA results showed that Fig.14 and Fig.15 In the acute phase of traumatic brain injury (7 days), compared with the control group (sh-NC+TBI group), the inflammation-related indicators in the brain tissue of the experimental group (sh-METTL1+TBI group) were significantly reduced. The qPCR results showed that Fig.16 and Fig.17 , the levels of various neurotrophic factors increased significantly in the chronic stage of traumatic brain injury (35 days). The above results suggest that specific knockdown of METTL1 in mouse astrocytes significantly reduced the inflammatory response caused by traumatic brain injury and promoted nerve regeneration.

[0075] In summary, in in vitro animal experiments, knocking down METTL1 in astrocytes can reduce the inflammatory response after central nervous system injury and promote nerve regeneration and motor function recovery.

[0076] Example 3: In vitro functional experiments after knockdown of METTL1 Take out the suckling mice within 7 days of birth, cut off the head of the suckling mice under sterile conditions, cut the skin and skull along the midline with fine scissors, be careful not to damage the cortex, then peel off the skull on both sides with fine forceps under a stereomicroscope, then remove the entire brain below the olfactory bulb, put it in pre-cooled HBSS (Hank's balanced salt solution), remove the meningeal tissue covering the skull and brain substance, cut the remaining tissue into pieces as much as possible with forceps / scissors, add 0.125% trypsin and DNase, digest it in a constant temperature water bath at 37℃ for 15 minutes, and shake it every 5 minutes. Digestion was terminated with DMEM complete medium, and the cells were centrifuged at 4°C and 1000 rpm for 5 min. Single-cell suspension was prepared after resuspending and filtering. The single-cell suspension was inoculated into a 10 cm cell culture dish containing DMEM complete medium (DMEM, 10% FBS, 1% double antibody), and differentially adhered to the wall in a 37°C incubator for 1 hour. After 1 hour, the upper layer of culture medium in the culture dish was moved to a 25T culture flask coated with PDL (polylysine) in advance to obtain primary astrocytes (Astrocyte, AST).

[0077] 3.1. Detection of inflammation-related indicators: After 5-6 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were divided into 4 groups: Si-NC CON, Si-NC TNF-α, Si-METTL1 CON, and Si-METTL1 TNF-α. When the cells were cultured in DMEM complete medium to 50%-60% confluence, siRNA transfection was performed. After 24 hours of continuous culture, TNF-α protein (working concentration was 10 ng / mL) was given to the TNF-α group, and the control group (CON group) was given an equal amount of PBS solution. After another 12 hours of culture, the cells were collected, and mRNA was extracted for qPCR experiments to detect inflammatory indicators (IL-1β, IL-6, CCL2, CXCL2).

[0078] Wherein: the siRNA nucleotide sequence of the Si-METTL1 group is shown in SEQ ID NO.3; The siRNA of the Si-NC group is a nonsense sequence, and its nucleotide sequence is shown in SEQ ID NO.4.

[0079] The qPCR results showed that Fig.18 , Fig.19 , Fig. 20 and Fig.21 In primary astrocytes cultured in vitro, the inflammation-related indicators in the experimental group (Si-METTL1 TNF-α) were significantly reduced compared with those in the control group (Si-NC TNF-α group).

[0080] 3.2. Detection of NF-κB pathway activation: After 5-6 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. DMEM complete medium was used to culture the cells to 50%-60% confluence, and then siRNA transfection was performed. The control group was Si-NC, and the experimental group was Si-METTL1. The corresponding siRNAs were the same as above. After 24 hours of continuous culture, serum-free medium was switched to starvation medium for 6 hours, and then TNF-α (working concentration was 10 ng / mL) was added to each well to stimulate the cells. Cell proteins were extracted at 0 min, 15 min, 30 min, and 60 min after the addition of TNF-α, and related indicators of the NF-κB pathway were detected by Western Blot.

[0081] Western Blot results showed that Fig. 22 Knockdown of METTL1 significantly reduced the expression level of p-p65, while the level of p65 remained basically unchanged, confirming that knockdown of METTL1 can inhibit the activation of the NF-κB pathway.

[0082] Example 4: Searching for METTL1 and m 7 G methylation relationship and verification: 4.1. METTL1 knockdown in cells 7 G level detection The method for extracting primary astrocytes is as described in Example 3. After 5-6 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were cultured with DMEM complete medium until the confluence reached 50%-60%, and siRNA transfection was performed. The control group was Si-NC, and the experimental group was Si-METTL1. The corresponding siRNA was the same as in Example 3. After 24 hours of continuous culture, mRNA was extracted and the mRNA concentration was determined. Sterile enzyme-free water was used to adjust the mRNA concentration to a uniform concentration, and then the mRNA was diluted to 400 ng / μL, 200 ng / μL and 100 ng / μL for Dot Blot detection. 7 G Methylation levels, membrane stained with methylene blue (MB) as a control.

[0083] Dot Blot results show that Fig.23 Knockdown of METTL1 can significantly reduce m 7 The level of G methylation.

[0084] 4.2. METTL1 content and m in inflammatory conditions in vitro 7 G level detection The method for extracting primary astrocytes is as described in Example 3. After 5-6 days of cell culture, the cells were inoculated into 6-well plates and placed in an incubator at 37°C, 5% CO2 and saturated humidity. The cells were cultured with DMEM complete medium and induced when they reached 50%-60% confluence. TNF-α (working concentration of 10 ng / mL) was added to each well to stimulate the cells. At 0h, 2h, 4h, 6h, 12h and 24h after the addition of TNF-α, the cells were collected to extract mRNA for qPCR experiment to detect the mRNA content of METTL1, and the extracted mRNA was subjected to DotBlot detection. 7 G Methylation levels, membrane stained with methylene blue (MB) as a control.

[0085] The qPCR results showed that Fig.24 , the mRNA expression level of METTL1 increased with the extension of TNF-α stimulation time, and DotBlot results showed that Fig.25 , m 7 The methylation level of G increased with the extension of TNF-α stimulation time, which inferred that under in vitro inflammatory conditions, METTL1 content and m 7 G methylation levels increased synchronously.

[0086] 4.3 METTL1 content and m after spinal cord injury in vivo 7 G level detection The mouse spinal cord injury model was established as in Example 1. The mice in the Sham group and the SCI group were anesthetized by intraperitoneal injection of 3% pentobarbital solution on the 7th day after spinal cord injury and then killed. Spinal cord tissues at the injured site were taken to extract mRNA for Dot Blot detection. 7 G Methylation levels, membrane stained with methylene blue (MB) as a control.

[0087] Dot Blot results show that Fig.26 , SCI group mice after spinal cord injury 7 The methylation level of G increased. Combined with the above-mentioned Example 1, the mRNA level of METTL1 in the SCI group mice gradually increased after spinal cord injury, which inferred that the METTL1 content and m 7 G methylation levels increased synchronously.

[0088] In summary, the present invention confirms that knocking down METTL1 can reduce m 7 The methylation level of G was also verified in vitro and in vivo after spinal cord injury. 7 The changes in G methylation levels were all synchronous.

[0089] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the claims of the present invention.

Claims

1. Use of METTL1 inhibitors in the preparation of drugs for treating inflammation following central nervous system injury.

2. The use according to claim 1, characterized in that The METTL1 inhibitor can inhibit the inflammatory pathway NF-κB pathway by inhibiting the expression of METTL1, inhibit p65 phosphorylation, and reduce the degree of inflammation after central nervous system injury.

3. The use according to claim 1, characterized in that The METTL1 inhibitor reduces m by inhibiting the expression of METTL1. 7 G methylation level, which plays a role in inhibiting inflammation.

4. The use according to claim 1, characterized in that The METTL1 inhibitor includes siRNA interfering with the expression of METTL1, or a virus packaged with shRNA interfering with the expression of METTL1.

5. The use according to claim 4, characterized in that The nucleotide sequence of the siRNA is shown in SEQ ID NO.

3.

6. The use according to claim 4, characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO.

2.

7. The use according to claim 4, characterized in that The virus is adeno-associated virus type 9, and the viral vector is AAV9-gfaABC1D-GFP-mir30-shMETTL1, wherein gfaABC1D is an astrocyte-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shMETTL1 is shRNA that interferes with the expression of METTL1.

8. The use according to claim 1, characterized in that The central nervous system injuries include spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, and neurodegenerative diseases.

9. Use of METTL1 inhibitors in the preparation of drugs for promoting nerve regeneration after central nervous system injury.

10. The use according to claim 9, characterized in that The METTL1 inhibitor can promote the expression of neurotrophic factors by inhibiting the expression of METTL1, thereby promoting nerve regeneration.

11. The use according to claim 9, characterized in that The central nervous system injuries include spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, and neurodegenerative diseases.

12. A drug for treating inflammation after central nervous system injury or a drug for promoting nerve regeneration after central nervous system injury, characterized in that: Including inhibitors of METTL1.

13. The drug according to claim 12, characterized in that The METTL1 inhibitor includes siRNA interfering with the expression of METTL1, or a virus packaged with shRNA interfering with the expression of METTL1.

14. The drug according to claim 13, characterized in that The nucleotide sequence of the siRNA is shown in SEQ ID NO.

3.

15. The drug according to claim 13, characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO.

2.

16. The drug according to claim 13, characterized in that The virus is adeno-associated virus type 9, and the viral vector is AAV9-gfaABC1D-GFP-mir30-shMETTL1, wherein gfaABC1D is an astrocyte-specific promoter, and its nucleotide sequence is shown in SEQ ID NO.1; shMETTL1 is shRNA that interferes with the expression of METTL1.

17. The drug according to claim 12, characterized in that The central nervous system injuries include spinal cord injury, traumatic brain injury, ischemic stroke, subarachnoid hemorrhage, and neurodegenerative diseases.

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